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THE ROLE OF COMPLEMENT IN ACUTE ANTIBODY-MEDIATED REJECTION
OF SKIN GRAFTS IN THE MOUSE
J. H. M. BERDEN
THE ROLE OF COMPLEMENT IN ACUTE ANTIBODY-MEDIATED REJECTION
OF SKIN GRAFTS IN THE MOUSE
PROMOTOR : D r . R . A . P . Koene
CO-REFERENT : D r . P . J . A . C a p e l
THE ROLE OF COMPLEMENT IN ACUTE ANTIBODY-MEDIATED REJECTION
OF SKIN GRAFTS IN THE MOUSE
PROEFSCHRIFT
TER VERKRIJGING VAN DE GRAAD VAN DOCTOR IN DE
GENEESKUNDE AAN DE KATHOLIEKE UNIVERSITEIT TE
NIJMEGEN, OP GEZAG VAN DE RECTOR MAGNIFICUS
PROF. DR. P. G. A. B. WIJDEVELD VOLGENS BESLUIT VAN
HET COLLEGE VAN DECANEN IN HET OPENBAAR TE
VERDEDIGEN OP 27 APRIL 1979 DES NAMIDDAGS TE
4 UUR
door
JOHANNES HENRICUS MARIE BERDEN
GEBOREN TE BLERICK
KRIPS REPRO - MEPPEL
Dit proefschrift werd bewerkt op de afdeling nierziekten (hoofd:
Prof.Dr. P.G.A.B. Wijdeveld) van de Universiteitskliniek voor
Inwendige Ziekten (directeur Prof.Dr. C.L.H. Majoor) van het St.
Radboud Ziekenhuis te Nijmegen.
Deze onderzoekingen werden gesteund door de Nier Stichting Neder
land en door de Stichting voor Medisch Wetenschappelijk Onderzoek
FUNGO. Het proefschrift werd uitgegeven met steun van de Nier
Stichting Nederland.
/¡ом. 9'Ш&.) iwf- ги, Ъщ.
Й-еЬи. IUAU<W O-UjdMJ.
CONTENTS
CHAPTER I
INTRODUCTION AND PROBLEM STATEMENT 9
References 14
CHAPTER II
THE COMPLEMENT SYSTEM 17
2.1. Introduction ig
2.2. Nomenclature of the complement system 20
2.3. Immunochemstry of complement 21
2.3.1. The classical activation unit 21
2.3.2. The alternative activation unit 24
2.3.3. The terminal attack unit 28
2.4. Immunobiology of complement 29
2.4.1. Alteration of the target complex 30
- Immune adherence 30
- Cytolysis 31
- Virus neutralization 32
- Solubilization of immune conplexes 32
2.4.2. Alteration of the micro-environment 33
- Permeability factors 34
- Chemotactic factors 35
2.4.3. Systemic effects of complement 35
- Mobilization of leukocytes 35
- Interrelations between the complement-, the clotting-,
the kinine generating- and the fibrinolytic system 36
2.5. Genetics of the complement system 39
2.5.1. Complement deficiency in man 39
2.5.2. Complement deficiency in animals 41
- C4 deficiency in guinea pigs 41
- C5 deficiency in mice 42
- C6 deficiency in rabbits 43
б
2.5.3. Relation between complément and major histocompatibility
complex 44
2.6. References 46
CHAPTER III
65
66
68
68
69
PARTICIPATION OF COMPLEMENT IN GRAFT REJECTION
A REVIEW OF THE LITERATURE
3.1. Introduction
3.2. Immunofluorescence studies of grafts
3.3. Prevention of complement fixation
3.4. Measurement of complement activity during graft rejection
3.5. Influence of complement depletion or congenital complement
deficiency of the host on allograft survival 75
3.6. Influence of complement depletion or congenital complement
deficiency of the host on xenograft survival 77
3 . 7 . C o n c l u s i o n 77
3 . 8 . R e f e r e n c e s 79
CHAPTER IV
ROLE OF ANTISERUM AND COMPLEMENT IN THE ACUTE ANTIBODY-MEDIATED
REJECTION OF MOUSE SKIN ALLOGRAFTS IN STRAIN COMBINATIONS WITH
INCREASING HISTOCOMPATIBILITY 89
Summary 91
Introduction 91
Materials and methods 92
Results 94
Discussion 96
References 98
CHAPTER V
THE ROLE OF COMPLEMENT FACTORS IN ACUTE ANTIBODY-MEDIATED REJECTION
OF MOUSE SKIN ALLOGRAFTS 99
Summary 100
Introduction 100
Materials and methods 101
Results 102
Discussion Ю З
References 104
CHAPTER VI
A SENSITIVE HAEMOLYTIC ASSAY OF MOUSE COMPLEMENT 105
Summary 107
Introduction 107
Materials and methods 108
Results 110
Discussion 115
References 116
CHAPTER VII
COMPLEMENT DEPENDENT AND -INDEPENDENT MECHANISMS IN ACUTE ANTIBODY-
MEDIATED REJECTION OF SKIN XENOGRAFTS IN THE MOUSE 119
Introduction 120
Materials and methods 121
Results I2 4
Discussion 133
Summary 136
References 137
CHAPTER VIII
SUMMARY 141
8
Chapter I
INTRODUCTION AND PROBLEM STATEMENT
9
Replacement of kidneys v/hich have irreversibly lost their
function is now a generally accepted form of treatment. The main
technical and surgical problems in kidney transplantation have
been solved. However, five year survival rates of transplanted
kidneys in centres participating in the Eurotransplant Organiza
tion are still not higher than 39.6% if there is a four antigen
mismatch and 56.6% if there are no HLA-A and В mismatches (Euro-
transplant Annual Report, 1977). The majority of losses is caused
by rejection of the graft.
After transplantation a dual response can occur which may lead
to rejection. The first type of response is mediated by specifi
cally activated cytotoxic Τ cells. It most often occurs within
a few days after transplantation, and leads to a so-called
cellular rejection of acute onset. In many instances this type of
rejection can be treated with success by increasing the dose of
immunosuppressive drugs. The second type of rejection is mediated
by antibodies specifically directed against the donor antigens.
These antibodies, if complexed with the antigens toward which
they are directed,can trigger a number of effector systems, which
can cause the eventual damage of the graft. One of the important
inflammatory effector systems triggered by antigen-antibody com
plexes is the complement sequence. The humoral type of rejection
has in most instances an insidious onset and responds very poorly
to immunosuppressive treatment. It therefore accounts for a con
siderable amount of graft losses. There exists a host of informa
tion concerning the pathogenic significance of humoral anti-donor
responses for the ultimate fate of the graft (reviewed by Carpen
ter et al, 1976). Anti-donor antibodies in the complement dependent
cytotoxicity assay or a positive antibody dependent lymphocyto-
toxicity were found during rejection episodes in almost 100% of
the cases (Stiller et al, 1976; Gailiunas et al, 1978). Histologic
examinations of grafts removed after chronic rejection show
intimai proliferation and media thickening (Kincaid-Smith, 196 7;
Busch et al, 1969), associated with deposition of IgG on the
vascular endothelium and in the tunica media (Busch et al, 1971).
10
The development of these lesions is associated with circulating
anti-donor antibodies (Jeannet et al, 19 70). The anti-donor
antibodies are not always detectable in the circulation, but this
can not serve as an argument against humorally mediated rejection,
because the antibodies formed can to a great extent be absorbed
by the graft. In these cases they are only detectable in the
circulation after removal of the grafted kidney (Milgrom, 1977) .
The most ideal approach to improve the results of human renal
allografting is the prevention of the above mentioned rejection
responses in the recipient by complete matching for the antigens
coded for by the major histocompatibility complex. This has been
done most successfully in the transplantation of kidneys from
living, МНС-identical sibling donors. In such donor-recipient
combinations graft survival rates between 90 - 100% have been
achieved. However, in cadaveric kidney transplantation complete
compatibility between donor and recipient is very rare because
of the great polymorphism of the МНС-system and the presence of
hitherto unidentified transplantation loci. This problem of
graft rejection calls for a continuing search for better and less
toxic forms of immunosuppression. The recently discovered strong
immunosuppressive activity of cyclosporin A might open new possi
bilities in this area (Borei et al, 1977; Green and Allison,
1978; Calne et al, 1978a> 1978b). In the humoral rejection process
the destructive effects of formed anti-donor antibodies might be
prevented by manipulating the host's complement system. There
exists a great deal of evidence in the literature that complement
participates in humoral graft rejection, especially in human
renal allografting. The literature concerning this aspect is dis
cussed in one of the subsequent sections (chapter III).
Before complement inhibition can become applicable we must
have a better understanding of the fundamental mechanisms that
are operative in this humoral rejection process. In 19 73 an ex
perimental animal model was developed in our laboratory in which
antibody-mediated destruction could be reproducibly elicited
(Koene et al, 1973). The model was based on previous studies in
which it was shown that mouse alloantisera are devoid of cyto
toxic activity versus donor lymphoid cells if fresh mouse serum
11
is used as a complement source. Cytotoxicity becomes only apparent
in the presence of guinea pig complement, and is highest with
rabbit complement (Koene and McKenzie, 1973). Similarly, mouse
skin allografts could not be destroyed by injection of anti-donor
antibodies. Acute antibody-mediated rejection (AAR) of established
skin grafts in immunosuppressed mice could, however, be induced
by the administration of anti-donor serum together with rabbit
complement. Administration of exogenous complement was not in all
donor-recipient combinations a prerequisite to induce antibody-
mediated destruction. Baldamus et al (1973) described a xeno
geneic skin graft model, in which rat skin grafted onto immuno
suppressed mice was destroyed by passive administration of mouse
anti rat serum alone. The mouse endogenous complement was suffi
ciently activated in this model, which was most likely a conse
quence of the greater antigenic disparity. We, therefore, decided
to investigate the influence of antigenic disparity on AAR in a
number of graft experiments in strain combinations with increasing
histoincompatibility. Because of the earlier mentioned differences
in the cytolytic efficiency of different complement sources in
lymphocytotoxicity assays their ability to mediate AAR in vivo
was compared to their cytotoxicity in vitro with the same anti
serum (chapter IV).
From earlier experiments it was known that, if rabbit comple
ment was inactivated prior to its administration along with anti
serum, AAR did not occur (Gerlag, 1975) . This effect has now been
studied in more detail and we have especially focused our attention
on the question which complement factors were necessary for the
induction of AAR. This was done by depleting the complement source
in vitro of distinct complement factors prior to its administration
and by the use of complement factor deficient sera. This approach
can provide information at which level the complement sequence
has to be inhibited in order to prevent AAR (chapter V).
Although manipulation of an administered exogenous complement
species can give fundamental insight into the mechanisms of AAR,
it is indispensable to complete the information thus gained by
studies of the endogenous complement system. Especially its con-
12
sumption during AAR, and on the other hand the influence of in
hibition of the complement sequence on AAR are important in this
regard. For this purpose we used the xenogeneic skin graft model
in the mouse, because in this system AAR is mediated by the en
dogenous complement of the mouse (chapter VII).
Before such a study was possible we had to develop a sensitive
assay for the accurate measurements of mouse complement levels,
since there was no method reported in the literature that had the
desired sensitivity (chapter VI) .
Prior to the presentation of the experimental data a review is
given concerning the immunochemical sequences and immunobiological
effects of the complement system. Special reference is given to
certain complement deficiencies in animals since we used sera of
these animals in a number of experiments (chapter II).
13
REFERENCES
B a l d a m u s , C A . , M c K e n z i e , I . F . С , Winn, H . J . , a n d R u s s e l l , P . S .
( 1 9 7 3 ) : A c u t e d e s t r u c t i o n by h u m o r a l a n t i b o d y o f r a t s k i n g r a f t e d
o n t o m i c e .
J . I m m u n o l . 110, 1 5 3 2 .
B o r e i , J . F . , F e u r e r , C , Magnée , С , a n d S t ä h e l i n , H. ( 1 9 7 7 ) :
E f f e c t s o f t h e new a n t i - l y m p h o c y t i c p e p t i d e c y c l o s p o r i n A i n
a n i m a l s .
Immunology 32, 1017.
Busch, G.J., Braun, W.E., Carpenter, C.B., Corson, J.M., Galvanek,
E.R., Reynolds, E.S., Merrill, J.P., and Dammin, G.J. (1969):
Intravascular coagulation in human renal allograft rejection.
Transplant. Proc. 1, 267.
Busch, G.J., Reynolds, E.S., Galvanek, E.G., Braun, W.E., and
Dammin, G.J. (1971): Human renal allografts. The role of vascular
injury in early graft failure.
Medicine (Baltimore) 50, 29.
Calne, R.Y., Rolles, К., White, D.J.G., Smith, D.P., and Herbert-
son, B.M. (1978a): Prolonged survival of pig orthotopic heart
grafts treated with cyclosporin A.
Lancet 1, 1183.
Calne, R.Y., Thiru, S., McMaster, P., Craddock, G.N., White, D.J.G.,
Evans, D.B., Dunn, D.C., Pentlow, B.D., and Rolles, К. (1978b):
Cyclosporin A in patients receiving renal allografts from cadaver
donors.
Lancet 2, 1323.
Carpenter, C.B., d'Apice, A.J.F., and Abbas, A.K. (1976): The role
of antibody in the rejection and enhancement of organ allografts.
Adv. Immunol. 22, 1.
14
Gailiunas, P., Suthanthiran, M., Person, Α., Strom, Τ.Β., Carpen
ter, C.B., and Garovoy, M.R. (1978): Posttransplant immunologic
monitoring of the renal allograft recipient.
Transplant. Proc. 10, 609.
Gerlag, P.G.G. (1975): Hyperacute afstoting van huidtransplantaten
bij de muis.
Thesis Katholieke Universiteit Nijmegen, pag. 61.
Green, C.J., and Allison, A.C. (1978): Extensive prolongation of
rabbit kidney allograft survival after short-term cyclosporin A
treatment.
Lancet 1, 1182.
Jeannet, M., Finn, V.W., Flax, M.H., Winn, H.J., and Russell, P.S.
(1970): Humoral antibodies in renal allotransplantation in man.
New Engl. J. Med. 282, 111.
Kincaid-Smith, P. (1967): Histological diagnosis of rejection in
renal homografts in man.
Lancet 2, 849.
Koene, R.A.P., Gerlag, P.G.G., Hagemann, J.H.F.M., van Haelst,
U.J.G., and Wijdeveld, P.G.А.В. (1973): Hyperacute rejection of
skin allografts in the mouse by the administration of alloantibody
and complement.
J. Immunol. Ill, 520.
Koene, R.A.P., and McKenzie, I.F.С. (1973): A comparison of the
cytolytic action of guinea pig and rabbit complement on sensitized
nucleated mouse cells.
J. Immunol. Ill, 1894.
Milgrom, F. (1977): Role of humoral antibodies in transplantation.
Transplant. Proc. 9, 721.
Stiller, C.R. (1976): Anti-donor immune responses in prediction
of transplant rejection.
New Engl. J. Med. 234, 978.
15
Chapter II
THE COMPLEMENT SYSTEM
17
2.1. INTRODUCTION
Complement is one of the major effector systems in the inflan-
matory and immunologic response. It consists of a number of
proteins, present in serum in non-active precursor forms (Table
1). Most of these obtain enzyme-like activity after activation,
leading to a sequential interaction of the individual complement
components. After activation by immunoglobulins, fungal or bac
terial products, multimolecular assemblages are formed, prefer
entially on the surface of biological membranes. This can lead
to the alteration of the target complex, and, depending on the
cell type involved, specialized cell functions are initiated
such as histamine release, enhancement of phagocytosis, con
traction of smooth muscles, aggregation and fusion of platelets.
Alternatively, lysis of the cell membrane may occur with conse
quent death of the target cell. Activated complement components
released in the fluid phase can also induce alterations of the
micro-environment such as Increased vascular permeability, and
Chemotaxis. Furthermore, the complement system shows interactions
with other plasma protein systems, e.g. the clotting sequence,
the fibrinolytic system, and the kinm-generating system.
The complement system is most conveniently divided into four
parts. This is schematically illustrated in figure 1. There are
two pathways for activation: the classical and alternative path
way which both lead to the generation of a C3-convertase. The
amplification loop is a positive feed-back cycle, in which
generation of C3b promotes the activation of the alternative
pathway. The terminal "attack" unit is a kind of final common
pathway, which generates Chemotaxis and induces cell lysis.
Spontaneous and generalized complement activation, with its
potentially dangerous effects, is prevented by the rapid intrin
sic decay of the formed enzymes and by the existence of several
control proteins. Table 1 summarizes some characteristics of the
various proteins involved in the complement system. The inrnmno-
chemistry and immunobiology of the various complement components
will be discussed in more detail in the subsequent sections.
18
TABLE I. Physiooaherrtcal aharaoteristzos of proteins of the oorrplement system
Approximate Electro- Serum Cleavage
molecular phoretic concentration fragments
weight(daltons) nobility (ug/ml)
Classiaal pathway
Clq
Clr
Cls
C4
C2
Alternative pathuay
Properdin
Factor D (C3PAse)b
390,000
188,000
86,000
209,000
117,000
223,000
25,000
Factor В (C3PA,GBG)100,000
C3 (BiO 185,000
Ύ2
6
аг
ei
ß2
Y2
«2
ß2
Bi
190
100
120
430
30
25
trace
240
1300
C4a,C4b,C4c,C4d
C2a,C2b(?C-kinin)
Bb(C3A,GGG),
Ba(GAG)
CSajCSb.CScißjA)
C3d(a2D)
Initiating factor IF
C3 nephritic factor
C3NeF
Attack sequence
C5
C6
C7
C8
C9
Control proteins
cT. INH
3iH
C3b.INA(KAF)
AI
170,000
160,000
206,000
95,000
120,000
153,000
79,000
105,000
150,000
100,000
310,000
6
Y
ei
ß2
ß2
<n a
<*2
e ß2
02
?
•p
75
60
55
40
10
180
600
25
7
C5a,C5b
a) Modified, according to Fearon and Austen, 1976.
b) Formerly used synonyms given between parentheses.
19
classical activation , unit r\ \ CA >
\ C2 J ca \ 1
/сзь J / в / 1 D / /
\ / Сэ- convertase
\
С 5-convertase
Ï
/ C 6
C7 ce с э У
terminal attack unit
alternative , activation | unit
\
J amplification loop
Figure 1. Schematic representation of the four functional parts
of the complement system.
2.2. NOMENCLATURE OF THE COMPLEMENT SYSTEM
In 1968 a standard nomenclature for the classical complement
sequence was chosen (Bull. WHO, 1968) to cope with the existing
confusion in terminology (see synonyms table 1). The complement
factors are notated by numbers in the same sequence as they
react in immune hemolysis. Each number is preceded by the symbol
C. For historical reasons an exception has been made for C4,
giving the sequence: C1-C4-C2-C3-C5-C6-C7-C8-C9. The first com
ponent CI comprises 3, possibly 4, distinct protein subcomponents
named Clq-Clr-Cls (and probably Clt). If a component becomes
activated, the activated state is denoted by a vertical bar over
the number for example: cTs. The postscript "i" represents the
inactive state of a previously active component (e.g. C2i) .
If a component is cleaved the postscript "a" is used for the
20
smaller fragment, and the postscript "b" for the bigger one
(C3a-C3b). C3b and C4b further cleave into two fragments each,
which are denoted as C3c-C3d and C4c-C4d. The factors involved
in the alternative pathway are notated in capitals (IF, B, D and
P). The antigen-antibody complex that initiates the classical
pathway is denoted as SA, while the activating substance for the
alternative pathway is represented by S. The control proteins of
the complement sequence are generally abbreviated as Cl.INH
(CI esterase inhibitor); C3b.INA (C3b inactivator); ßlH; and AI
(anaphylatoxin inactivator).
2.3. IMTIUNOCHEMISTRY OF COMPLEMENT
2.3.1. The classical activation unit (fig. 2)
Activation of the factors of this unit leads to the formation
of a C3 convertase. The activation is initiated by binding of the
CI molecule to antigen-antibody complexes, or to non-specifically
aggregated immunoglobulins (IgG and IgM). CI consists of 3 sub-
units Clq, Clr, and Cls presumably in equimolar ratio in the
presence of one molecule Ca which is necessary for the struc
tural integrity of the CI molecule (Lepow et al, 1963) . Recently
a fourth subunit of CI has been described, called Clt (Assimeh
and Painter, 1975). This subunit is not necessary for the hemo
lytic activity of CI, and its precise function is as yet unknown.
The process is started by binding of Clq to the immunoglobulin.
The binding site for Clq resides in the IgG molecule in the
second constant domain (CH2) of the Fc region (Ellerson et al,
1972) , and in the fourth constant domain (CH4) of the IgM mole
cule (Hurst et al, 1975). For the activation of Clq at least
2 IgG antibodies are necessary, which moreover should be located
in a certain spatial relationship, while in the case of IgM one
molecule is sufficient (Borsos and Rapp, 1965). The Clq subunit
consists of 6 identical non-covalently linked subunits, with an
N-terminal collagen-like region and a C-terminal globular segment
(Reíd and Porter, 1976). The six subunits are joined at their
21
SA
^-Clqrs
SA C i
= = inhibition by Cî INH
• S.CÎbiô classical Сз convertase
C4c-C4d
Figure 2. The classical activation unit
N-terminal regions to form a stem fibril from which branch the
individual C-globular segments (fig. 3). Each of these six glo
bular portions can bind to one IgGl, IgG2, IgG3 or IgM antibody.
Binding of at least two globular portions of the Clq molecule
leads to steric changes in the Clr subunit (Reid and Porter,
1976), which results in the formation of cTr, that has an esterase
activity (Naff and Ratnoff, 1968). The natural substrate of cTr
is the Cls subunit, that obtains esterase activity after cleavage.
cTs and cTr are susceptible to the naturally occurring cT in
hibitor (CT.INH) (Pensky et al, 1961). cT.INH stoichiometrically
inhibits the enzymatic function of cTr and cTs (Gigli et al, 1968) .
Only if Clr is formed in such amounts that it locally exceeds the
inhibitory capacity of cT.INH, the next activation step follows.
The substrates of CTs are the complement factors C4 and C2. C4 is
cleaved into two fragments by Cls, a larger fragment C4b which
binds to cell membranes and immune complexes, (Müller-Eberhard
and Biro, 1963) and a smaller fragment C4a that is released into
the fluid-phase. This latter fragment has weak smooth muscle
contractile properties (Schreiber and Müller-Eberhard, 1974).
Bound C4b has also an affinity to immune adherence receptors,
22
present on various cells, to which C3b is preferentially bound
(Cooper, 1969). The binding of C4b to the cell membrane is very
labile; most molecules dissociate and are released into the
fluid phase, where they are rapidly cleaved by C4b-inactivator
into C4c and C4d. This leads to loss of both hemolytic
activity and the capacity to bind to immune complexes or cell
antibody binding site
globular portion (C terminal)
end fibril j „„(аде,,.,.!« J portion
stem fibril Μ N terminal)
Figure 3. Ultrastructural composition of human Clq (after Reid
and Porter, 1976) — , portions of the moleoule pointing
towards the reader; , portions of the moleoule
pointing away from the reader.
membranes. This C4b inactivator is very probably identical with
C3b.INA (Cooper, 1975). The next step after cleavage of C4 is
the splitting of C2 by cTs in C2a and one or more minor fragments
(Polley and Müller-Eberhard, 1968). Before C2 is cleaved, it is
loosely bound to the C4b molecule, which requires the presence
of Mg (Sitomer et al, 1966). The presence of C4b on the cTs
molecule enhances the splitting of C2 by Cls, by an allosteric
modification of cTs or C2 (Gigli and Austen, 1969). The C2a
molecule forms on the cell membrane a bimolecular complex with
C4b, yielding the classical C3 convertase C4b2a, which cleaves
and activates C3 (Müller-Eberhard et al, 1967) . For this C3
23
Splitting activity Cis is not required. Within the C4b2a complex
C4b is responsible for the binding to the target cell membrane
(Müller-Eberhard et al, 1967) while the enzymatic properties
reside in the C2a fragment. After cleavage of C3 and assembly of
C3b into the complex, forming the classical C5 convertase C4b2a3b,
C2a also carries out the enzymatic cleavage of C5 (Cooper, 1971;
Shin et al, 1971). Once formed C3 convertase has a relatively
short half-life (5 min. at 37 C), because C2a easily dissociates
into the fluid phase, where it is readily inactivated into C2i.
The C2i molecule is not re-usable for formation of a C4b2a com
plex in contrast to C14b that again can react with a native C2
molecule to restore C4b2a.
2.3.2. The alternative activation unit (fig. 4)
Activation of the alternative pathway also yields C3 conver
tase activity, but without the use of the classical activation
factors CI, C4 and C2. Activation of the alternative pathway can
be initiated by IgA immunoglobulins, polysaccharides (zymosan,
inulin, endotoxins of gram negative bacteria), dextran sulphate,
T-cell independent antigens, B-cell mitogens (pokeweed), and other
substances (Götze and Müller-Eberhard, 1977). No specific chemi
cal structure responsible for this activation has hitherto been
recognized. However, all activating substances have a polymeric
structure, consist of repeating subunits and contain poly
saccharides. On the surface of the activating substance, a very
labile C3 convertase is formed which is an Mg dependent complex
of the initiating factor (IF), factor B, factor D, and native C3.
The existence of the IF has been discovered after recognition of
a certain protein present in sera of some patients with hypo-
complementaemia and membranoproliferative glomerulonephritis
(Spitzer et al, 1969) or partial lipodystrophy (Sissons et al,
1976). This protein, called C3 nephritic factor (C3MeF), generates,
in the presence of factor B, factor D, C3, and tig , a stable
soluble C3 convertase (Schreiber et al, 1975). Antisera prepared
against C3NeF, remove a factor from normal serum, after which
activation of the alternative pathway with for instance zymosan
24
52-СЭЬРВЬ
Ρ stabilized Сэ convertase
C 3 № F stabilized Сз convertase
C3b dependent Сз convertose (labile)
C5 convertase (labile)
Ρ stabilized CS convertase
Figure 4. Activation sequence of the alternative pathway (Modified, after Götze and Müller-Eberhard, 1977),
сап no longer be induced (Schreiber et al, 1976a). The factor
removed by anti C3NeF antisera was called initiating factor (IF).
However, this IF is a distinct protein, not identical with C3NeF.
The nature of interactions between the activating substances and
IF are completely unknown. However, the initiation is started
by the formation of an initial C3-convertase. After formation of
this convertase on the surface of the activating substance, the
enzyme deposits formed C3b in its vicinity. The formation of C3b
is a prerequisite for the further activation of the alternative
pathway. Soluble or bound C3b has an affinity to factor B, with
which it forms, in the presence of Mg , a bimolecular complex
(Medicus et al, 1976a). This complexed factor В becomes susceptible
to factor D (Vogt et al, 1976) which cleaves factor В in two
fragments Bb (retained in the complex) and Ba (dissociated into
the fluid phase). Complex СЗЬВЬ is the alternative pathway C3
convertase (Müller-Eberhard and Götze, 1972). The formation of
this alternative C3 convertase is further enhanced by C3b (the
so-called amplification loop). The СЗЬВЬ enzyme has when present
in the fluid phase only C3 splitting properties (Medicus et al,
25
1976b) but in the solid phase it also has a slight C5 splitting
activity (Medicus et al, 1976a), which is greatly enhanced when
more than one molecule C3b is present in the СЗЬВЬ complex
(S2-C3b(n)Bb) (Medicus et al, 1976c). This is quite similar to the
classical pathway where the C3 convertase (C4b2a) obtains C5
splitting activity after incorporation of a C3b molecule (C4b2a3b).
Both C3b(n)Bb and СЗЬВЬ have properdin (P) activating activity.
After binding,? undergoes conformational changes by which acti
vated properdin (P) is formed (Götze et al, 1976). The presence
of Ρ stabilizes the C3b(n)Bb complex and prolongs its half-life
by inhibiting the spontaneous dissociation of Bb from the complex
(Fearon and Austen, 1975; Medicus et al, 1976c). Furthermore Ρ
indirectly makes the СЗЬВЬ complex less susceptible to C3b in-
activator (C3b.INA) because C3b.INA cannot cleave C3b into C3c
and C3d as long as C3b is complexed with Bb (Fearon et al, 1976).
In contrast to earlier ideas properdin itself does not play a
central role in the alternative pathway. It is activated rather
late in the sequence, and its sole function seems to be to
stabilize the С3b(n)Bb complex.
As can be seen in figure 4 there exists also another molecular
sequence in the alternative pathway. After formation of S2-C3b
activated fluid phase properdin (P) can bind to the C3b molecule.
In the presence of factor В and D this enzyme obtains C3 splitting
activity which subsequently leads to the formation of a Ρ stabi
lized C5 convertase. (Götze and Müller-Eberhard, 1974). The formed
СЗЬР is susceptible to C3b.INA which splits the complex in S2-
C3d and СЗсР. The latter complex can dissociate with recovery of
P. However, this sequence seems to be less important and there are
even serious doubts whether it exists under physiological condi
tions (Götze and Müller-Eberhard, 1977).
As already mentioned sera of some patients with membrano-
proliferative glomerulonephritis contain a protein called C3NeF.
The presence of C3NeF is accompanied by depressed serum concen
trations of C3, B, and P, but relatively normal concentrations CI,
C4 and C2 (Gewürz et al, 1968; McLean and Michael, 1973) . This
proves that only activation of the alternative pathway has taken
place. If C3NeF containing serum is added to normal serum C3
26
activation will occur. C3NeF has tvo functions, first it induces
the formation of a C3 convertase (Schreiber et al, 1975), second
it stabilizes the C3 convertase so that it becoires insusceptible
to the control proteins of the alternative pathway C3b.INA and
B1H globulin (Daha et al, 1976).
Recently it has been reported that C3NeF is an antibody pro
bably directed against alternative C3 convertase СЗЬВЬ (Davis et
al, 1977). However, this report is in contrast with the ideas of
the group of Müller-Eberhard, who think that C3NeF represents a
permanently active form of IF (Schreiber et al, 1976a, 1976b).
Further elucidation of the structure, function and reaction pro
ducts of IF is necessary for a better delineation of the role of
C3Ner m disease, (see addendum: page 45).
Finally e few remarks should be made regarding the control
systems in the alternative pathway. An important control mechanism
is the lability of the formed enzymes. By their rapid decay a
prolonged and sustained action is prevented. The intrinsic decay
of the СЗЬВЬ complex is the result of the rapid dissociation of
Bb out of the complex. There also exists an extrinsic decay due
to C3b.INA and B1H (fig. 5). C3b.INA was first recognized by
GENERATION STABILIZATION CONTROL
D ι
Сзь.в-^Сзь Bb- 1 -Bo
- W СЗЬ Ρ Bb -
Intrinsic decay
η—Ç*C3b-Βι
CîNeF ^ - » С з ь В ь C3NeFH
Ρ IH extrinsic decoy
СЗЬ INA inactivatran
-!*СЭс »СЗй
- ^ C 3 b P - > C 3 c P . C 3 d
Escape
Сзь Bb СЭМеГ
Figure S. Stabilization and control of alternatzve C3 aonvertase
(after Weiler et al, 1976).
27
Tamura and Nelson (1967), who found that it blocked C3b in immune
adherence and hemolysis. Later on it was found that СЗЬ.ІЫА
cleaved C3b into two fragments C3c and C3d (Ruddy and Austen,
1971). The action of СЗЬ.ІМЛ leads to four effects: destruction
of the C5 convertase activity of C4b2a3b, inhibition of binding
of factor В to C3b, inhibition of immune adherence, and of phago
cytosis. It is the major control protein of the amplification
loop, which can be derived from the observation that a patient
with a genetic deficiency of C3b.lNA had very low levels of C3
and factor В (Alper et al, 1972a). As already mentioned C3b
cannot be cleaved by СЗЬ.ІМА if it is complexed with Bb. The in
trinsic decay of СЗЬВЬ is inhibited by P. The stabilizing effect
of Ρ is antagonized by the second control protein BiH (Weiler et
al, 1976), whereafter Bb can dissociate again out of СЗЬВЬ.
Subsequently C3b can be cleaved by C3b.INA (fig. 5). If the com
plex is stabilized by C3NeF it is much more (approximately 200
fold) resistant to BiH and thus escapes from the control mecha
nisms. This results in sustained C3 cleavage (Daha et al, 1976).
2.3.3. The terminal attack unit (fig. 6)
This phase of the complement sequence is less well understood
than the preceding steps. After formation of the classical or al
ternative C3 convertase, C3 is cleaved into a major fragment C3b
which can be incorporated into the C3 convertase, and a minor
fragment C3a which evokes important immunobiological effects that
will be discussed in the following section. After incorporation
of C3b into the C3 convertase the specificity of the enzyme is
changed, such that it now has C5 splitting activity (Shin et al,
1971; Daha et al, 1976). C5 is cleaved into a major fragment C5b
which interacts physicochemically with C6 and C7 and a minor
fragment C5a that has a variety of immunobiological properties
that will be discussed later. The binding of C6 to C5b must occur
immediately after the generation of C5b, otherwise C5b will decay
into cytolytically inactive C5i (Nilsson and Müller-Eberhard,
1967a). Binding of C6 and later on also C7 can occur both on the
28
classical CS altematn* Cs convertase convertase
С4Ь 2o ЗЬ Cs Сзь п РВь 1 >4 1
^ C 5 a
CSb—»CSI
^ С б С 7
C 5 b 6 7 — » С 5 І 6 7
^ - С
С5Ь-в
^ 6 С 9
• Csb^g
cell lysis
Figure 6. Activation sequence of the terminal attack unit.
membrane and in the fluid phase. The formation of the trinolecular
complex C5b67 is non-enzymatic, v/hich implies that no cleavage
occurs and that its formation is the result of non-covalent assem
bly. The C5b67 complex binds to membranes via its C5b part. If the
fluid phase C5b67 does not encounter a membrane, the binding site
decays and cytolytically inactive С5І67 is formed. The fluid phase
C5b67 complex can also bind via C5b to membranes not sensitized
with antibody, thereby extending the cytolytic effects of comple
ment to "innocent bystander" cells. This mechanism is known as
"reactive lysis" (Thompson and Lachmann, 1970). After formation
of the trimolecular complex C5b67 one molecule C8 becomes attached
whereafter six C9 molecules can bind to C8 to form a decamolecular
complex (Kolb and Müller-Eberhard, 1973). This complex causes
cytolysis by an impairment of osmotic regulation of the cell.
2.4. IMMUNOBIOLOGY OF COMPLEMENT
The biological activities that are generated during the comple
ment cascade are mainly a consequence of activation of C3 and the
terminal attack sequence. These activities can be divided into
three types :
29
1. alteration of the target complex (e.g. immune adherence, cyto-
lysis)
2. alteration of the nicro-environment (anaphylaxis, leucotaxis)
3. systemic effects (interaction with coagulation system etc.).
2.4.1. Alteration of the target complex
Immune adherence
One of the most important biological activities generated
during complement activation is the immune adherence phenomenon.
Immune adherence is the binding of C3b coated immune complexes,
cells, or particles to cells bearing a C3b receptor on their sur
face. Such C3b receptors are present on human polymorphonuclear
leukocytes, macrophages, erythrocytes, В lymphocytes, monocytes
and renal glomerular basement membrane. Presumably an intact C3b
molecule is required, because it was shown that the phenomenon
is inhibited by C3b.INA. (Tamura and Nelson, 1967; Gigli and
Nelson, 1968). Bound C4b shows also an affinity to the same re
ceptors (Cooper, 1969). Binding to the membrane receptor acts as
a signal to start certain cellular processes, and the resulting
biological effects depend on the cell type involved. Immune ad
herence to phagocytic cells results in an enhancement of phago
cytosis (Gigli and Nelson, 1968). The biological importance of
this event can be derived from the observation that in patients
who are genetically deficient in C3 repeated and severe bacterial
infections can occur (Alper and Hosen, 1974). If immune adherence
with non-phagocytosable particles takes place extracellular
release of lysozymes occurs, which can contribute to inflammatory
damage (glomerulonephritis, graft-rejection) (Henson, 1972).
Immune adherence to В lymphocytes seems to be important for antibody
production, because C3 depletion leads to a delayed antibody pro
duction towards thyrrus-dependent antigens but not to thymus-
independent antigens (Pepys, 1974). It seems therefore very likely
that C3 and C3 receptors play a role in the T-B cell cooperation.
There exists also evidence indicating that C3 plays a role in
В cell proliferation. It has been even suggested that the Τ cell
30
independency of certain antigens is caused by the fact that they
can initiate the alternative pathway leading to the formation of
C3b on the surface of these antigens. This can result in trigger
ing of the В cell without Τ cell help (Dukor et al, 1974) . How
ever the fact that in a patient with a genetic deficiency of C3
a normal response to antigens was found, strongly suggests that
C3 is not an absolute requirement for antibody production (Alper
et al, 1972b). Immune adherence to macrophages activates the
macrophage and induces the release of several lysosomal enzymes,
like hydrolases and proteinases. One of these enzymes has C3
splitting activity (Schorlemmer and Allison, 1976) . Since macro
phages synthesize factor C3 (Lai A Fat and van Fürth, 1975) and
factor В (Bentley et al, 1976), and activated macrophages even
release C3b (Allison, 1978), СЗЬВЬ convertase can be formed in
the vicinity of the macrophage. This convertase enhances further
C3b generation which can lead to subsequent activation of other
macrophages, so that serial activation can take place. These in
vitro findings have probably immunopathological significance in
the production of chronic inflammatory responses evoked by bac
terial or fungal cell walls, or other polyanions. They can induce
prolonged macrophage activation, by generation of C3b via the
alternative pathway (Schorlemmer et al, 1977). Furthermore C3b
activated macrophages produce C3a (Ferluga et al, 1978) which has
cytolytic properties against tumor cells (Ferluga et al, 1976).
Immune adherence of C3b to human monocytes gives a marked increase
in the generation of tissue thromboplastin by the monocyte
(Prydz et al, 1977). C3b receptors have not been found on human
platelets, but in experimental animal studies immune adherence
to platelets can lead to secretion of coagulation active and vaso
active intracellular products (Henson and Cochrane, 1971) and to
aggregation of platelets.
Cytolysis
Generation of the membrane attack unit with formation of the
multimolecular C5-C9 complex on the membrane induces cytolysis
31
of cells and gram negative bacteria. Kinetic studies revealed
that cytolysis is a one hit phenomenon, or in other words that
one C5-C9 complex on a membrane is sufficient for cytolysis
(Kitamura et al, 1976). This makes it very unlikely that cyto
lysis is caused by enzymatic attack, because this process is a
multi-hit process. Work by the group of Mayer (Mayer, 1972; 1977;
Hammer et al, 1975; 1977) indicates that the lesion produced is
a transmembrane channe], which is formed by a hydrophobic outer
ring and an hydrophilic inner core. During the formation of the
C5-C9 complex conformational changes of the C8 and C9 molecule
lead to the exposure of hydrophobic polypeptide regions from the
interior of the molecule. If this occurs in the immediate vici
nity of the lipid bilayer of the membrane, insertion of the mole
cule will occur with formation of the transmembrane channel.
Through this transmembrane channel salt and water will flow into
the cell because of the Donnan effect. This causes swelling which
leads to bursting of the cell membrane and release of macro-
molecules .
Virus-neutralisation
Under certain conditions antisera against viruses have little
or no ability to neutralize viruses, unless CI, C4, C2 and C3 are
present, presumably to cover the surface of the virus (Daniels
et al, 1970) .
Solubilization of immune complexes
Immune complexes can interact with the complement system at
several levels. Clq has 6 binding sites for immunoglobulins, while
C4b and C3b can bind to immune complexes.
Recently, Miller and Nussenzweig (1975) described a new function
of the complement system that consists of solubilization of pre
cipitated Ag-Ab complexes. This solubilization is dependent on
C3, which is the effector molecule in this release reaction
(Takahashi et al, 1977). The complex release activity (CRA) is
generated via the alternative pathway, and this can occur in the
32
absence of the classical activation unit (Miller and Nussenzweig,
1975). However, the presence of C2, C4 and Ca leads to a more
efficient release (Czop and Nussenzweig, 1976) . The late compo
nents (C5-C9) play no role in CRA since it was found unaltered
with C5 or C6 deficient sera. The reaction leading to CRA is
probably started by binding of Clq to the Ag-Ab complex, and sub
sequently some C3b molecules are deposited on the immune complexes
via the classical C3 convertase. Via the amplification loop C3b
dependent alternative C3 convertase is formed on the immune com
plexes which greatly enhances further deposition of C3b. The C3b
molecules somehow interact with the complexes and lead to dis
ruption of the lattice formation. After dissociation of antigen
from antibody the C3 molecules prevent reassociation and cross-
linking with antigen (Takahashi et al, 1976). Although this re
lease activity was studied in vitro, it has probably relevance
in vivo, where by this mechanism complement can prevent deposi
tion of immune complexes in tissues. It could explain the finding
that in patients with a congenital deficiency of the complement
system, immune complex mediated diseases are frequently found
(see 2.5.1.) .
2.4.2. Alteration of the micro-environment
Alterations of the micro-environment may be brought about
primarily by complement-mediators formed during complement acti
vation and released in the fluid phase (C3a, C5a). They increase
vascular permeability, contract smooth muscle and attract phago
cytic cells. Alternatively, alterations are induced by secondary
mechanisms, such as release of lysozymes from neutrophils after
immune-adherence with non-phagocytosable particles or surfaces,
or exposure of platelets to vascular basement membrane after
complement mediated damage of endothelial cells. Both primary and
secondary mediators are important in inflammatory processes
associated with immune reactions.
33
Permeability factors
The most important factors that lead to an increased vascular
permeability, generated during complement activation are the split
products of C3 and C5: C3a and C5a. These effects are mediated by
histamine liberated from mast-cells and basophils. Moreover, these
factors have smooth-muscle contractile activity, which is inde
pendent of histamine release (Vallota and Müller-Eberhard, 1973).
The effects of C3a and C5a on smooth muscle and vascular permea
bility promote sludging and margination of leukocytes, thereby
producing optimal conditions for Chemotaxis, which is an addition
al activity of C3a and C5a. The vasoactive properties of C3a and
C5a are susceptible to rapid enzymatic inactivation by anaphyla-
toxin inactivator (A.I.) which cleaves arginine (СЗа), or arginine
or lysine (C5a) from the molecule (Bökisch and Müller-Eberhard,
1970) .
As already mentioned, it has been shown in animal experimental
studies that vasoactive peptides can be released by thrombocytes
after binding of C3b to their C3b receptor.
In sera from patients with heriditary angioneurotic edema
(HANE), which is caused by an autosomal dominant deficiency of
Cl.INH, a complement factor is found which is called "C-kinin".
This protein has vasoactive properties, and is responsible for the
clinical manifestations in HANE patients (Donaldson et al, 1969).
During an attack pathologically increased CI activity can be found
in sera from these patients, accompanied by very low levels of
C4 and C2. (Lepow, 1971). The high cT activity is due to the lack
of its inhibitor Cl.INH, which leads to an increased cleavage of
the substrates of cT i.e. C4 and C2. Intradermal injection of cT
causes a local increase in vascular permeability in normal indi
viduals, an extensive area of angioedema in HANE patients, while
no reaction is seen in C2 deficient patients (Klemperer et al,
1968) . A peptide having the same vasoactive properties has been
isolated from incubation mixtures of CI, C4 and C2. It is very
likely that the polypeptide, (called "C-kinin") responsible for
the clinical features of HANE-patients, is liberated from C2 by
the action of cTs in the presence of C4.
34
Chemotaatia factors
Three products generated during the complement sequence attract
leucocytes by their chemotactic properties: C3a, C5a and probably
fluid phase C5b67 (Ward et al, 1965; Ward, 1972). The most potent
among these three is C5a, C3a has a much lower activity. Although
chemotactic activity of C5b67 has been described in in vitro ex
periments, serious doubts exist whether this effect is of signi
ficance in vivo. Thus, C6 or C7 deficient sera have unimpaired
chemotactic properties (Snijderman et al, 1969; Stecher and Sorkin,
1969; Hannema et al, 1975; Lim et al, 1976). The chemotactic
factors C3a and C5a are not only formed during complement acti
vation but are also liberated through the action of plasmin, tryp
sin and tissue- and neutrophil proteinases (Ward, 1972). Chemo
tactic factors do not only attract neutrophils but also eosino
phils and monocytes. After exposure to chemotactic molecules,
the cell becomes unresponsive to further attraction. This keeps
the cell at the inflammatory focus, so that other cell functions
can come to expression. One of the properties of neutrophils is
to release a proteinase that can form C5a, so that vascular
permeability will increase even more and further accumulation of
leucocytes and monocytes will occur (Snijderman et al, 1971a).
The chemotactic properties are abolished by a serum factor called
chemotactic-factor inactivator. This inactivator is distinct
from anaphylatoxin inactivator, that abolishes the vasoactive
activities of C3a and C5a (Till and Ward, 1975) .
2.4.3. Systemic effects of complement
МоЪгЪъгаЬгоп of leucocytes
During cleavage of C3 a factor is formed (called leucocyte
mobilizing factor) which mobilates leucocytes from the bone-
marrow reserves into the circulation (Rother, 1972). The relevance
of this factor in vivo can be derived from the fact that in pa
tients with a genetic deficiency of C3, leucocytosis does not
occur during bacterial infections (Alper and Rosen, 1974).
35
Inter-relations between the complement-, the alotting-3 the кгпгп
generating-, and the fibrinolytic system
Activation of the clotting system can directly lead to comple
ment activation. The mechanisms involved will be discussed in this
section. Although direct activation in the onposite direction does
not exist, certain effects produced by complement activation
(release of platelet factors due to immune adherence, lysis of
platelets, release of coagulatory enzymes from leucocytes, etc.)
can secondarily induce activation of the coagulation sequence.
A central place in the relation between these enzymatic cascades
is the Hageman factor (F XII), as can be seen in figure 7.
FURTHER ACTIVATION CLOTTING SEQUENCE m
ACTIVATION KININ SEQUENCE
S a -
XI
/ activation
Activated Hageman
(actor Ш a
plasminogen proactivator
^ plasminogen activator
plasminogen •
• positive feed back inhibition by CÎ.INH
plasmin
prekallikrem
* СЗЬ . C3a
Figure 7. Inter-relations between the clotting-, kinin generating-,
fibrinolytic-, and complement system.
36
Activation of Hageman factor leads to four different functions
(Kaplan and Austen, 1975):
a) Initiation of the intrinsic pathway of coagulation, by acti
vating factor XI, which induces further activation of the
clotting sequence.
b) Initiation of fibrinolysis by formation of plasmin, the fibri
nolytic enzyme. Activated factor XII activates plasminogen-
proactivator to plasminogen activator which converts plasmino
gen to plasmin. Plasmin cleaves fibrin and fibrinogen into
smaller fragments.
c) Activation of the kinin-system, by liberation of kallikrein
from prekallikrein. The formed kallikrein cleaves kinins from
kininogen. The formation of kallikrein by activated factor XII
is enhanced by plasmin, which splits Xlla into smaller frag
ments (Xll-f), that have a much greater capacity than Xlla to
form kallikrein out of prekallikrein. Furthermore plasmin can
directly, without F XII, convert prekallikrein to kallikrein.
d) Activation of the complement system. Activated Hageman factor
can also via plasmin, trigger the complement sequence at two
different levels. First plasmin has the capacity of cleaving
CI into its active form cT; second it can split C3 in the fluid
phase into its biologically active fragments C3a and C3b.
The membrane attack unit is not activated by Hageman-factor.
Activation of Hageman factor can occur by contact with bio
logical materials presenting dense negative charges. These inter
act with positively charged aminoacids in the molecule (e.g.
arginine) leading to a rearrangement into the molecule, with
uncovering of the enzymatic site. Collagen, vascular basement
membrane, uric acid, pyrophosphate and a number of insoluble
materials (glass, kaolin, etc.) belong to these activating sub
stances. It has been found that antigen-antibody complexes and
immune aggregates can activate F XII after binding of Clq (which
exhibits a chemical composition similar to that of collagen)
(Austen, 1974). Furthermore, complement-mediated immunologic tissue
injury is very often primarily localized at the surface of blood
vessels. The alterations of the vascular basement membrane induced
37
by this injury can result in activation of Hagenan factor. There
is however as yet no experimental evidence that immune complexes
are able to activate F XII directly.
At several levels inhibition by Cl.INH can occur (see fig. 7),
so that all effects of activated factor XII can become blocked.
Deficiency of Cl.INH, as in patients with heriditary angioneurotic
edema, leads to unappropriate activation of factor XII, resulting
in kinin generation and complement activation, which are res
ponsible for the clinical symptoms.
Activation of factor XII leads to a number of biological
activities. Plasminogen activator and kallikrein both have chemo-
tactic properties (Kaplan et al, 1972; 1973), cleavage products
of fibrinogen and fibrin have also chemotactic properties, and
can enhance vascular permeability (Marder, 1971; Barnhart et al,
1971), while the formed kinins are potent vasoactive peptides.
So far we have presented some data about the influence of the
coagulation system on complement. There exists very little evi
dence that there are also direct interactions in the opposite
direction. Deficiencies of virtually all classical complement
pathway factors have been reported in man, but defects in coa
gulation were not mentioned in these reports (Brown, 1975).
However, in rabbits congenital deficient in C6 (Zimmerman et al,
1971), and in guinea pigs deficient in C4 (Dodds et al, 1977) a
decrease in coagulation activity was found that normalized after
reconstitution of the deficient factor. The fact that coagulation
is normal in humans with C6 deficiency (Heusinkveld et al, 1974) ,
while C6 deficient rabbits have a clotting defect is probably
the result of a peculiar property of the rabbit platelet, in that
it requires all complement factors for the release of initiating
clotting factors (Rosen, 1975), and for aggregation (Fong and
Good, 1975). As already mentioned earlier, in many experimental
animals (including the mouse), but not in man, an indirect rela
tion exists between complement and the coagulation system via the
platelets, because of their C3b receptors. Binding of C3b to these
receptors leads to platelet aggregation with subsequent release
of platelet procoagulants (Henson and Cochrane, 1971). If the
complement cascade is further activated up to C9 on the surface
38
of the platelet, platelet lysis does even occur. This is accom
panied by a substantial increase in the release of platelet
factors (Henson, 1970) . However, even this link between comple
ment and coagulation is incomplete, because it does not lead to
a direct triggering of the coagulation system. It only can
accelerate an already initiated clotting sequence. Probably the
most important interaction between complement and coagulation
takes place on the endothelium of small vessels via a number of
biological activities generated during complement activation.
Generation of C3a and C5a causes increased vascular permeability
by releasing vasoactive amines from basophils and mast cells,
and by inducing release of serotonin from platelets via platelet
aggregation factor liberated fron basophils (Benveniste, 1974).
Neutrophils and monocytes marginate at the sites of altered vas
cular permeability, leading to endothelial cell damage (Stewart
et al, 1974), release of lysosomal enzymes (Janoff, 1970), and
release of a leucocyte procoagulant which can initiate coagula
tion (Kociba et al, 1972; Prydz et al, 1977). Local coagulation
is further enhanced by platelet aggregation on exposed subendo-
thelial surfaces (Baumgartner, 1974), and by Hageman factor
activation by the altered vascular basement membrane.
2.5. GENETICS OF THE COMPLEMENT SYSTEM
2.5.1. Complement deficiency in man
Genetic defects have now been described for almost all the
complement components in man (see table II). In most cases the
defects are transmitted as autosomal recessive traits. Some of
these deficiencies predispose to certain diseases, which are
listed in table II. As mentioned previously deficiency of Cl.INH
is associated with heriditary angioneurotic edema. (Donaldson and
Evans, 1963). The disease consists of recurrent attacks of non
inflammatory swelling localized in the subcutaneous tissues,
intestinal wall, airways and lungs. Laryngeal edema may often be
fatal (Donaldson and Rosen, 1966) . The deficiency is most pro
bably transmitted as an autosomal dominant trait. Attacks are
39
о TABLE il. Deficienaies of the complement system in man*
Deficient factor Clinical findings
Clq
Clr
Cls
C4
C2
C3
C5
C5 dysfunction
C6
C7
С
combined immunodeficiency state
SLE, glomerulonephritis, reumatoid disease
SLE
SLE
none, discoid LE, SLE, anaphylactoid purpura, derraa-
tomyositis, glomerulonephritis
infections
SLE
Leiner's syndrome (eczema and secondary infections)
none, infections
none, Raynaud
gonococcal infection, SLE
Deficient control protein
Cl.INH
С3b.IMA
heriditary angioneurotic edema, SLE
infections
* Based on Day and Good, 1976; Alper and Rosen, 1976, Agnello, 1978
evoked by an unappropriate activation of Cl, possibly via plasmin
generated after activation of Hageman factor. One patient has
been described with a deficiency of another control protein,
C3b.INA (Alper et al, 1972a). In the serum of this patient very
low C3 levels have been found. Clinically the deficiency is accom
panied by a life long history of severe infections.
2.5.2. Complement deficiency in animals
In animals also a number of complement deficiencies (C4, C5
and C6) have been found. Because we made use of these animals in
our experiments (see chapter 5 and 7) the immunobiological effects
that are found in association with these complement deficiencies
will be described here in more detail.
C4 deficienoy in guinea pigs
In 1970 Ellman et al described a strain of guinea pigs that
lacked C4. This deficiency is transmitted as an autosomal reces
sive trait (Ellman et al, 1970). The serum of these animals lacks
hemolytic complement activity, which can be restored by the
addition of normal guinea pig serum or of purified guinea pig C4.
Serum concentrations of the other complement components in C4-
deficient guinea pigs are normal except for C2 (50 percent of
normal value). The alternative pathway of complement is entirely
normal (Frank et al, 1971). A number of immunological mechanisms
have been studied in these animals (Ellman et al, 1971). Passive
cutaneous anaphylaxis, contact and delayed hypersensitivity, and
the cellular exsudative response to foreign body were normal.
Somewhat striking was the finding that the direct and reverse
passive Arthus reaction are unimpaired, while in C6 deficiency
(Biro, 1966) and in experimental C3 depletion (Cochrane et al,
1970) these reactions are diminished. This difference is the
result of the fact that guinea pig antibodies are able to acti
vate the alternative pathway, so that via this bypass mechanism
the late acting complement components can become activated (Sand-
berg and Osier, 1971). C4 deficient guinea pigs show furthermore
41
a decrease in the clearance of autologous erythrocytes coated
with antibodies. Again this can be restored by the addition of
normal guinea pig serum. Clearance of bacteria v/hich is dependent
of alternative pathway activation is normal in C4 deficient guinea
pigs. Induction of Chemotaxis in C4 deficient guinea pigs is
normal, but the kinetics of the production of chemotactic factors
shows a latency period which is characteristic for alternative
pathway activation (Clarck et al, 1973).
CS deficiency in mice
Genetically defined complement deficiency in mice was found in
many inbred mouse strains. The locus coding for the presence or
absence of a β globulin was originally designated He' (Erickson
et al, 1964) or MuBl (Cinader et al, 1964) . Later on it was found
that the product of this locus was C5 (Nilsson and Müller-Eberhard,
1967b). This deficiency is transmitted as an autosomal recessive
trait. The serum of these mice completely lacks hemolytic comple
ment activity. In general C5 deficient mice are as healthy as
normal mice, although there are some immunobiological deficiencies.
C5 deficient mice are more susceptible to infections with Coryne-
bacterium Kutscher! (Caren and Rosenberg, 1966). The clearance of
erythrocytes coated with antibodies and bacteria (S. typhimurium
and E.coli) was equal to normal mice (Stiffel et al, 1964) .
The finding that Chemotaxis for leucocytes was not produced by
C5 deficient serum after incubation with immune complexes or
zymosan, has lead to the identification of the central function
of C5a in Chemotaxis (Ward et al, 1965). This defect was also
found to exist in vivo. Injection of endotoxin into the peritoneal
cavity of C5 normal mice resulted in the generation of a factor
that was chemotactic for mouse polymorphonuclear leucocytes and
was followed by the accumulation of these cells in the peritoneal
fluid. By contrast the same procedure carried out in C5 deficient
mice did not reveal chemotactic activity tested in vitro nor
accumulation of polymorphonuclear leucocytes (Snijderman et al,
1971b). There exist two brief reports in the literature concerning
the Arthus phenomenon in C5 deficient mice, and it was found to
42
be unimpaired in comparison to normal mice (Linscott and Cochrane,
1964; Crisler and Frank, 1965). With immunofluorescence studies
C3 was found in Arthus lesions in C5 deficient mice (Linscott and
Cochrane, 1964). From these findings it might be concluded that
activation up till C3 is sufficient to induce the Arthusphenomenon,
however in C6 deficient rabbits an impaired Arthus reaction has
been found. Since nephrotoxic serum nephritis (Lindberg and Rosen
berg, 1968; Unanue et al, 1967) and autoimmune disease occur in
C5 deficient mice (Howie and Helyer, 1965) it is likely that these
immunologic inflammatory reactions can occur without activation
of the terminal complement attack sequence. Cellular immune res
ponses are normal in C5 deficient mice since normal delayed hyper
sensitivity reactions and skin allograft rejection have been ob
served (Caren and Rosenberg, 1965; Crisler and Frank, 1965) .
Св deficiency in rabbits
In 1961 Rother discovered an inborn defect in the complement
system in certain rabbits, which turned out later to be caused by
a deficiency of C6 (Rother and Rother, 1961; Rother et al, 1966a).
The characteristic is inherited as a single autosomal recessive
trait. The serum of these animals lacks hemolytic complement and
bactericidal activity which are both restored after addition of
C6 (Rubin et al, 1967; Rother et al, 1964a). Passive Arthus
reaction in C6 deficient rabbits was macroscopically absent or
incomplete (Rother et al, 1964b). However, on histologic examina
tion, it was found that the leucocyte infiltration was only slight
ly less than in normal rabbits. The most striking difference was
the complete absence of hemorrhagic necrosis (Biro, 1966), that
is considered to be a hallmark of the Arthus reaction. As already
mentioned these observations are in apparent contrast to those of
unimpaired Arthus reactions in C5 deficient mice (Linscott and
Cochrane, 1964; Crisler and Frank, 1965). However, these latter
studies only mention leucocyte infiltration and fail to give in
formation on the presence of hemorrhagic necrosis. If one assumes
that necrosis was also absent in the C5 deficient mice, the fin
dings can be explained by the hypothesis that, for leucocyte
43
accumulations, activation up to C3 suffices, while activation of
the late complement components is necessary for the development
of hemorrhagic necrosis. Clearance of bacteria, and phagocytosis
of antibody sensitized erythrocytes was unimpaired in C6 deficient
rabbits (Rother and Rother, 1965; Rother et al, 1966a), which
evidently shows that complement-dependent functions up to C5 are
normal. This is also supported by the finding that anaphylatoxin
generation and histamine release were the same for normal and C6
deficient rabbit serum (Giertz et al, 1964) . Nephrotoxic serum
nephritis could be induced in C6 deficient rabbits and had the
same characteristics as in normal rabbits (Rother et al, 1966b;
1967). Reports regarding the cellular immune reactivity in C6
deficient rabbits are contradictory. Delayed hypersensitivity was
found to be absent in one study (Volk et al, 1964), whereas it
was completely normal in another (Biro, 1966). Equally contro
versial findings have been reported on allograft rejection, that
was impaired in a study by Volk et al, 1964 but found to be normal
in another study (Biro, 1966) . As already mentioned slight dis
turbances in the clotting system of C6 deficient rabbits have been
found.
2.5.3. Relation between complement and the major histocompatibili
ty complex
In man the inheritance of a number of isolated complement
deficiencies is linked to the HLA-system (reviewed by Jersild et
al, 1976; and Rittner, 1976). This linkage has been shown for C2,
C4 and probably C8 deficiency. C2 deficiency which is the most
frequently occurring complement deficiency, even shows linkage
with two particular haplotypes (AW25, B19, DW2 and AIO, B18, DW2).
A number of other complement deficiencies (Clr, cT.INH, C6 and
probably C5) does not segregate with HLA. Furthermore the electro-
phoretic polymorphism that has been found for factor В is linked
to HLA. By contrast a comparable polymorphism of factor C3, shows
no linkage to HLA.
Also in the mouse a correlation has been found between H-2
haplotype and hemolytic complement activity (Demant et al, 1973).
44
The genes controlling complement activity are mapped in the S-
region of the H-2 complex. Initially it was found that some mouse
strains had high concentrations of a certain serum protein (called
Ss-protein) and others showed consistently lower levels. This
characteristic was controlled by the S-region in the H-2 complex.
In 1975 Lachmann et al demonstrated that the product of this S-
region was complement factor C4. Furthermore, there is evidence
that at least one gene within the H-2 complex influences C3
levels, but also one or more genes are involved that are not
coded for by H-2 (Ferreira and Nussenzweig, 1975). Recently it
was shown that not only variation in levels of C4 but also CI and
C2 levels are governed by the S-region, since mice with the Ss
high genotype have higher functional levels of CI, C4 and C2 than
strains of the Ss low genotype (Goldman and Goldnan, 1976). The
only known complement deficiency in mice, C5, is coded for by a
single gene which is not located within the H-2 complex (Herzen
berg et al, 1963). A further relationship between H-2 and comple
ment is found in its influence on C3b receptors on lymphocytes.
Complement can trigger a number of cellular responses via the C3b
receptor present on various cells. The density of these C3b
receptors on the lymphocyte membrane is controlled by two genes,
and one of these is linked to H-2 (Gelfand et al, 1974).
In conclusion we can say that there is growing evidence that
especially the early components of complement both in man and in
mice are controlled and influenced by genes of the major histo
compatibility complex (MHC). Within the MHC also genes (SD and
la) are located that code for several responder and stimulator
characteristics of the immune response. Thus, there seems to be
a functional relationship between immune recognition, mediation
and response which is governed by a set of closely linked genes
on the chromosomal region of the MHC.
Addendum (to page 27): Recently it was convincingly demonstrated
that C3NeF is an autoantibody against СЗЬВЬ (Daha, M.R., Austen,
K.F., and Fearon, D.T., J. Immunol. (1978) 120, 1389).
45
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Pepys, M.B. (1974): Role of complement in induction of antibody
production in vivo.
J. Exp. Med. 140, 126.
Polley, H.J., and Müller-Eberhard, H.J. (1968): The second com
ponent of human complement: its isolation, fragmentation by C'l
esterase and incorporation into С'З convertase.
J. Exp. Med. 128, 533.
57
Prydz, H., Allison, A.C., and Schorlemmer, H.U. (1977): Further
link between complement activation and blood coagulation.
Nature 270, 173.
Reid, K.B.M., and Porter, R.R. (1976): Subunit composition and
structure of subcomponent Clq of the first component of human
complement.
Biochem. J. 155, 19.
Rittner, Ch. (1976): Genetic loci of components of the classical
and alternate pathway of complement activation: A new dimension
of the immunogenetic linkage group (HLA) on chromosome 6 in man.
Hum. Genet. 35, 1.
Rosen, F.S. (1975): Immunodeficiency.
In: Immunogenetics and immunodeficiency.
Ed.: В. Benacerraf
M.T.P. (London) 1975, p. 229.
Rother, U., and Rother, К. (1961): Über einen eingeborenen
Komplementdefekt bei Kaninchen.
Z. Immunitätsforsch. Exp. Ther. 121, 224.
Rother, K., Rother, U., Petersen, K.F., Gemsa, П., and Mitze, F.
(1964a): Immune bactericidal activity of complement: separation
and description of intermediate steps.
J. Immunol. 93, 319.
Rother, К., Rother, U., and Schindera, F. (1964b): Passive
Arthus-reaktion bei Komplement-defekten Kaninchen.
Ζ. Immunitäts-Allergieforsch. 126, 473.
Rother, К., and Rother, U. (1965): Studies on complement defec
tive rabbits. IV. Blood clearance of i.v. injected S. typhi by
the reticuloendothelial system.
Proc. Soc. Exp. Biol. Med. 119, 1055.
Rother, К., Rother, U., Müller-Eberhard, H.J., and Nilsson, U.R.
(1966a): Deficiency of Сб in rabbits v/ith an inherited comple-
58
ment defect.
J. Exp. Med. 124, 773.
Rother, К., Vassalli, P., Pother, U., and McCluskey, R.T. (1966b):
Masugi neohritis in С'б defective rabbits.
Fed. Proc. 2S, 309.
Rother, К., Rother, U., Vassali, P., and McCluskey, R.T. (1967):
Nephrotoxic serum nephritis in C6 deficient rabbits. I. Study of
the second phase of the disease.
J. Immunol. 98, 965.
Rother, К. (1972): Leukocyte mobilizing factor: a new biological
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Eur. J. Immunol. 2, 550.
Ruddy, S., and Austen, K.F. (1971): C3b inactivator of man.
II. Fragments raroduced by C3b inactivator cleavage of cell-bound
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J. Immunol. 107, 742.
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Fed. Proc. 26, 602.
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J. Immunol. 107, 1268.
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Immunology 31, 781.
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Complement activation by the alternative pathway and macrophage
enzyme secretion in the pathogenesis of chronic inflammation.
Immunology 32, 929.
59
Schreiber, P.D., and Müller-Eberhard, H.J. (1974): Fourth com
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J. Exp. Med. 140, 1324.
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J. Exp. Med. 14 2, 760.
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J. Exp. Med. 144, 1062.
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Scand. J. Immunol. 5, 70 5.
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J. Immunol. 106, 473.
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N. Engl. J. Med. 29 4, 461.
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adsorption of С'З by EAC'4 : Role of Mg
Immunochemistry 3, 57.
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as the major chemotactic factor.
Infect. Immun. 1, 521.
60
Snijderman, R. , Shin, H.S., and Hausiran, M.H. (1971a): Λ chemo-
tactic factor for mononuclear leukocytes.
Proc. Soc. Exp. Biol. Med. 128, 387.
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Biological activity of complement in vivo. Role of C5 in the
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J. Exp. Med. 134, 1131.
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Science 164, 436.
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XII. Generation of chemotactic activity for polynorphonuc]ear
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Immunology 16, 231.
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Aro. J. Path. 74, 507.
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61
complex.
J. Exp. Med. 146, 86.
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62
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63
Chapter III
PARTICIPATION OF COMPLEMENT IN GRAFT REJECTION
A REVIEW OF THE LITERATURE
65
3.1. INTRODUCTION
Organs and tissues transplanted across a histocompatibility
barrier elicit as a rule an immune response directed against the
graft, which in most instances results in its destruction. This
process may be mediated by cells that are cytotoxic to the graft
(cellular immunity) or it can be initiated by antibodies speci
fically directed against the graft antigens (humoral immunity).
Which of these reactions will occur or will predominate depends
on a host of variables such as the animal model, the type of allo
graft, the presence of specific presensitization, or the concomi
tant use of immunosuppressive treatment.
The rejection of first set allografts in unmodified recipients
is predominantly cellular, although signs of humorally mediated
destruction (vascular lesions, granulocyte invasion, platelet
aggregation, and fibrin deposits) can be found (Carpenter et al,
1976). However, if an individual is sensitized and has antibodies
against the graft, then the destruction after transplantation is
clearly a humoral process. Depending on the concentration of
circulating antibody the graft shows signs of hyperacute rejection.
This phenomenon occurs in primarily vascularized grafts in
several experimental systems and also in human transplantation.
It is most dramatically illustrated by the grafting of a human
kidney to a recipient who has circulating antibodies against the
HLA-antigens of the donor. Within minutes after restoration of
the vascular connections a violent and rapid destruction occurs.
(Kissmeyer-Nielsen et al, 1966; Williams et al, 1968; Patel and
Terasaki, 1969) . Comparable events take place in first set,
primarily vascularized xenografts, where rejection is caused by
naturally existing antibodies against the graft (Perper and
Najarían, 1966; Giles et al, 1970; Rosenberg et al, 1971).
The rejection of skin-grafts is somewhat different from that
of other organ grafts, since in both first- and second set grafts
cellular rejection has already developed before vascular con
tinuity and consequent accessibility to humoral antibodies are
established.
66
In certain experimental models humorally mediated graft des
truction can be induced by the passive administration of anti
bodies. Klassen and Milgrom (1971) and Holter et al (1972)
induced hyperacute rejection of kidney allografts in rabbits by
injection of antidonor serum. In this kind of experiments skin
grafts again react somewhat differently, since they become only
sensitive to the destructive action of antibodies after the graft
has become vascularized, which occurs 4-5 days after transplanta
tion (Gerlag et al, 1975). To study antibody mediated rejection
of skin grafts cellular rejection has to be prevented by immuno
suppressive treatment (neonatal thymectomy, anti lymphocyte serum).
In such systems it has been shown that administration of anti
bodies can indeed induce acute rejection of both xenografts and
allografts of skin (Winn et al, 19 73; Baldamus et al, 19 73; Koene
et al, 1973).
The tissue injury induced by antibodies is a consequence of
the activation of secondary mediator systems of which the comple
ment system is the most important. Activation of the complement
sequence not only generates a number of immunobiological acti
vities important for the inflammatory reaction, but can also
trigger other effector pathways such as coagulation and kinin
generation (see chapter 2). Since this study focuses on the role
of complement in antibody mediated graft destruction we will here
review the relevant literature concerning its participation in
the rejection process. In the subsequent section we will discuss
the literature concerning the participation of antibodies and
complement in the rejection of various grafts as studied by
immunofluorescence techniques. The following section deals with
the reports of the absence of destruction in experimental animal
systems, if non-complement fixing antibodies are administered.
The literature in which complement activity is measured during
graft rejection is also reviewed, and finally attention will be
given to graft experiments in recipients with congenital comple
ment deficiency, or experimental complement depletion.
67
3 . 2 . IIIMUNOFLUORESCENCE STUDIES OF GRAFTS
By immunofluorescence techniques complement components,
particularly C3 and Clq, can frequently be demonstrated in asso
ciation with IgG and IgM m rejected kidney allografts both m
man(Porter et al, 1968; McKenzie and Whittingham, 1968; HcPhaul
et al, 1970; Andres et al, 1970) and in experimental animals
(Lmdquist et al, 1968; Pbbas et al, 1974). The presence of these
deposits does not by itself mean that these immunoglobulins and
complenent have participated in the allograft rejection. However,
their pathogenetic role is supported by a number of observations.
These deposits in the glomerular capillaries are frequently found
at sites where active destruction of the graft occurs and where
the density of HLA antigens is maximal (Sijbesma et al, 1974) .
Immunoglobulin eluates of these grafts react m vitro with donor
cells (Hager et al, 1964; Hampers et al, 1967; Goldman et al,
1971; Pedersen and Morris, 1974) or, alternatively, produce a
lesion indistinguishable from rejection when injected into ani
mals of the donor strain (Spong et al, 1968). Furthermore it was
found that deposits of IgM and C3 in vessel walls in one hour
biopsies correlated in 89% of the cases studied with 1 month
graft failure, while, if these deposits were absent, only 11% of
the grafts failed after one month (Tourville et al, 1977).
Although it is likely that similar mechanisms play a role in
the humoral destruction of skin grafts, this cannot be substanti
ated, since reports on immunofluorescence studies of such grafts
are lacking.
3.3. PREVENTION OF COMPLEMENT FIXATION
A number of experiments is reported which suggest that des
truction cannot occur if the administered antibodies are unable
to activate complement. This was found with F(abl)2 fragments of
antibodies and with antibodies or antibody subclasses that do not
or very poorly fix complement. These preparations can even block
the destructive action of complement fixing antibodies by their
68
prior binding to the antigenic target. Thus, allografts perfused
with donor specific F(abl)2 antibodies prior to transplantation,
did not show acute rejection after transplantation in specifically
sensitized monkeys (Kobayshi et al, 1972; Habal et al, 1973) or
rabbits (Holter et al, 1973). In experiments with rat skin grafts
in mice Winn and coworkers (1973) found that injection of F(ab,)2
fragments of mouse anti rat serum did not induce acute rejection
in contrast to intact antibodies, which gave rise to a violent
humoral destruction. Moreovor, if F(ab1)2 was given prior to the
intact antibodies the rejection process could be blocked. Similar
results were found in our laboratory in an allogeneic skin graft
model (Capei et al, 1979). Results analogous to those with F(ab,)2
fragments were found with fowl-antibodies which do not fix
mammalian complement e.g. chicken anti rat serum in rat skin grafts
in mice (Winn et al, 1973), and with non-complement-fixing 7S Igl
mouse alloantibodies in skin allografts in mice (Jansen et al,
1975) .
These studies convincingly demonstrate that complement acti
vation plays an important role in graft destruction by passively
administered antibodies.
3.4. MEASUREMENT OF COMPLEMENT ACTIVITY DURING GRAFT REJECTION
Another approach to determine whether complement is involved
in graft rejection, is the measurement of total complement acti
vity (CH50 titer) and the determination of isolated complement
factors with functional assays or with immunochemical methods
(radial immunodiffusion assay) during graft rejection. However,
interpretation of the results is difficult for a number of reasons.
Certain complement factors can already be decreased while the
CH50 titer is still unaltered. Furthermore, the activity found
is the resultant of synthesis, catabolism, and tissue distribution.
Thus, a decrease in complement activity does not by itself mean
that there exists an accelerated catabolism, while alternatively
increased catabolism may go undetected when there is also an in
crease in synthesis. Some complement factors react as acute phase
69
-J о
TABLE I. Complement activity during graft réfection
Author Transplantation model
Grafted organ Recipient species
Complement alterations
A. ALLOGRAFT EXPERIMENTS
A.l. Animals
Simonsen, 1953
Guinea et al, 1964
Gewürz et al, 1966
Coppola and Villegas, 1967
Rother et al, 1967b
Coppola et al, 196Θ
Glovsky et al, 1973
Kux et al, 1973
kidney
skin
kidney
skin
skin
skin
skin
kidney
dog
rat
dog
guinea pig
rat
guinea pig
guinea pig
rabbit
no changes
no changes (neither in first- nor in
second set grafts)
no changes
depression of CH50 titer until
rejection was complete
no changes
depression of CH50 and C3 up to C9,
C2 levels unaltered
no changes
intrarenal complement consumption
(arterio-venous differences in CH50)
A.2. Human
Guinea et al, 1964 kidney slight reduction of CH50 titer, more
pronounced reduction of C2
Austen and Russell, 1966
Gewürz et al, 1966
Carpenter et al, 1967
Carpenter et al, 1969
Shehadeh et al, 1970
Kux et al, 1974
Fearon et al, 1977
Ooi et al, 1977
kidney
kidney
kidney
kidney
kidney
kidney
kidney
kidney
nan
man
man
man
man
man
C2 depression
C2 and CH50 depressed in cases with
"accelerated" rejection
initial rise, followed by a depression
of C3
hypercatabolism of C4 and C3
initial rise of C3 and C4, later on
depression
intrarenal complement consumption
indicative for subsequent rejection
modest depressions of factor В and 01H
depression of factor C3 and C4
B. XENOGRAFT EXPERIMENTS
Gewürz et al, 1966 rabbit kidney
sheep kidney
dog
dog
depression of CI, C4, C2, C3 and CH50
depression of CH50
Table II. Influenae of congenital complément def-iciency or complement devletion on allograft survival
Author Transplantation model
Grafted
organ
Recipxent
species
Presensiti-
zation
Method of decomple-
nentation or defi
cient factor
Effect on graft sur
vival
Ä. COMPLEMENT DEPLETION
A.l. Fvrst-set grafts
Fuji et al, 1966
Gewürz et al, 1967
Pepys, 1972
Glovsky et al, 1973
A.2. Second-set grafts
Maillard and Zarco, 1968
Dempster and Brown, 1970
Kux et al, 1971
skin
kidney
skin
skin
skin
skin
dog
chicken
mouse
guinea pig
guinea pig
skin
kidney
kidney
rat
dog
dog
sodiumcopper-
chlorophyllin
CoVFb)
С о VF
CoVF
CoVF
fumaropimaric acid
slight prolongation
no effect
no effect
prolongation during
C3 depression
c) prolongation of MST
from 8.1 to 10.6 days
no effect
skin grafts CoVF
skin graft CoVF
kidney graft citrate intra-
artenally
Kobayashi et al, 1970 kidney monkey skin grafts CoVF
no effect
no effect
prolongation in 50%,
no rejection in 50%
during citrate in
fusion
no effect
Thomas et al, 1977 kidney rabbit
Whittum and Lindquist, 1977 heart rat
B. COMPLEMENT DEFICIENCY
Volk et al, 1964 skin rabbit
Crisler and Frank, 1965
Caren and Rosenberg, 1965
Biro, 1966
skin
skin
skin
mouse
mouse
rabbit
Rother et al, 1967a skin rabbit
Salerno, 1969 spleen cells mouse
Weitzel and Rother, 1970 skin mouse
a) induction of donor specific antibodies
b) CoVF = cobra venom factor
c) MST = median survival time
skin grafts CoVF
skin grafts CoVF
C6
none
none
skin grafts
C5
C5
C6
C6
none
none
C5
C5
no rejection during
complement depletion
prolongation of MST
from 1.04 to 66 hours
delayed rejection in
50%, graft acceptance
in 50%
no effect
no effect
no effect on first,
second and third
set grafts
no effect in 50%,
prolongation in 35%,
acceptance of graft
in 15%
no effect
slight prolongation
reactants, and can be raised by a number of non specific stimuli.
On the other hand complement factors can be lower as a consequence
of immunosuppressive treatment with prednison (Atkinson and Frank,
1973; Sneiderman and Wilson, 1975), during infection, and in
uremic patients (Kult et al, 1974).
It is therefore not surprising that on considering the litera
ture concerning complement activity during graft rejection one
finds contradictory results. The observations are summarized in
table 1.
In experimental allografts in animals in most instances no
changes in complement activity are found, which is in agreement
with the observation that first-set allografts are predominantly
rejected by cellular mechanisms. In all reports on clinical trans
plantation, however, a decreased complement activity has been
found, especially of factor C2. There is no proper explanation
for this difference. Possibly this observed difference is due to
the fact that in clinical situations attempts are made to treat
the rejection, which is not done in the reported experimental
animal systems. Treatment of rejection in man consists mainly of
increasing the dosis of corticosteroids and it has been shown
that these drugs, especially in high doses, depress the levels of
all complement components (Atkinson and Frank, 1973). A further
argument for this might be that in most instances there is a
delay between the onset of clinically diagnosed rejection and the
decrease of complement levels. However, not all complement altera
tions during graft rejection can be contributed exclusively to a
decreased synthesis since Carpenter et al (1969) found an in
creased catabolism of radiolabelled C4 and C3 during rejection.
It is also possible that humorally mediated rejection is more
important in renal allografts in man because the immunosuppressive
therapy tends to suppress cellular immunity more strongly than the
humoral response (Russell and Winn, 1970). As stated above, immuno
suppression was not used in the reported animal systems.
During the rejection of primarily vascularized xenografts in
animals a clearcut and impressive decrease in complement activity
has been observed. We have found analogous results in a xenogeneic
skin graft system and these will be presented in chapter 7.
74
3.5. INFLUENCE OF COMPLEMENT DEPLETION OR CONGENITAL COMPLEMENT
DEFICIENCY OF THE HOST ON ALLOGRAFT SURVIVAL
The best method to prove participation of complement in graft
rejection, is trying to influence graft survival by manipulation
of the host's complement system. For this purpose cobra venom
factor (CoVF) has been mostly used. CoVF is a factor from certain
snake venoms that, via activation of the alternative pathway,
induces a stable C3 convertase, which is not susceptible to the
control mechanisms. It therefore leads to continuous C3 cleavage
and induces C3 depletion (Cochrane et al, 1970).
The results reported in the literature concerning complement
depletion and graft rejection are rather confusing (see table 2).
For first set allografts three authors (Fuji et al, 1966; Pepys,
1972; Glovsky et al, 1973) report prolongation of graft survival,
while Gewürz et al, 1966, and Glovsky et al, 1973 did not find
any delay in rejection. At first sight it is surprising that in
second set grafts, where humoral rejection is generally more domi
nant, three of six authors do not find prolongation after comple
ment depletion of the recipients.
Also, when recipients with congenital complement deficiencies
are used, almost all authors find an unchanged rejection pattern.
The reason for this becomes however clear, if one realizes that
all authors except one made use of skin grafts in their experi
ments. As we already mentioned, humoral rejection is only seen in
skin grafting experiments if the cellular immune response is
suppressed until vascular connections between graft and recipient
are established. If no immunosuppression is given the rejection
process will be predominantly cellular, and therefore not influenced
by complement deficiency. Two authors (Volk et al, 1964; Rother
et al, 1967a) reported prolonged survival of skin grafts, carried
by C6 deficient rabbits, but they also had in their groups a
number of animals that accepted their grafts indefinitely. This
raises serious doubts whether there existed equal differences in
histoincompatibility between all donors and recipients, and for
this reason it must be questioned whether the observed prolongation
75
σι TABLE III. Influenae of complement depletion, or congenital complement deficiency on xenograft survival
Author Transplantation model Method of deconple- Effect on graft survival
Graft Recipient species mentation or defi
cient factor
A. COMPLEMENT DEPLETION
Snijder et al, 1966
Gewürz et al, 1966,1967
L m n et al, 1970
Moberg et al, 1971
Kux et al, 1971
Mejia-Laguna et al,1972
Winn et al, 1973
pig kidney
rabbit kidney
pig kidney
pig kidney
pig kidney
dog kidney
rat skin
dog
dog
dog
dog
dog
rabbit
mouse
CoVF
CoVF
aggregated IgG
carrageenin
citrate
CoVF
citrate
CoVF
CoVF
prolongation from 9 to 123 m m .
prolongation from 5 to 72-150 min.
prolongation from 5 to 22-90 min.
prolongation from 5 to 51 m m .
20 fold prolongation
prolongation from 5 to 30-60 m m .
no rejection during citrate infusion
prolongation from 17 to 74 min.
delayed or no rejection depending
on the dose of CoVF and passively
administered antibodies
B. COMPLEMENT DEFICIENCY
Mejia-Laguna et al,1970 dog kidney rabbit
Winn et al, 1973 rat skin mouse
C6
C5
no effect
delayed rejection depending on the
amount of passively administered
antibodies
was rightly attributed to the C6 deficiency.
3.6. INFLUENCE OF COMPLEMENT DEPLETION OR CONGENITAL COMPLEMENT
DEFICIENCY OF THE HOST ON XENOGRAFT SURVIVAL
In the studies reported (see table 3) acute rejection could be
prevented by depletion of the complement system (in most instances
C3 depletion). However the protection was not complete, since
rejection eventually occurred m all cases.
The few studies that were performed in animals with congenital
complement deficiencies suggest that rejection of xenografts
occurred in spite of a deficiency in the terminal complement com
ponents. Thus, in a study with dog kidneys transplanted to C6
deficient rabbits the rejection was the same as in normal rabbits
(Me^ia-Laguna et al, 1970). Comparable results were obtained by
Winn et al (1973) with established rat skin grafts m C5 deficient
mice. With high dosis of mouse anti rat antiserum humoral destruc
tion of the grafts could be induced.
3.7. CONCLUSION
On reviewing the literature we have come to the conclusion
that complement clearly is involved in the rejection of xenografts.
This is supported by the histologic findings in rejected xeno
grafts. These show the characteristic picture of an Arthus reac
tion, that is known to be complement dependent. From the results
of xenograft studies in complement deficient recipients it seems
likely that not all complement factors are necessary, and that
generation of factors C3a and C5a, with their biologic important
functions, suffices to cause graft damage. However, complement
inhibition did never prevent a rejection, which suggests that
complement independent processes are also involved.
The literature concerning complement participation in allo
graft rejection is contradictory. With regard to skin allografting
in non-immunosuppressed animals most of the available data indi
cates that humoral rejection, and thus complement involvement, is
77
of minor importance.
The reports on primarily vascularized allografts in animals
do not allow a definite conclusion, although the results in sen
sitized recipients make it likely that humoral mechanisms contri
bute to graft damage.
In human kidney transplantation complement participation is
probably of greater importance than in analogous experimental
animal systems. Most rejected human kidneys show characteristics
of antibody mediated graft destruction. This is possibly due to
the fact that the recipients are treated with immunosuppressive
agents which better suppress cell-mediated than humoral immunity.
Manipulation of the complement system might, therefore, be a means
to interfere with this destructive process.
78
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83
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84
coagulation and antibody synthesis.
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N. Engl. J. Med. 280, 735.
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86
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87
Chapter IV
ROLE OF ANTISERUM AND COMPLEMENT IN THE ACUTE ANTIBODY-MEDIATED
REJECTION OF MOUSE SKIN ALLOGRAFTS IN STRAIN COMBINATIONS WITH
INCREASING HISTOINCOMPATIBILITY
J.H.M. Berden, P.G.G. Gerlag, J.F.H.M. Hagemann, and R.A.P. Koene
Department of Medicine, Division of Nephrology, Sint Radboud
Ziekenhuis, University of Nijmegen, Nijmegen
published in: Transplantation (1977) 24, 175
89
ROLE OF ANTISERUM AND COMPLEMENT IN THE ACUTE ANTIBODY-MEDIATED REJECTION OF
MOUSE SKIN ALLOGRAFTS IN STRAIN COMBINATIONS WITH INCREASING
HISTOINCOMPATIBILITY1
J. H. M B E R D E N , P. G G GERLAG, J F H M H A G E M A N N , A N D R A Ρ
KOENE
Department of Medicine, Division of Nephrology, Sint Radboud Ziekenhuis University of Nijmegen, Nijmegen, The Netherlands
SUMMARY
Acute antibody-mediated rejection (AAR) of mouse skin allografts was studied in nine donor-recipient combinations with increasing histoincompatibihty ranging from an Η-Y to a complete H-2 plus non-H-2 disparity AAR was induced by the injection of specific alloantiserum along with a heterologous complement on day 7 after grafting Sera from rabbits, guinea pigs, and from a human volunteer were used as complement sources The recipients were treated with antilymphocyte serum on days 0, 2, and 4 to postpone cell-mediated rejection With increasing histoincompatibihty the mean survival time of untreated grafts decreased, the in vitro cytotoxic activity of the alloantiserum rose, and AAR could be induced with lower amounts of antiserum The higher efficiency of rabbit complement compared with guinea pig complement and human complement, that is known to exist in in vitro cytotoxicity, was also found in vivo Rabbit complement could induce AAR m combination with relatively weak histoincompatibihty (H-2K, H-2D, or non H-2 differences), where guinea pig complement and human complement were ineffective All three complement species elicited AAR if there was a disparity for H-2D plus non-H-2, H-2K plus non-H-2, H-2, or H-2 plus non-H-2 The rules for immunogemcity of the different histocompatibility loci as they have been described for cell-mediated graft destruction also apply to this humorally mediated rejection process
Since the first description of hyperacute rejection of human renal allografts (14), there has been a growing interest in the role of humoral antibodies in graft destruction Primarily, vascularized grafts are clearly susceptible to antibody-mediated damage, not only in man, but also in many experimental animals (2) The role of antibody in the destruction of skin grafts is more controversial Until recently it was assumed that skin grafts were resistant to antibody and that their destruction was brought about by cellular mechanisms However, several models have now been described In which antibodies are responsible for acute skin graft destruction Jooste and Winn ill) and Jooste et al (12) were able to induce acute
1 This work was supported by grants from the Netherlands Foundation of Medical Research (FUNGO) and from the Netherlands Kidney Foundation
destruction of rat skin grafted onto immuno-suppressed mice by the injection of mouse anti-rat serum or of rat antiserum specifically directed against the alloantigens of the donor In a subsequent study they showed that allogeneic rat skin grafted onto immunosuppressed rats could also be destroyed by alloantibody (11) Furthermore, they found evidence that the final damage is caused by the activation of mouse complement (27) In a previous report we have shown that mouse skin allografts can be successfully destroyed by alloantibody provided a heterologous complement, ι e , rabbit complement, is administered along with the antiserum (16) Apparently the mouse endogenous complement is inefficient in this situation and this correlates well with the low efficiency of this complement species in in vitro cytolysis systems (9) In the mouse the study of acute rejection of allografts by antibody is of particu-
91
TRANSPLANTATION
lar interest because its many inbred strains permit detailed genetic analysis The genetics of cell-mediated skin grail destruction has been extensively studied in this species, and it has become evident that there exists a close relationship between the time required for graft, destruction and the extent of histocompatibility differences (7,20,21) Our model of acute antibody-mediated, complement-dependent rejection (AAR) enabled us to study whether an analogous correlation also exists for humorally mediated allograft damage To this end we have studied the occurrence of AAR in nine donor-recipient combinations with increasing histoincompatibihty
Complement activation is necessary for AAR and whether this occurs seems to depend upon the degree of histoincompatibihty between donor and recipient The studies of Jooste and Winn ill) and Jooste et al (12) have shown that mouse complement can be come activated in a xenogeneic system and also in certain allogeneic combinations In our system with limited antigenic differences, rabbit complement was required for the de struction of allografts by antibody Analogous results have been obtained in renal grafts in rats August ra t kidneys in AS recipients were not destroyed by antibody, unless guinea pig complement was administered with the antiserum (6) On the other hand, DA grafts in Lewis recipients, which represent a stronger histoincompatibihty than August to AS, could be destroyed by administration of antibody alone (5) Differences in efficiency of various complement species have also been observed in many in vitro cytolytic systems (4, 9, 10, 17, 26) With mouse alloantibody rabbit complement is superior to guinea pig complement, while rat and mouse complement hardly ever induce complement-dependent cytotoxicity (9, 17) The second aim of this study was, therefore, to test several complement species rabbit complement (RC), guinea pig complement (GPC), and human complement ( H O , for their ability to evoke AAR of skin grafts m combinations with increasing histoincompatibihty and to compare these results with the complement activity in in vitro cytotoxicity
MATERIALS AND METHODS
Mice Inbred lines of BIO D2/new Sn, A/HeJ, BIO Br, and BIO LP mice were originally ob
tained from the Jackson Laboratory, Bar Harbor, Maine C57BL6/Rij mice were obtained from the Radiobiological Institute TNO, Rijswijk, The Netherlands BALB/c and DBA/2 mice were originally obtained from the National Institutes of Health, Bethesda, Maryland CBA/J mice were bought from a commercial animal farm (G L Bomholtgard Ltd, Rij, Denmark)
In our animal laboratory these mouse strains were kept by continuous brother-sister mat-ings (C57BL6/R1J x A/HeJJF, (B6AF1) hybrids were raised in the laboratory All of these strains were analysed with various monospecific alloantisera (obtained from National Insti tutes of Health, Bethesda, Maryland) against private and public specificities of the H-2 complex to rule out the possibility of differences between the presumed and real haplotypes of these strains
Selection of donor-recipient combination Information concerning H 2 haplotypes, H-2 recombinants, and non H 2 differences used in selecting donor-recipient combinations came from Demant (3), Graff and Bailey (7), Graff and Snell (8), and Klein (15) The nine combinations chosen and their antigenic differences are listed in Table 1 The first three combinations comprise only non-H-2 differences in an increasing number Combination 3 represents disparity of almost all known non H 2 loci Combinations 4, 6, and 8 differ in the H-2D region, the H 2K region, and the complete H-2 complex, respectively Since C57BL10 congenie strains served as donors on C57BL6 recipients there is also a difference at the H 9 locus (8) This difference, however, is weak so that there will be no cumulation of immunogemcity between the non-H-2 and H-2 differences ( 7) Furthermore, absorption of the C57BL6 anti-B10 D2 serum with C57BL10 cells did not change the titer of the antiserum in vitro For practical reasons we therefore consider these combinations as differing only at the H 2 complex In combinations 5, 7, and 9 there exists not only a difference at the H 2 complex (H-2D end, H 2K end, and complete H-2, respectively), but there are also a number of non-H-2 differences
Production of alloantisera Alloantisera were prepared by injecting female recipient mice at weekly intervals, ι ρ , with a suspension of approximately 5 χ IO7 donor lymphoid cells in complete Freund's adjuvant For the
92
BERDEN ET AL.
TABLE 1. Antigenic differences of strain combinations used
Combination
1 2
3
4 5
6 7 θ
9
Donor (M)
B6AF, (M) C57BL6
B 1 0 D 2
BIO Br A/HeJ
B10.D2 DBA/2 B 1 0 D 2
DBA/2
• Only "classic" H-2 4 According toU).
Recipient (F)
B6AF, (F) BIO LP
BALBc
B6AF, CBA/J
B6AF, B6AF, C57BL6
C57BL6
specificities
Haplotype
a χ b -> a χ 6
ь — b
d->d
k — a χ 6 0 - *
d - » α χ 6 d -» a χ 6
< i - > 6
H-2 coi
Private
-
H-2D 32 II-2D 4
H.2K 31 H-2K 31 H-2K31 H-2D4
H-2K 31 H-2D4
nplex
Specifici ties"
Public
-
H-2 6, 10, 13, 14, 27, 29, 35, 36, 40, 41, 43.44
H-2 34 H-2 34 H-2 3 ,8 , 10, 13,34, 4 1 , 4 2 . 4 3 , 4 4 , 4 7 , 4 9
H-2 3, 8, 10, 13, 34, 41, 42, 43, 44, 47, 49
28, 42,
40,
40,
are listed Specificties 34 and 47 are probably la specific
Non-H-2 loci
H-Y H-3, H-9, H-13 and probably
two other*
H-1.H-3,H-4,H-7,H-8,H-' 13, H-15, tlm 30, Il-X
H-9 H-I, H-4, H-7, H-9 and prob
ably also H-3 and H-8
H-9 HI, H-3, H-4, H-8, H-13 H-9
HI, H-3, H-4, H-8, H-13
ι ties
anti-H-Y antiserum B6AF, sperms cells were used instead of lymphoid cells. After five injections most animals developed ascites and could be tapped every 1 to 2 weeks. The production of several weeks was pooled and, before use, all antisera were heated at 56 С for 45 min and sterilized by passing through a sterile 0.20-Γημ filter. The cytotoxic titer of the ascites fluid was the same as the titer in the serum of these mice. Antisera were stored at -20 С.
Anhlymphocyte serum (ALS). ALS was prepared by a s.c. injection of 5 to 8 χ IO7 C57BL6 lymphoid cells (thymus and mesenterial lymph nodes) in complete Freund's adjuvant into a goat. This priming dose was followed by weekly i.v. boosters of the same number of C57BL6 lymphoid cells. The animals were bled 1 week after the fourth immunization, and the serum was heated at 56 С for 45 min and absorbed once with C57BL6 RBC (5 volumes of serum/1 volume of packed cells). This ALS was not toxic to the recipients if injected i.p.
Complement. Fresh or fresh frozen sera from New Zealand White rabbits, random-bred SPF Albino guinea pigs, and from a human volunteer were used as sources of complement. Before use all sera were tested for toxicity in vitro (a 1/4 dilution should give less than 10% cytoly-sis of the various donor lymphocytes) and in vivo by injecting 0.5 ml of serum i.v. Blood was obtained in rabbits and guinea pigs by cardiac puncture, in the human by venous puncture. After clotting for 45 min at room temperature
and centnfugation, the serum was divided in aliquots of 0 5 ml and, if not immediately used, stored at -30 С Complement was used only once after thawing.
Serology. Cytotoxic tests were performed using the trypan blue exclusion test. Except in the B6AF! female anti-male antiserum, where B6AF, sperm cells were used, spleen cells were used as the targets. They were prepared free of RBC with Tris-NH,C1. One hundred microliters of cells (5 x 105) in Hank's balanced salt solution (BSS) were incubated with equal volumes of antibody dilutions and RC, GPC, or HC (diluted 1/4) for 30 min at 37 С The titer was defined as the antiserum dilution giving 50% lysis. All tests were performed in triplicate and the following controls were performed: (1) cell control, 100 μ\ of cells in 200 μΐ of Hank's BSS; (2) antibody control, 100 μΐ of cells, 100 μΐ of undiluted antiserum, and 100 μΐ of Hank's BSS; and (3) complement control, 100 μΐ of cells, 100 μΐ of complement, and 100 μΐ of Hank's BSS. In these controls cytolysis should not exceed 10%.
Skin grafting technique. Donor tail skin was grafted onto the flank of the recipients by a modification of the "fitted graft" technique. Instead of plaster bandage we used a band-aid to cover the graft. In all experiments except in combination 1, donor and recipient were females and between 6 to 10 weeks old. The band-aid was removed on the 6th day after transplantation. The fate of the grafts was followed by
93
TRANSPLANTATION
daily macroscopic inspection The grafts were considered to be rejected when no viable epidermis remained The median survival time (MST) and the standard deviations were calculated according to the method of Litchfield (ÍS)
Hyperacute rejection studies All experiments were performed on well established grafts that showed no signs of rejection In all combinations, except combinations 1 and 2, the survival of these grafts was prolonged by nonspecific immunosuppression with ALS (0 25 ml ι ρ ) on days 0, 2, and 4 after grafting Seven days after grafting, the mice received 0 01 to 2 0 ml of alloantiserum directed against the donor antigens and 0 25 ml of RC, 0 5 ml of GPC, or 0 5 ml of HC by ι ν injection through the tail vein The choice of the volumes of complement was based on dose response studies in combination 8, where we found that these amounts provided excess of complement if injected with excess antibody Volumes of alloantiserum lower than 0 5 ml were given in an appropriate dilution in saline, with an end volume of 0 5 ml In each donor-recipient combination control experiments were performed by injecting antiserum alone, complement alone, and 0 25 ml of RC with a particular for that combination nonspecific alloantiserum AAR was defined as the complete necrosis of the skin graft 24 to 72 hr after ι ν injection In most cases this process was completed within 48 hr
Histological examination In the two combinations where we did not see macroscopic signs of acute rejection, histological examinations were performed Vz, 2, and 8 hr after ι ν injec
tion of alloantiserum and complement The skin graft and surrounding recipient skin were removed and fixed in 4% buifered formalin The paraffin sections were stained with hematoxylin and eosin
RESULTS
Cytotoxic titers of alloantisera The titers of the various alloantisera are given in Table 2 In all cases we could detect cytotoxic antibodies, even in the three combinations with only non-H-2 differences Antiserum 1 induced cytolysis of B6AF| sperm In antiserum 2, prepared across an H-3, H-9, and H-13 difference, we could demonstrate cytotoxic activity by increasing the sensitivity of the test with the use of less target cells (W instead of 5 χ IO5) With many non-H-2 antigenic differences present, as is the case in combination 3, the antiserum became highly cytotoxic (i/128 with RC)
Antisera produced across H-2 differences showed a gradual rise in cytotoxicity as the antigenic difference increased The titer was lowest with only an H-2D difference, higher with an H-2K difference, and highest when there was incompatibility for the whole H-2 complex (antisera 4, 6, and 8) Addition of non-H-2 differenceb to each of these H-2 differences (antiserum 5, 7, and 9) gave a further nse in cytotoxic activity with GPC and HC, whereas this increase was absent in the tests with RC The situation is somewhat special with antiserum 5, since this serum does not simply represent an extension of antiserum 4 with non-H-2 antibodies The H-2D 32 difference of combina-
TABLE 2 Cytotoxic titers of alloantisera with different complement species
Combination Alloantiserum
B6AF, (F) anti-BSAF, BIO LP anti-C57BL6 BALB/c anti-BIO D2 B6AF, anti-B10 Br CBA/J anti-A/HeJ B6AF, anti BIO D2 B6AF, anti-DBA/2 C57BL6 anti-BIO D2 C57BL6 anti-DBA/2
(M)
Cytotoxic titer
RC GPC
+° + ••
1/128 1/16 1/512 1/512 1/512 1/3072 1/4096
ND» ND + « 0 1/16 1/12 1/32 1/64 1/128
HC
ND ND + « 0 1/16 1/8 1/32 1/48 1/96
α Cytolysis of sperm cells up to 90% in dilution 1/2 * ND, not done r Cytolysis up to 60% above controls in dilution 1/2, with lower number of target cells (10 /̂100 μΐ), controls
less than 10% lysis '* Cytolysis up to 30% in dilution 1/2
9 4
BERDEN ET AL
tion 4 has been replaced here by an H-2D 4 difference, which is more immunogenic (20) and, furthermore, many public specificities are introduced This could explain the relatively high rise in cytotoxic activity of antiserum 5 as compared with antiserum 4
In accordance with previous studies (4,9,10, 17), we found that in all instances the cytotoxicity was highest if measured with RC as compared with GPC or НС To test the activity of mouse complement, each test was also performed with fresh recipient mouse serum, but cytolysis clearly above control levels was never observed
Acute antibody-mediated rejection The results are summarized in Table 3 Control grafts placed on recipients that received ALS survived with MST's ranging from 17 8 ± 1 2 to 33 9 ± 1 4 days Thus, in no case were there signs of rejection on the day of ι ν injection (day 7) and the following 3 days Not shown are the control experiments performed in each donor-recipient combination with administration of alloantise-rum alone, RC, GPC, or HC alone, or nonspecific alloantiserum along with RC In no case was AAR observed and the MSTs of these ex
periments did not differ significantly from the MST's of the ALS-treated animals
Non-H-2 differences (combinations 1, 2, and 3) We were not able to induce AAR across an Η-Y barrier nor across an H-3, H-9, and H-13 barrier In the B6AF, male onto female combination it was, however, possible to reduce the survival t ime of the skin grafts from 126 to 99 8 days by administration of alloantiserum and RC Microscopic examination 11г, 2, and 8 hr after ι ν injection of antiserum and RC did not show any signs of rejection In the second combination, C57BL6 onto BIO LP, where macro-scopically no signs of rejection occurred, we found microscopically 2 hr elfter administration of antibodies and RC a slight increase of granulocytes in the blood vessels of the graft and in the surrounding tissues of the graft with focal extravasation of erythrocytes Eight hours after the injection, these rejection signs were already less extensive This suggests the induction of a reaction that was not severe enough to cause visible rejection of the graft When skin was grafted across a great number of non-H-2 differences (combination 3), the MST of the untreated animals came into the range of
TABLE 3 AAR of mouse skin allografts in strain combinations with increasing histoincompatibihty
Combi nation
1 2
3
4
5
6
7
8
9
Donor -» Recipient (M-.F)
B6AF, (M) - B6AF, (F) C57BL6 — BIO LP
BIO D2 -» BALB/c
BIO Br - B6AF,
A/HeJ - CBA/J
BIO D2 ^ B6AF,
DBA/2 -. B6AF,
BIO D2 — C57BL6
DBA/2 -> C57BL6
MST in untreated (I) and immunosuppreseed
(II)
No of animals
HO
из 112
II 20
no II 20 110
1110
115 II 18
no ino
no и i3
115 II 14
animals"
Days ±
>120 15 4 ±
10 4 ± 24 0 ±
12 8 ± 31 9 ±
9 8 ± 24 0:!:
10 0 ± 21 2 ±
9 5 ± 22 0 ±
9 3 ± 19 0 ±
85 ± 17 8 ±
SD
5
1 1 2
RC
Antiserum (ml)
2 5" 2 5"
0 5 10 15 10 15 0 01 0 05
0 01 0 05
0 01 0 05
0 005 0 01
0 005 0 01
b
AAR'
0/15 0/8
0/6 0/11 6/6 0/12 8/8 0/6 6/6
0/6 6/6
0/6 11/11
0/8 8/8
0/8 6/6
JFC
Anti serum
(ml)
15
15
05 10 15 05 10 15 2 0 05 10 15 0 01 0 05
0 01 0 05
ND' ND
ъ
AAR'
0/7
0/β
0/7 0/6 7/7 0/7 0/8 0/9 0/6 0/6 0/6 6/6 0/5 8/8
0/5 6/6
HC'
Anti serum (ml)
15
15
15
15
15
05
05
ND
ND
AAR'
0/5
0/5
8/8
0/8
8/8
10/10
8/8
" Immunosuppression by ι ρ injection of 0 25 ml of GAMLS on day 0, 2, and 4 after grafting 6 AAR was induced by ι ν injection of listed volumes of antiserum along with 0 25 ml of RC, 0 5 ml of GPC, or 0 5 ml of
HC on day 7 after grafting r Number of mice that show complete graft necrosis within 24 to 72 hr after injection/number of mice tested d 1 5 ml was given ι ν and 1 0 ml ι ρ ' ND, not done
9 5
TRANSPLANTATION
MST's of grafts acrctes an H-2 b a r n e r In this combination AAR could be induced with 1 5 ml of antiserum and RC Lower doses of antiserum did not cause AAR, although there was a decrease of several days in the MST of the skin grafts With GPC and HC no AAR could be induced
H-2 differences (combinations 4, 6, and 8) Induction of AAR across an H-2D incompatibility was only possible with 1 5 ml of alloantise-rum and RC, while across an H-2K incompatibility it was obtained with 0 05 ml of alloantise-rum If there existed a complete H-2 complex disparity, the amount of alloantiserum necessary for induction of AAR with RC was further reduced to 0 01 ml In this latter combination it was also possible to induce AAR with GPC and HC but the dose of alloantiserum had to be higher than with RC
H-2 and non-H-2 differences (combinations 5, 7, and 9) AAR with RC of skin grafted across H-2D plus non-H-2 differences or across H-2K plus non-H-2 differences was obtained with 0 05 ml of alloantiserum whereas only 0 01 ml was necessary for rejection of a graft differing at the whole H-2 complex plus non-W-2 loci Thus, the introduction of non-H-2 differences did not lead to a lower amount of alloantibody necessary for AAR with RC The A/HcJ -» CBA/J combination formed an exception, but as already mentioned in this combination not only H-2 differences are introduced but there is also a change in the H-2 difference compared with BIO Br -> B6AF, For GPC and HC we found that the amount of alloantiserum required for AAR decreased when non-H-2 differences were introduced In BIO D2 onto BBAF, we could not induce AAR with 2 0 ml of alloantiserum, but in the DBA/2 onto B6AF, combination 1 5 ml of antiserum given with GPC or HC was effective This must be the result of the cumulative effect of non-H-2 differences In the in vitro lymphocytolysis we observed a similar pattern The addition of non-H-2 differences to H-2 differences increased the cytotoxicity if the antiserum was titrated with GPC and HC, but the titer with RC did not change (Table 2) Figure 1 gives a graphical summary of the results of this study
DISCUSSION
Our results show that the sensitivity of skin grafts to the destructive effects of alloantibody and heterologous complement increases with
the extent of antigenic differences between donor and recipient In a combination with a few non-H-2 differences, we found only microscopic signs of tissue damage, even if a high amount of specific antiserum was administered In the combinations with H-2 differences, К end-in-compatible grafts were more easily destroyed than D end-incompatible grafts, whereas with complete H-2 and non-H-2 differences a violent rejection could be induced with minimal amounts of antiserm This increase in sensitivity was accompanied by a gradual decrease in MST of the skin grafts in untreated animals in which rejection is presumed to be mediated mamly by cellular mechanisms The decrease in MST that occurs when the antigenic differences become greater has been described earlier (7, 20, 21), and it has been shown in those studies that the MST can even be used as a measure for the degree of immunogemcity of the graft The rules of immunogemcity that have been found for these cellular immune responses (7) also apply to our model where the destruction is mediated by humoral mechanisms
A second aspect that deserves to be mentioned is the finding that there were clear-cut differences in the activity of a given antiserum if tested with different complement species The lower in vitro activity of GPC compared with RC has been described by several authors (4,9, 10,17) and is confirmed in this study Furthermore, we found that HC behaves more or less like GPC in this respect In all of the combinations studied, we found similar differences in complement activity in vivo RC was always more active than GPC or HC These two latter complement species only did work in combinations with strong antigenic differences The argument about the relationship between in vitro and in vivo complement activity extends to mouse complement This complement species is very inefficient in vitro, and was apparently not sufficiently activated in our in vivo system to induce antibody-mediated destruction
The correlation between in vitro and in vivo activity raises the question whether the mechanism of AAR is exactly the same as the in vitro cytolysis, or in other words, whether the complement pathway has to become completely activated to induce AAR Winn et al (27) found in their model, where mouse complement was the active complement, that in C5-deficient mice the process was delayed, while severe de-
9 6
BERDEN ET AL
Minimal volume of a n t i s e r u m ( m l )
lo
i s 1
O l 0 05
О 01
• RC
D GPC
Ш не
и (cytotoxic t i t e r ) ' of a n t i s e r u m
4 0 9 6 -
2 0 4 8
1024 -
512 -
256
12Θ -
6 4
32
16
8 -
4 -
2 -<2 -
Histocompatibil ity barr ier
Η-Y H-3 H-2D multiple H 2K H 2D H-2K H 9 non H-2 plus plus H-13 ηοπΗ2ηοπΗ-2
H-2 H-2 plus
non H-2
MST untreated animals (days) >126 15 4 12 8 10 4 10 0 9 8 9 5 9 3 8 5
FIGURE 1 Acute antibody-mediated rejection in strain combinations with increasing histoincompatibil-ity Relationship between MST of grafts in untreated recipients, in vitro cytotoxic titer of antiserum, and minimal amount of antiserum required to induce AAR with different complement species (RC, GPC, and HC)
pletion of granulocytes could abolish rejection.
They concluded from these observations that
the role of complement is merely one of gener
ating the chemotactic substances C3a and C5a
Histological studies show that in our model
similar mechanisms play a role, since we found
earlier a dense accumulation of granulocytes in
the graft after injection of antibody and comple
ment (76) It is, however, not excluded that, in
our experiments with heterologous comple
ment, direct lysis of the graft attributable to
complement activation beyond C5 played an
important role. Further support for this as
sumption stems from our unpublished findings
that with complement from C6-deficient rab
bits, antibody-mediated graft destruction could
not be induced and that the destructive capac
ity was reestablished after reconstitution of
this serum with C6. It might be argued that C6
also contributes to the leucotactic activity in
the C567 complex (25), but there is evidence
(23, 24) that the leucotactic property of C6-
deficient serum is unimpaired For the time be
ing, this problem remains unresolved and
awaits studies with sera that are deficient only
in the terminal components of the complement
9 7
TRANSPLANTATION
system, such as C9.
In clinical transplantation little is known about the role of non-HLA differences in the acceptance of allografts There are a few reports of severe rejection episodes of renal grafts from HLA mixed lymphocyte culture-identical siblings U9, 22). Kano et al. (13) reported that in recipients with a rejection of an HLA-identical kidney, antibodies could be detected against non-HLA antigens. From models in mice it is known that, if there exist multiple non-H-2 differences, skin graft survival can be equivalent to that of grafts differing at the complete H-2 complex (7). In our study it was possible to induce AAR across only non-H-2 differences. These differences can, therefore, not only cause cellular graft rejection but can also be responsible for antibody-mediated graft damage. This phenomenon should be bome in mind when the antibody-mediated rejection in clinical transplantation is studied.
Acknowledgments The technical assistance of the staff of the animal laboratory (Head; Dr. W. J. I v. d Gulden) is gratefully acknowledged We thank Mrs. J. Koene-Bogman for performing the histological examinations.
LITERATURE CITED
1. Bevan M J· 1976 Immunogenetics 3: 177 2 Carpenter CB, d'Apice AJF, Abbas AK: 1976
Adv Immunol 21 1 3. Demant Ρ- 1973 Transplant Rev 15: 162 4 Detnck-Hooks B, Borsos T, Rapp HF: 1975 J
Immunol 114: 287
5. Fabre JW, Morris PJ 1974 Transplantation 18: 429
6 French ME. 1972 Transplantation 13: 447 7. GrafTRJ, Bailey DW: 1973 Transplant Rev 15- 26 8 Graff RJ, Snell GD: 1969 Transplantation 8: 861 9 Grant CK: 1976 Transplantation 21: 323
10 Haughton G, McGhee MP. 1969 Immunology 16: 447
11. Jooste SV, Winn HJ: 1975 J Immunol 114: 933 12. Jooste SV, Winn HJ, Russell PS: 1973 Trans
plant Proc 5· 713 13. Капо К, Kussmaul CA, Milgrom F: 1975 Trans
plant Proc 7· 185 14. Kissmeyer-Nielsen F, Olsen S, Petersen VP, et
al· 1966 Lancet 2: 662 15. Klein J: 1973 Transplantation 15 137 16. Koene RAP, Gerlag PGG, Hagemann JFMH, et
al 1973 J Immunol 111: 520 17. Koene RAP, McKenzie IFC: 1973 J Immunol
111: 1894 18. Litchfield JT: 1949 J Pharmacol Exp Ther 97: 399 19. Lundgren G, Magnussen G, Moller E, et al: 1972
Tissue Antigens 2: 32 20. McKenzie IFC, Snell GD: 1973 J Exp Med 138:
259 21. McKenzie IFC, Snell GD: 1973 Transplantation
17. 328 22. Salaman JR, Godfrey AM, Russell RB, et al:
1976 Tissue Antigens 8: 233 23. Snijderman R, Phillips J, Mergenhagen SE:
1970 Infect Immun 1: 521 24. Stecher VJ, Sorkin E: 1969 Immunology 16: 231 25. Ward PA, Cochrane CG, Muller-Eberhard HJ:
1966 Immunology 11: 141 26. Winn HJ: 1965, ρ 133 In Wolstenholm GEW,
Knight J (eds). Ciba foundation symposium on complement. Churchill Ltd, London
Received 18 January 1977. Accepted 5 May 1977.
Chapter V
THE ROLE OF COMPLEMENT FACTORS IN ACUTE ANTIBODY-MEDIATED
REJECTION OF MOUSE SKIN ALLOGRAFTS
J.H.M. Berden, P.J.A. Capel, and R.A.P. Koene
Department of Medicine, Division of Nephrology, Sint Radboud
Ziekenhuis, University of Nijmegen, Nijmegen
published in: European Journal of Immunology (1978) S, 158
99
J.H.M Berden, Ρ J.A. Capel and
R.A.P. Koene
Department of Medicine, Division of
Nephrology, Sint Radboud Ziekenhuis,
University of Nijmegen, Nijmegen
The role of complement factors in acute antibody-mediated rejection of mouse skin allografts*
Established, Η 2 incompatible, mouse skin allografts are desiroyed in 2 4 - 4 8 h by a single intravenous injection of specific alloantibody and heterologous complement (C) from rabbits (RC), guinea pigs (GpC) or humans (HC) Mouse complement (MC) itself is inefficient in this respect since grafts are not destroyed by the injection of antibody alone This is in keeping with the low efficiency of MC in in vitro cytolysis
We have studied the role of different С factors in this model Two patterns were observed (a) Three C-deficient sera (GpC-R4, GpC-R3, RC-R6) showed neither activity m vitro (C-mediated lymphocytoloxicily in a trypan blue assay) nor did they induce graft destruction Activity in vitro and in vivo could be completely reconstituted with the appropriate С component (b) Two C-deficient sera (GpC-R 1 and HC-R9) were inactive ш in vitro cytotoxicity but, nevertheless, induced destruction of the skin grafts if injected with antibody Thus, in vivo, the mouse seemed to provide the deficient factor, and this was confirmed by the observation that m vitro cytotoxicity could be reconstituted not only with the appropriate С component, but alto with normal mouse serum The results show (hat the inefficiency of MC m vitro and in vivo does not reside in the factors CI or C9 Furthermore, in our model С is activated via the classical pathway, and at least exogenous C4, C3 and C6 are required to induce acute antibody-mediated graft destruction
From the experiments with C6-deficicnt serum which has normal chemo-tactic properties, it is concluded that activation of chemotactic factors with attraction of granulocytes is not sufficient to induce rejection, but that the activation of the membrane attack unit is required
1 Introduction
The rejection of skin grafts in the mouse is considered a primarily cellular immune event The antibody-mediated destruction of mouse skin allografts is of less importance, as is shown by the failure of passively administered allo-antibodies to induce graft rejection [ I ] This is at least partly due to the inefficiency of the mouse complement (MC) system We have previously shown that well-established H-2-incompatible allografts earned by immunosuppressed mice can be quickly (within 24 h) destroyed by an intravenous (ι ν ) injection of alloantiscmm together with rabbit complement (RC) [ 1 ] There are several arguments which support the assumption that the exogenous С is necessary for this antibody-mediated graft rejection First, the histology of the grafts shows the charactenstics of an Arthus reaction [ 1 ] which is known to be C-dependent Second, after inactivation of the С system, by heating, treatment with zymosan, carra-
[I 1873]
* Tins study was supported in part by grants from the Netherlands Poundation of Medical Research FUNGO, and from the Netherlands Kidney Foundation
Correspondence: J Η M Berden, Department of Medicine, Division of Nephrology, Sint Radboud Ziekenhuis, Geert Grooteplein Zuid 16, Nijmegen, The Netherlands
Abbrevia lions: AAR Acute antibody-mediated rejection ALS: Anti-lymphocyie serum GpC· Guinea pig complement GVB++: Veronal-buffered saline with gelatin, Ca++ and Mg++ Hanks' BSS. Hanks" buffered salt solution HC- Human С MC: Mouse С MST: Median survival time NMS: Fresh normal mouse serum RC* Rabbit С Ri С deficient in Cl R4· С deficient in C4 R3: С deficient in СЭ R6: С deficient in C6 R9: С deficient in C9 RSO Relative salt concentration SD. Standard deviation S RBC: Sheep red blood cells
geenan or aggregated human IgG, the rabbit serum no longer induced acute graft destruction if injected with antibody [2] Third, acule rejection was only seen with C-fixing antibodies (7 S IgGj) and not with 7 S IgG l antibodies that do not fix
с[з] Heterologous С is apparently not required in all skin grafting systems in mice Winn et al [4, S] could destroy rat skin grafted onto mice by injection of mouse anti-rat serum or by rat alloantiserum without exogenous С However, m none of our allogeneic skin graft experiments was MC sufficiently activated, since injections of alloantibodies alone never induced graft destruction [6] This is in keeping with the very low activity of MC in m vitro lymphocytotoxicity tests with mouse alloantibodies [7]
The donor recipient combination (BIO D2 ->C57BL/6) used in the current study, consists of a complete H 2 complex disparity in which we can induce acute antibody-mediated rejection (AAR) by the injection of RC, guinea pig complement (GpC), or human complement (HC) together with alloantibody [6]
We have in this model tested various sera which were made deficient in isolated С factors, and sera from animals that were congenitally deficient in certain С factors We have used these С factor-deficient sera (R sera) to study the role of individual С components in our model of AAR Furthermore, this approach could delineate which components m the mouse endogenous С system are responsible for its low efficiency in cytotoxic tests and in AAR For this purpose we compared the m vivo results with the lymphocytotoxic activity of the various R sera in the presence or absence of normal mouse serum (NMS)
100
Complement factors in humoral rejection of skin grafts
2 Materials and methods
2 1 Animals
Inbred BIO D2/new Sn (H-2d) mice were originally obtained from the Jackson Laboratory (Bar Harbor, ME) C57BL6/Rij (II 2 b ) mice were obtained from the Radiobiological Institute TNO (Rijswijk, NL) C4 deficient guinea pigs came from the National Institutes of Health, (Bethesda, MD) The C6 deficient rabbits were provided by Dr Lachmann (Cambridge, G B )
2 2 Production of alloantiserum
Alloanliserum was prepared by injecting female C57BL/6 mice at weekly intervals, intrapenloneally (ι ρ ), with a suspension of approximately 5 χ IO7 BIO D2 lymphoid cells in complete Freund's adjuvant After five injections, most animals developed ascites and could be tapped every 1 to 2 weeks The ascitic fluid of several weeks was pooled and, before use all antisera were heated at 56 0 C during 45 mm and sterilized by passing them through a sterile 0 20 mji filter The cytotoxic titer of the ascitic fluid was the same as the titer in the serum of these mice Antisera were stored at - 20 0 C
2 3 Anti-lymphocyte serum (ALS)
This was prepared by a subcutaneous injection of 5 χ IO 7 -8 χ IO7 C57BL/6 lymphoid cells (thymus and mesenterial lymph nodes) in complete Freund's adjuvant into a goat This priming dose was followed by weekly ι ν boosters of the same number of C57BL/6 lymphoid cells The animals were bled one week after the fourth immunization, the serum heated at 56 0 C for 45 mm, and absorbed once with C57BL/6 red blood cells (5 vol of serum/1 vol of packed red blood cells) After absorption, this ALS was not toxic to the recipients if injected ι ρ
2 4 Complement
Fresh or fresh frozen sera from New Zealand white rabbits, random bred SPF Albino guinea pigs, and from a human volunteer were used as sources of С Blood was obtained in rabbits and guinea pigs by cardiac puncture, in the human by venous puncture After clotting for 45 mm at room temper ature, and centnfugation the serum was divided into aliquots of 0 5 ml and, if not immediately used stored at - 3 0 0 C for maximally 3 weeks С was used only once after thawing Be fore use all sera were tested for toxicity m vitro (a 1 4 dilution should give less than 10 % cytolysis of В10 D2 lympho cytes), and in vivo by ι ν injection of 0 5 ml serum
2 5 Reagents
Veronal-buffered salme (GVB + + ), pH 7 35, containing 0 1 %
gelatin 0 15 mM Ca',"r and 0 5 m M Mg+ +, relative salt concentration (RSC) 0 147, was prepared accordmg to Mayer [8] The buffer was freshly prepared each day The rabbit anli-sheep red blood cell antiserum and rabbit anti-human C9 were commercially obtained from Behrmgwerke, Marburg, FRG Sheep red blood cells (SRBC) were drawn monthly from the same sheep, and collected in an equal volume of
Alsever's solution Before use, SRBC were washed four times in ten volumes of G V B + +
2 6 Production of R sera
The Rl serum was prepared from pooled guinea pig serum by euglobulin precipitation (pH 7 5, RSC 0 04) accordmg to the method of Nelson [9] The precipitate was redissolved m a volume of GVB + + equal to the original serum volume The С1 free supernatant was brought back to a RSC of 0 15 with NaCl after concentration to the original volume R4 serum was obtained from C4-deficient guinea pigs R3 serum was prepared by incubation of guinea pig serum with boiled zymosan (Koch Light Laboratories, Colnbrook, Bucks , GB), 15 mg/ml pooled guinea pig serum, for 60 min at 37 0 C After incubation, the zymosan was spun down in a refnger-ated centrifuge The R6 serum was obtained from C6-de fluent rabbits R9 serum was prepared by absorption of C9 from human serum by Sepharose coupled rabbit anti-human C9 antiserum The absorption procedure was performed at 0 0C in the presence of 0 01 M fcDTA and was repeated until, after reconstitution with Ca + + and Mg't"t\ hemolytic activity was no longer detectable R9 serum was concentrated to the original volume at 0 0 C The R sera were sterilized by passage through a 0 20 π\μ filter AU R sera, when tested in a microhemolytic assay, were unable to lyse sensitized SRBC, while they were functionally active after reconstitution with the appropnate factor(s)
2 7 Reconstitution of the R sera
The reconstitution experiments were performed with functionally pure GpC and HC factor preparations from Cordis Ltd (Miami, FL) In reconstitution experiments, Rl was reconstituted with the in G V B + + dissolved euglobulin precipitate, R4 with GpC4, R3 with guinea pig R4, R6 with GpCó and R9 with HC9 The R sera were serially diluted in GVB + + or Hanks' buffered salt solution (BSS) to which a constant amount of the deficient С factor had been added R l , R3 and R9 serum and the reconstituted sera were always compared in the same test with the batch of С from which they were prepared Th R4 serum was compared with pooled normal guinea pig serum, and the R6 serum with pooled normal rabbit serum In the lymphocytotoxicity tests an attempt was also made to reconstitute the R serum with NMS This was done in a two stage procedure to eliminate the anticomplementary activity that is often present in NMS No lymphocytotoxicity was found if NMS alone was used as a source of С
2 S Serology
A microhemolytic assay was performed on microliter plates Twenty five μ\ of the test sample were serially diluted in 2 5 y l G V B + + Thereafter, 25 ді of SRBC (2 χ 10B/ml) sensitized with anti SRBC antiserum (diluted 1 600) were added After mixing, the plates were incubated for 30 mm at 37 0 C After incubation, the plates were placed on ice to stop the reaction ¿nd centnfuged for 1 min in the cold The 5 0 % lysis point was taken as the titer R sera were also tested in a lymphocytotoxicity assay using the trypan blue exclusion test BIO D2 spleen cells were used as targets prepared free of red cells with Tm-NH^CI One hundred μΐ of cells (5 χ 105) in Hanks' BSS were incubated during 30 mm at 37 0 C with
101
J H M Berden, Ρ J A Capel and R A Ρ Koene
equal volumes of CS7BL/6 anti В10 D2 antiserum (diluted 1 16) and senal dilutions of whole C, R serum, reconstituted R serum, or С factors The titer was defined as the dilution giving 50 % cell lysis Tests were done in triplicate, and the following controls were performed (a) cell control 100 μ) cells in 200 μΐ Hanks' BSS, (b) antibody control 100 μ\ anti serum (diluted 1 16), 100 μΐ Hanks' BSS and 100 μΐ cells, (с) С control 100 μΐ undiluted C, R serum, reconstituted R serum, or pure С factor with 100 μΐ Hanks' BSS and 100 μΐ cells In these controls cytolysis did not exceed 10 %
2 9 Skin grafting technique
Female BIO D2 tail skin was grafted onto the nght dorsal flank of female C57BL/6 recipients by a modification of the fitted graft technique The graft was fixed by Nobecutane spray Instead of plaster badage, we used band-aid to cover the graft The donor and recipient mice were between 6 and 10 weeks old The band aid was removed on day 6 after transplantation and the fate of the grafts followed by daily macroscopic inspection Grafts were considered to be rejected when no viable epithelium remained Median survival time (MST) and the standard deviation (SD) were calculated by the method of Litchfield [ 10] The level of significance was calculated with Student's t-test
2 10 Acute rejection studies
The experiments were performed on well established grafts that showed no signs of rejection Survival of grafts was prolonged by nonspecific immunosuppression with ALS (0 25 ml ι ρ on days 0, 2 and 4 after grafting) from 9 3 + 1 1 days to 19 0 ± 1 1 days (Table 1) Thus, an ongoing cellular rejection could not interfere with the antibody-mediated destruction induced at day 7 after grafting and completed within 2 4 - 7 2 h On day 7 the recipients received 0 5 ml C57BL/6 anti-B10 D2 antiserum and 0 5 ml C, R serum, С factor, or reconstituted R serum by an ι ν injection through the tail vein Control experiments were performed by injecting antiserum, C, R serum and reconstituted R serum, alone, or the deficient С factor and alloantiserum AAR was defined as the complete necrosis of the skin graft 2 4 - 7 2 h after the iv injection
3 Results
3 1 Lymphocytotoxic activity of the R sera in vitro
The titration curves of the va nous R sera are given in Fig I
Rl serum The R l serum showed no cytotoxicity towards BIO D2 cells sensitized with alloantiserum After reconstitution with CI (100 μΐ of the redissolved euglobulin precipitate/ 100 μΐ R l ) , the cytotoxic titer became 1 24 This is close to 1 32, the original titer that was found with the batch of pooled guinea pig serum from which the R1 and С1 fractions were prepared The GpCl fraction itself had no cytotoxic activity If the cells were incubated with alloantiserum and NMS and washed, subsequent addition of GpC R l also produced a cytotoxicity of 1 24 Reversal of the procedure, ι e
incubation of cells with alloantiserum and GpC-RI, followed by washing and addition of NMS, did not result in cytotoxicity This confirms earlier reports that the reconstituting
factor in NMS is С1 [16], and is further supported by our finding that an euglobulin precipitate of NMS was as active in the reconstitution of GpC-RI as whole NMS
>90-Θ 0 -
6 0 -
4 0 -
20-<10-
• — ^ ^ч
^ 5 β ι , α ^ ν ,
В
ч
•ν *л х Л л\ Ч
RI
»90-80·
6 0 -
4 0 -
20-<10-
S "Ч итз ч и
\ \
R3
ч,
- ! 1 7—I 1 1 Г-
НИ* ti
Figure 1 Lymphocytotoxic activity of the various R sera against BIO 02 spleen cells sensitized with C57BL/6 anü BIO D2 antiserum Each diagram represents the activity of normal C, R serum, reconsti tuted R serum, R serum in the presence of NMS, or the deficient factor
102
Complement factors in humoral rejection of skin grafts
R4 serum Neither the R4 serum nor punfted GpC4 (12S0 CH50 U/ml), or GpC-R3 showed cytotoxic activity against BIO D2 spleen cells Reconstitution of 100μ] GpC-R4 with 100 μΐ GpC4 ( 1250 CHso U/ml) or with 100 μΐ GpC-R3 restored cytotoxicity up to 1 24 It was not possible lo reconstitute GpC-R4 with NMS
R3 serum With the R3 serum no cytotoxicity was found, while addition of excessive amounts of GpC-R4 restored the cytotoxicity to the same titer as that of pooled GpC Reconstitution of the R3 serum with NMS was not possible R6 serum Neither the R6 serum norGpC6 (2500 CHSo U/ml) were able to lyse sensitized B\0 D2 spleen cells Retonbtitu-tion of 100 μΐ Rt-R6 with 100 jul GpC6 ( 1250 CHso U/ml) restored the cytolytic activity up to 1 12, while normal RC in this (est system had a cytotoxic titer of I 32 Thus, we found a functional synergism between RC-R6 and ОрСб, and this is analogous to the findings of Rother et al who could reconstitute RC R6 with purified HC6 [11] Reconstitution with NMS was not possible
R9 serum In the experiments with R9 serum the same pattern was found as with GpC-Rl The R serum itself had no cytotoxic activity, neither had pure HC9 (5000 CHso U/ml) When 100 μ1€9 (5000 tH S o U% I) were added to 100 μΐ R9, the cytotoxicity was restored up to a titer of I 12, while nor mal HC had a titer of 1 16 NMS was also able to restore the cytolytic activity of human R9 up to a titer of 1 12, if NMS was added after incubation of sensitized target cells with the R serum
3.2 Ability of R sera to induce AAR
By the administration of 0 S ml RC, GpC, or HC together with 0 5 ml alloanlisemm, AAR could be induced within 48 h,
while alloantiserum alone, or the various С species alone gave negative results The MST of the control groups did not differ significantly from the ALS-treated group (Table 1) The in vivo results with the different R sera are summarized in Table 2 It was possible to induce AAR with GpC-R 1, while injection of R serum without alloantiserum was ineffective A similar pattern was observed with the HC-R9 serum
Table 1. Occuircnce of AAR and MST of BIO D2 skin giafu on C57BL/6 recipients m va no us control groups
No of ¡murali
150 13-1)
10
16 10
10 β
10 11
C57BL/6 anti-BIO D2 antlierum1)
-
+ +
+
+
Complément·)
-
-RC RC
GpC GpC
HC HC
AARb)
-+
+
+
MSTiSD
9 3 i 1 1 1 9 0 i 1 1
23.0 1 1 1
18 6 1 1.1
2 1 5 1 1 1
19 4 t 1.1
a) 0 5 ml ι ν on day 7 after grafting b) Defined as complete necrosis of the skin graft 24-72 h after 1 ν
injection c) Untreated animals d) Recipients received 0 25 ml ALS ι ρ on days 0, 2 and 4 after graft
ing
Table 2 Induction of AAR by 1 ν injection of the various R sera and alloantiserum on day 7 after grafting
R serum AARa) R serum and R senim Reconstituted Reconstituted alloandbody only R serum and R serum only
alloantibody
GpCRl 7/ 7b) 0/8 HC-R9 6/ 6 0/6
GpC-R4 0/12 11/11 0/8 GpC-R3 Q/ll Π/11 0/8 RC-R6 0/11 10/10 0/8
a) Defined as complete necrosis of the graft 24 72 h aften ν injection
b) Number ot animals that showed complete necrosis of Mie graft/ number animal* tested
Administration of GpC R4 with alloantiserum did not induce AAR Addition of pure GpC4 to the R4 serum before the injection did not reconstitute the destructive capacity of the R serum Even doses as high as 5000 CH50 U were ineffective This was somewhat surprising since m vitro, the lymphocyto-toxicity of the R4 scrum was completely restored by C4 A possible explanation might be that after the injection of the reconstituted R serum, GpC4 is rapidly inactivated in the mouse circulation [12] However, AAR was obtained with R4 serum that had been reconstituted with guinea pig R3 serum Injection of this mixture without alloantiserum did not lead to AAR
The R3 serum did noi induce AAR if injected together with alloantiserum Active C3 is not available since its isolation leads to inactivation Thus, in this instance wc were unable to reconstitute the defect with the appropriate factor How ever, in the experiments with R4 serum it had already been shown that combination of R3 and R4 resulted η compie mentation and led to induction of AAR
Induction of AAR with R6 serum and alloantiserum was also impossible Reconstitution of 0 5 ml R6 serum with an equal amount of guinea pig C6 ( 1250 CH50 U/ml) lead to destruction of the graft if the mixture was injected with alloantiserum In the control experiments, injections of only C6 with alloantibody or reconstituted R serum alone did not result in rejection
4 Discussion
Our results show that for the induction of AAR in our model not all heterologous С factors have to be administered GpC Rl and HC R9 were as effective as normal GpC and HC. showing that the mouse can reconstitute the defect On the other hand, the limiting effect of MC does not he at a single point in the С sequence since three sera deficient in different С components were only able to induce rejection after rcconsti tution with the appropriate factor
In all but one experiment (R4 reconstituted with C4) we found in R sera a close correlation between С activity m intro and tn vivo AAR occurred only if the R serum had cytotoxic activity in vitro after reconstitution with the deficient factor,
103
J H M Berden, Ρ J A Capel and R A Ρ Koene
or with NMS This suggests that the С system is activated similarly in vivo and in vitro From the expenments with GpC*R4 it can be concluded that m vivo С activation in our model is initiated via the classical pathway, as it is ш іл vitro cytotoxicity of cells sensitized with antibody If alternative pathway activation alone were sufficient to induce AAR, then administration of the R4 serum should have induced rejection, since activation of the alternative pathway is normal in this serum [13] At least three murine С factors, C4, C3 and C6, are relatively inefficient in this system since NMS could not reconstitute these R sera neither m virro nor m vivo With regard to C4 and C3 these findings are in accord with those of Rice, who in a hemolytic system found that CpC-R4 and R3 were not reconstituted by NMS [14] Mouse CI, on the other hand, seems not to be at fault NMS and also its euglobuhn precipitate, reconstituted the GpC-RI serum in vitro This complémentation confirms previous findings of other investigators who obtained similar results in a hemolytic system with sensitized SRBC [ 1 4 - 1 6 ] Since the Rl serum tould induce graft rejection without reconstitution with exogenous С1, we must conclude that also in vivo the mouse effectively provided the deficient CI factor
It has been suggested by Winn [4] that for antibody-mediated graft destruction, activation of the complete С pathway is unnecessary He assumed that the generation of chemotactic С components is sufficient for the destruction of the graft This was based on expenments with CS-deficient mice that rejected rat xenografts after the administration of mouse anti-rat serum These mice are completely deficient in CS, and thus activation beyond this component is impossible Care was taken to prevent administration of exogenous С components with the anti-rat serum On the basis of his findings, we expected that our R6 serum would also induce rejection, but this was clearly not the case This cannot be the result of deficient chemotactic properties of the R6 serum, Since it has been shown to generate normal Chemotaxis [ Π Ι 9] and lacks only lytic activity We have preliminary unpublished data from histological studies that after administration of R6 serum, the granulocyte infiUration in the graft is as abundant as after the injection of whole RC Despite this, destruction did not occur with R6 We therefore assume that in our model complete С activation with generation of not only chemotactic but also membranolytic activity is necessary to induce AAR
We think that the only possible explanation for the induction of AAR by R9 serum is that the mouse endogenous C9 is efficient enough to complement exogenous R9-serum especially after this is what happened in the in virro test It is highly unlikely that graft destruction occurred without activation of C9, but this possibility could not be formally excluded through lack of a negative control in the m vivo experiments This would require a mouse completely deficient in C9, and in such an animal the R9 serum would fail to induce AAR
We may thus conclude that for the induction.of AAR, the complete С pathway has to become activated via the classical pathway, and that generation of chemotactic С components alone is not sufficient Our study gives some indications for a useful approach to influence the occurrence of antibody-mediated graft destrULtion by manipulation of the С system If it becomes possible to inhibit С activity in vivo it seems wise to concentrate on the early factors such as С1 or C4 because deficiency of these factors might lead to inhibition of humor ally mediated rejection Such an approach would leave the alternative pathway fully intact so that the C-dependent defense mechanisms against bacterial infections remain unaltered This would be of great ad\antage in clinical transplantation
The skillful technical assistance of Ms Jacqueline Hage mann and the staff of the animal laboratory (Head Dr WJ/ van de Cuiden) is gratefully acknowledged
Received August 30 1977
5 Reference*
1 Koene, R A P , Gerlag Ρ G G , Hagemann, J h H M , van Haelst, UJG andWydcveld.PG A B , / Immunol 1973 JIJ 520
2 Gerlag, Ρ G G , Thesis University of Nijmegen 1975 Nijmegen
3 Jansen, J L J Koene, R Α Ρ , van Kamp, G J , Tamboer W Ρ M andWijdeveld, PC AB,У Immunol 1975 /75 3β7
4 Winn, Η J , Β al da mu s, С А , Jóos te, S V and Russell P S , / Exp Med 1973 137 893
5 Jooste.SV and Winn, H J, ƒ Immunol 1975 J14 933
6 Berden J H M Gerlag, Ρ G G , Hagemann, J F H M and Koene, R AP, Transplantation 1977 24 175
7 Grant CK , Transplantation 1976 21 323
β Kabat, E A and Mayer, M M , Experimental Immunochermstry, 2nd Edit, Thomas, С Γ , Springfield 1961
9 Nelson R A , Jensen, J Gigli, I and Tamura, N , Immunochem istry 1966 S 111
10 Litchfield, J Τ, У Pharmacol Exp Ther 1949 97 399
11 Rother, К , Rother, Ь Muller Eberhard, Η J and Nilsson, U R , J Exp Med 1966 ¡24 773
12 Mandle, R J , McConell Mapes, J A and Nilsson, L R J Immunol 1977 ¡19 180
13 brank, M M , May J , Gaither, Τ and Ellman. L J Exp Med 1971 J 34 176
14 Rice.CE andCrowson C N , y Immunol 1950 65 201
15 Brown, G С J Immunol 1943 46 319
16 Winn H J in Wolstenholme, G E W andKmght.J (fcdsi.Oto Foundation Symposium on Complement Churchill Ltd , London 1965.ρ 133
17 Stecher, VJ and Sorkin, E .Immunology 1969 ¡6 231
18 Snyderman, R , Philips J and Mergenhagen, S Ь , Infect Immun 1969 У 521
19 Lim, D . Gewürz A Lint, Τ F , Ghaze, M , Scphen В and Gewürz, H, J Pediat 1976 89 42
104
Chapter VI
A SENSITIVE HAEMOLYTIC ASSAY OF MOUSE COMPLEMENT
J.H.M. Berden, J.F.H.M. Hagemann, and R.A.P. Koene
Department of Medicine, Division of Nephrology, Sint Radboud
Ziekenhuis, University of Nijmegen, Nijmegen
published in: Journal of Immunological Methods (1978) 23, 149
105
A SENSITIVE HAEMOLYTIC ASSAY OF MOUSE COMPLEMENT *
J.H.M. BERDEN, J.F.H.M. HAGEMANN and R.A.P. KOENE Department of Medicine, Division of Nephrology, St. Radboud Ziekenhuis, University of Nijmegen, Nijmegen, The Netherlands (Received 1 March 1978, accepted 20 March 1978)
Under appropriate conditions the haemolytic activity of mouse complement can be measured in a 5ICr release assay. Modifications that increase the sensitivity are the following: (a) mouse 7 S anti-SRBC antibodies as amboceptor; (b) low amount of target cells; (c) ionic strength of 0.13 M NaCl in the test medium; (d) incubation temperature of 30oC; (e) incubation time of 90 min; (f) total reaction volume of 1 ml.
INTRODUCTION
In tests that are generally used to measure the haemolytic activity of human and guinea pig complement, mouse serum seems to lack complement activity (Brown, 1943; Rice and Crowson, 1950). In 1952 McGhee reported that in a system with a very low number of sensitized sheep red blood cells (SRBC) one CH50 U/ml could be detected in some mouse sera, while other sera from mice of the same strain were still unable to induce haemolysis.
Several authors claimed that some of the then known complement factors (CI, C4, C2, C3) were absent from the mouse complement system, and that, therefore, no haemolytic activity could be demonstrated (Ritz, 1911; Brown, 1943; Rice and Crowson, 1950; Muschel and Muto, 1956). In 1961, however, Borsos and Cooper found, by using cellular intermediates, that all these factors were present in at least the mouse strain they tested. Later on it became evident that some inbred strains lack haemolytic activity due to lack of C5 (Nilsson and Müller-Eberhard, 1967). Although this C5 deficiency could explain the inability to demonstrate haemolytic activity of mouse complement in these strains it did not explain why in other strains that were not C5 deficient, С activity was so difficult to measure.
In 1962 Rosenberg and Tachibana described an assay for the measurement of haemolytic activity of mouse complement, but its sensitivity was rather low. In 1975 Stolfi et al. mentioned a method that used spectrophotometric determination of the haemoglobin release but details of the method have not been published. Mouse sera are always spontaneously, and sometimes
* This study was partly supported by grants from the Netherlands Foundation for Medi cal Research FUNGO and from the Netherlands Kidney Foundation.
107
strongly, haemolysed. This interferes with the measurement of haemoglobin release due to complement activity, and makes the above mentioned approach less attractive. To overcome this problem we have developed an assay that makes use of slCr-labelled sheep red blood cells. This technique has a greater sensitivity than the determination of haemoglobin release (Weinrach et al., 1958). We describe here the optimum conditions for this 51Cr-release test. We have also studied how the haemolytic activity of mouse complement is influenced by the use of different amboceptors and by variation of the reaction conditions.
MATERIALS AND METHODS
Mice Inbred lines of B10.D2/old Sn and A/HeJ mice were originally obtained
from the Jackson Laboratory (Bar Harbor, Maine, U.S.A.). C57BL6/Rij mice were obtained from the Radiobiological Institute TNO (Rijswijk, The Netherlands). DBA/2 and A/J mice were brought from Zentral Institut für Versuchstierzucht, Hanover, G.F.R. In our animal laboratory these mouse strains were maintained by continuous brother sister matings. (C57BL6/Rij X A/HeJ)Fi (=B6AF1) hybrids were raised in the laboratory.
Production of mouse amboceptor Mouse amboceptor was prepared in C57BL6, A/J and B6AF1 mice in two
different ways. The first method consisted of weekly intraperitoneal injections of 4 X 107 SRBC in 0.1 ml 0.9% saline. One week after the 5th immunization the mice were bled from the retro-orbital venous plexus. After clotting for 45 min at room temperature, the separated serum was heated for 45 min at 56°C to destroy complement activity. The sera were stored at —30oC. For the production of larger amounts of amboceptor in the same strains we gave weekly intraperitoneal injections of 4 X 107 SRBC in 0.1 ml 0.9% saline mixed with 0.1 ml complete Freund's adjuvant. After 5 injections most animals developed ascites, which was tapped every week. The product of several weeks was pooled. Before use, the ascites fluid was heated at 56°C for 45 min, and sterilized by passing through a sterile 0.20 nm filter. The haemolytic titer of the ascites fluid was the same as that of the serum of these mice. The sera were divided into 1 ml aliquots and stored at —30oC.
Complement All experiments were performed with pooled mouse sera. The mice were
bled from the retro-orbital venous plexus. After clotting for exactly 45 min at room temperature, the serum was collected by centrifugation at 40C, and stored in liquid nitrogen. Fresh serum from special pathogen free random bred Albino guinea pigs served as a source of guinea pig complement.
Reagents If not otherwise stated all titrations were performed in dextrose gelatin-
108
veronal buffer (DGVB2+) containing 0.00015 M Ca2+ and 0.0005 M Mg2+
(pH 7.35), ionic strength of 0.13 M NaCl, and prepared according to Rapp and Borsos (1970). The ionic strength was determined by measuring specific conductance at 20oC. The buffer was freshly prepared each day. Rabbit amboceptor was obtained commercially from Behring Werke.
SRBC were obtained aseptically from the same sheep each month. Blood was collected in an equal amount of Alsever's solution, and stored at 40C. Before labelling with s l Cr (Sodium Chromate, 50—400 mCi/mg/Cr, Radiochemical Center, Amersham, U.K.) the SRBC were washed four times with DVGB2+. One millilitre SRBC (containing 8 X 107 cells) was labelled with 6 μΟ 51Cr, by incubation during 60 min at 37°C. This resulted in 90% binding of the radioactivity (Ebaugh et al., 1953). With this dose of sodium Chromate no injury to erythrocytes occurs (Necheles et al., 1953). After incubation the labelled SRBC were washed three times with 10 volumes of DGVB2+, and the pellet resuspended in the original volume. To prepare sensitized cells (EA), an equal volume of an appropriate dilution of amboceptor in DGVB2+ was slowly pipetted into the labelled SRBC suspension with continuous mixing. The EA cell suspension was then incubated for 10 min at 370C with frequent mixing. The spontaneous release of 5 1Cr from this cell suspension during the first 48 h was 2—4% of the total activity bound.
Separation of mouse amboceptor in IgG-IgM antibodies This was performed with isokinetic sucrose gradients, prepared by addi
tion of 33% (w/w) sucrose in phosphate buffered saline (PBS) to 12 ml 5% (w/w) sucrose in PBS in a mixing chamber at constant volume (Noll, 1973). Test serum (0.5 ml) was layered on top of a 12 ml gradient and centrifuged in a SW 41 rotor (IEC) at 35,000 rev./min for 16 hours at 40C. The gradient was sampled from the bottom; 0.8 ml fractions were collected and each fraction was tested for agglutinating and haemolytic activity.
Serologic tests Basic outline of the haemolytic assay of mouse complement. The tests
were performed in sterile plastic tubes. The mouse serum to be tested was diluted 1 : 10 in DGVB2+ and 10—800 μΐ was put in tubes containing varying amounts of DGVB2+ to produce dilutions with a final volume of 800 μΐ. The dilutions were chosen such that at least 4 dilutions gave partial lysis. After adding 200 μΐ of 51Cr-labelled EA suspension (4 Χ 107 cells/ml) the tubes were incubated in a waterbath at different temperatures, and for different incubation periods. After incubation the tubes were placed on ice, and 2 ml of ice-cold DGVB2+ were added to quench the reaction. The tubes were then centrifuged for 5 min at 3,000 rev./min at 4°C. From each tube 2 ml supernatant was collected to measure the released s l C r activity. Each test included the following controls (in triplicate): (a) 200 μΐ of EA suspension in 800 μΐ DGVB2+ to determine spontaneous s l C r release; (b) 200 μΐ of EA suspension
109
lysed in 800 μΐ water to measure 100% lysis; (c) 200 μΐ of SRBC incubated with the highest amount of mouse serum used in that test, to exclude the presence of naturally existing mouse antibodies against SRBC. The released s l Cr activity in each tube was measured for 10 min in a gamma-ray well type scintillation counter (MS 588, Beard Atomic). The fraction of SRBC lysed in each dilution was calculated according to the formula:
cpm test serum—cpm control cpm complete lysis—cpm control
The data were plotted according to the logarithmic transformation of the Von Krogh equation to determine the CH50 titer per ml of undiluted mouse serum.
Antibody titrations of different amboceptor species. We studied the influence of the amboceptor species by comparing rabbit amboceptor in a limited complement system with different mouse amboceptors (A/J, C57BL6 and B6AF1). One hundred microliters of amboceptor were diluted in 2-fold steps in 100 μΐ DGVB2 +. To these serial dilutions 100 μΐ of 51Cr-labelled SRBC (8 X 107/ml) were added. After incubation for 10 min at 370C, 6μ1 of MC (C57BL6 male mouse serum) and 794 μΐ DGVB2+ were added, and incubated for 90 min at 30oC. Percentage lysis (fraction X100) was calculated for each amboceptor dilution.
Antibody titrations of different anti-SRBC antibody classes. After ultra-centrifugation of the different mouse amboceptors on a sucrose gradient 25 μΐ of each collected fraction was diluted in 2-fold steps in 25 μΐ DGVB2* in microtiter plates. To test the agglutinating activity 25 μΐ of SRBC (108/ ml) were added, and after mixing, the plates were incubated for 30 min at 370C. To test the haemolytic activity 25 μΐ SRBC (108/ml) were added to the amboceptor dilutions, incubated for 10 min at 370C, and thereafter 25 μΐ guinea pig serum or 25 μΐ B10.D2 male serum were added. After mixing, the plates with GPC were incubated for 30 min at 37CC, and those with MC for 90 min at 30CC. The plates were centrifuged for 1 min at 1000 rev./min at 40C, and read macroscopically. The titer was defined as the last dilution that gave complete haemagglutination or lysis. All tests were performed in duplicate.
RESULTS
Amboceptor species and class of anti-SRBC antibody As can be seen in Fig. 1, mouse and rabbit amboceptor give comparable
haemolysis. Both antisera exert strong anticomplementary effects, and maximal lysis requires relatively high concentrations of antibodies (dilution of 1/8 to 164). Mouse amboceptors had the advantage of giving less haemagglutination during sensitization of the SRBC than rabbit amboceptor. A difference in activity was observed between the three mouse amboceptors. With C57BL6 amboceptor maximal lysis occurred at a lower dilution than
110
·/. cell lysis
ЮОл
- ι 1 1 1 1 г 16 64 256 1024 256 1024
(amboceptor dilution)"
Fig. 1. Titration of three mouse and one rabbit amboceptor in a limited complement system (measured with 6 μΐ C57BL6 male MC). Each point represents the mean value of 4 different tests, the vertical bars give the S.E. M.
hemagglutinin — ( t i t e r ) - ' ,9s
4 0 9 6
1024
256
64
16
4
hemolytic ( t i t e r ) - 1
19S
I
I ' ' I ' I I ' ' ' I
5 10 15 fraction number
Fig. 2. Haemagglutinin and haemolytic titers of A/J, B6AF1 and C57BL6 amboceptor fractions, collected after separation on an isokinetic sucrose gradient. Haemolysis was measured with GPC and MC.
I l l
with B6AF1 and A/J, and the maximal lysis reached was also lower. Silver et al. (1972a; 1972b) reported that the antibody response to SRBC in C57BL6 mice was different from that in mice of the A strain'. In C57BL6 they found a predominantly 2-ME sensitive (IgM) response, while in A mice the antibodies formed were 2-ME resistant (IgG). Furthermore, Winn (1965) observed that mouse complement (MC) was better activated by IgG anti-SRBC antibodies, while by contrast, GPC was better activated by IgM anti-SRBC antibodies. To study whether the higher haemolytic activity of A/J amboceptor as compared to C57BL6 amboceptor in our system was due to the class of antibody present in these amboceptors, we separated IgG and IgM antibodies of A/J, C57BL6 and B6AF1 amboceptor with isokinetic sucrose gradients. The results are shown in Fig. 2. In the haemagglutination test the A/J amboceptor contains more 7 S anti-SRBC antibodies, while C57BL6 amboceptor contains more 19 S antibodies. The values for B6AF1 amboceptor are intermediate between those of the other two strans. A comparable pattern was found in the haemolytic test with MC, where in A/J and B6AF1 serum only 7 S antibodies were haemolytic, while in the C57BL6 serum lysis by 19 S was predominant. It is also clear from this figure that MC is better activated by IgG than by IgM antibodies, since the haemolytic titer of 7 S A/J antibodies with MC was 1 : 32, while with 19 S C57BL6 antibodies, of comparable haemagglutinating activity, the titer was only 1 : 2. For GPC a better activation by 19 S anti-SRBC was found, which is in agreement with findings of other investigators (Winn, 1965; Borsos and Rapp, 1965; Ishizaka et al., 1968). The results thus suggest that for the optimal determination of MC amboceptor containing 7 S antibodies (e.g., A/J anti-SRBC serum) should be preferentially used to sensitize SRBC.
C H 5
120-
100-
8 0 -
6 0 -
4 0 -
2 0 -
0 titer ( U/ml)
/ r
15 22 30 37 incubation temperature (°C)
Fig. 3. Haemolytic activity of B6AF1 male MC from a single batch measured at different incubation temperatures. Each point represents the mean of 4 different tests; the vertical bars are the S.E.M.
112
с·, ьсн
ι •30 J 120н
Т Ю -
•oc-9 0 -
ao-7 0 -
r
•e^
* ^
С Э 5
U г- ι
/ ^Y
0 0 /
X
0 0 9
У /
C-i
/ \ \
013
IO"iC s
\ \
0 1 5
' e r g i · ·
"
(•vi NaCi
Fig. 4. Haemolytic activity of B6AF1 male MC from a single batch at different ionic strengths. Each point represents the mean value of 4 different tests; the vertical bars are the S.E.M.
Incubation temperature It has been shown for several complement species that a lower incubation
temperature than 370C gives better haemolysis (Leon, 1956; Borsos et al., 1961; Colten et al., 1967; Cooper and Müller-Eberhard, 1968; Cunniff and Stollar, 1968; Sakamoto, 1975). To find out which incubation temperature was optimal for mouse complement we performed a haemolytic titration of the same batch of B6AF1 MC m quadruplicate at 15°C, 2 2 ° ^ 30oC and 370C (Fig. 3). The figure shows that for MC an incubation temperature of 30oC is optimal.
Ionic strength Lepow et al., (1956) stated that a lower ionic strength of the test medium
resulted in a higher haemolytic activity of human complement (HC). Later many reports confirmed these findings for GPC and HC (Becker and Wirtz, 1959; Rapp and Borsos, 1963; Inoue and Nelson, 1965; 1966; Gölten et al., 1968; Shin et al., 1971; Segerling and Muller, 1976). To find out whether this also holds true for MC we measured the CH50 titer of the same batch of B6AF1 MC (in quadruplicate) at different ionic strengths. The buffers were prepared by adding appropriate amounts of veronal buffered dextrose 5% (with 0.1% gelatine, 0.0005 M Mg2+, 0.00015 M Ca2+) to GVB2+. We found an optimum at 0.13 M NaCl (Fig. 4).
Incubation time The time necessary to obtain maximal lysis was measured in the presence
of one CH50 unit of MC (9 μΐ B6AF1 male serum) at 300C with 8 X 106
sensitized SRBC, total reaction volume 1 ml. After 90 min of incubation no further increase in haemolysis wis found (Fig. 5). In the presence of a lower amount of MC (7 μΐ B6AF1 serum) the time necessary to obtain maximal lysis increased to 120 min.
113
yss
6 0 1
2 0 -
1Θ0 210 ?40 mcubalion time (min)
Fig. 5. Percentage lysis of SRBC measured in a limited complement system at increasing incubation times. (I) Measured with 9 μΐ B6AF1 male mice MC, (II) with 7 jul B6AF1 MC. Each point represents the mean percentage lysis of four different tests. The S.E.M. for each point was < 1%.
Reaction volume By decreasing the total reaction volume haemolytic tests become more
sensitive (Mayer et al., 1946), and less serum is necessary to determine the CH50 titer. This is of special advantage in measuring haemolytic complement activity of mouse serum, because in most instances there is only a small volume of serum available. The same batch of mouse complement measured in total reaction volumes of 1 and 3 ml respectively gave a CH50 titer of 148.6 ± 2.0 and 69.9 ± 1.5 U/ml (means ± S.E.M. of 4 determinations).
Number of sensitized SRBC To determine the influence of the number of sensitized SRBC on the
CH50 titer ( U/ml)
300
250
200
150
100 H
50
10° 510 10' 510 Ю sensitized SRBC
Fig. 6. Relation between CH50 titer (U/ml) of male C57BL6 MC and number of labelled SRBC added. Each point gives the mean titer of four measurements, the vertical bars are the S.E.M.
1 1 4
TABLE 1
COMPLEMENT LEVELS IN DIFFERENT INBRED MOUSE STRAINS *
B10.Br C57BL6 B6AF1 A/HeJ DBA/2
Ss-low Ss-high Ss-high X C5 deficient C5-deficient CS-deficient
48.1 ± 4.1 167.5 ±10 .7
77.3 ± 4.1 0 0
Strain Complement status CH50 titer »* ± SEM U/ml Male Female
34.9 ±4 .9 63.8 ± 3.9 45.9 ± 1.7
0 0
* All mice were 6 weeks old. ** Each value is the mean of determinations in 6 mice.
CH50 titer we measured the CH50 titer of the same batch of C57BL6 MC to which different numbers of sensitized SRBC were added, under otherwise similar test conditions. We found a linear relationship between the CH50 titer and the number of SRBC if the results were plotted semi-logarithmi-cally (Fig. 6). It should be noted that although sensitivity is highest with 106
SRBC the reproducibility is less at this concentration.
Reproducibility of the test The intra-assay precision, determined by the coefficient of variation of
25 measurements of sera with complement levels ranging from 4—300 CH50 U/ml varied from 0.3—5.1%. Inter-assay variation with the same sera was 3.1—7.1%.
Complement levels in different inbred mouse strains Since the investigations of Demant et al. (1973) it has become evident
that a correlation exists between complement activity and H-2 haplotype, especially with the Ss-region allotype. To demonstrate the applicability of the method we measured complement levels in Ss-high and Ss-low mice. Furthermore we measured complement activity in some C5 deficient mice, and in a hybrid of a C5 positive and C5 negative parental strain (Table 1). In agreement with Nilsson and Müller-Eberhard (1967) the results also show a difference in complement level between male and female mice.
DISCUSSION
Mouse complement is very difficult to measure in standard haemolytic assays and test conditions have therefore to be modified. An important improvement in the system is the replacement of measurement of haemoglobin release by that of s lCr release. The first advantage of this is that the number of target cells may be decreased which leads to higher sensitivity. For example, the CH50 titer of C57BL6 mice measured with our method
1 1 5
was 10 times higher than in the method of Rosenberg and Tachibana (1962). Second, the error introduced by the fact that many mouse sera are haemo-lysed is eliminated. A third advantage is that in spite of the increased sensitivity of the assay, its coefficient of variation is very small.
The results of antibody titrations with amboceptors from different mouse strains stress the importance of the antibody class used. Mouse complement is much more readily activated by IgG than by IgM. We found that C57BL6 mice make predominantly IgM antibodies, while A/J mice produce mostly IgG after multiple immunization with SRBC.
Our findings confirm those of Silver et al. (1972a; 1972b), although there are quantitative differences. These might be accounted for by differences in technique since we used sucrose-gradient centrifugation instead of 2-mercapto-ethanol sensitivity to distinguish the two classes. The differential activity of 7 S and 19 S amboceptors with regard to the activation of mouse complement has practical implications because a sample of the same batch of MC tested with SRBC sensitized with A/J amboceptor gave a CH50 titer approximately 50% higher than with C57BL6 amboceptor.
On the basis of our findings we may summarize practical guidelines for the use of the assay as follows: (a) number of sensitized SRBC: 8 X 106 cells; (b) amboceptor class: IgG; (c) incubation temperature: 30oC; (d) ionic strength: 0.13 M NaCl; (e) incubation time: 90 min; (f) reaction volume: 1 ml. With this test substantial differences in complement activity between different mouse strains were found. The method offers an opportunity to measure sensitively complement levels in an animal experimental system widely used in immunological research.
REFERENCES
Becker, E.L. and G.H. Wirtz. 1959, Biochim. Biophys. Acta 35, 291. Borsos, T. and M. Cooper, 1961, Proc. Soc. Exp. Biol. Med. 107, 227. Borsos, T. and H.J. Rapp, 1965, Science 150, 505. Borsos, T., H.J. Rapp and M.M. Mayer, 1961, J. Immunol. 87, 326. Brown, G.C., 1943, J. Immunol. 46, 319. Colten, H.R.,T. Borsos and H.J. Rapp, 1967, Protides Biol. Fluids 15, 471. Gölten, H.R., T. Borsos and H.J. Rapp, 1968, J. Immunol. 100, 799. Cooper, N.R. and H.J. Müller-Eberhard, 1968, J. Immunol. 101, 813. Cuniff, R.V. and B.D. Stollar, 1968, J. Immunol. 100, 7. Demant, P., J. Capkova, E. Hinzova and B. Voracova, 1973, Proc. Natl. Acad. Sci. U.S.A.
7 0 , 8 6 3 . Ebaugh, F.G., C.P. Emerson and J.F. Ross, 1953, J. Clin. Invest. 32, 1260. Inoue, K. and R.A. Nelson, 1965, J. Immunol. 95, 355. Inoue, K. and R.A. Nelson, 1966, J. Immunol. 96, 386. Ishizaka, T., T. Tada and K. Ishizaka, 1968, J. Immunol. 100, 1145. Leon, M.A., 1956, Proc. Soc. Exp. Biol. Med. 91, 150. Lepow, I.H., O.D. Ratnoff, F.S. Rosen and L. Pillemer, 1956, Proc. Soc. Exp. Biol. Med.
92, 32. Mayer, M.M., A.G. Osier, O.G. Bier and M. Heidelberger, 1946, J. Exp. Med. 84, 535. McGhee, R.B., 1952, Proc. Soc. Exp. Biol. Med. 80, 419.
1 1 6
Muschel, L.H. and T. Muto, 1956, Science 123, 62. Necheles, T.F., I.M. Weinstein and G.V. Le Roy, 1953, J. Lab. Clin. Med. 42, 358. Nilsson, U.R. and H.J. Müller-Eberhard, 1967, J. Exp. Med. 125, 1. Noll, H. 1967, Nature 251, 360. Rapp, H.J. and T. Borsos, 1963, J. Immunol. 91, 826. Rapp, H.J. and T. Borsos, 1970, The Molecular Basis of Complement Action (Appleton,
Century and Crofts, New York). Rice, C E . and C.N. Crowson, 1950, J. Immunol. 65, 201. Ritz, H., 1911, Ztschr. f. Immunitaetsf. 9, 321. Rosenberg, L.T. and D.K. Tachibana, 1962, J. Immunol. 89, 861. Sakamoto, M., 1975, Japan. J. Exp. Med. 45, 183. Segerling, M. and F. Müller, 1976, Immunochemistry 13, 117. Shin, H.S., R.J. Pickering and M.M. Mayer, 1971, J. Immunol. 106, 480. Silver, D.M., I.F.C. McKenzie and H.J. Winn, 1972a, J. Exp. Med. 136, 1063. Silver, D.M. and H.J. Winn, 1972b, Cell. Immunol. 7 237. Stolfi, R.L., R.A. Fugmann, L.M. Stolfi and D.S. Martin, 1975, J. Immunol. 114, 1824. Weinrach, R.S., M. Lai and D.W. Talmage, 1958, J. Inf. Dis. 102, 60. Winn, H.J., 1965, in: Ciba Foundation Symposium on Complement, eds. G.E.W. Wolsten·
holm and J. Knight (Churchill, London) p. 133.
1 1 7
Chapter VII
COMPLEMENT DEPENDENT AND -INDEPENDENT MECHANISMS IN ACUTE
ANTIBODY-MEDIATED REJECTION OF SKIN XENOGRAFTS IN THE MOUSE
Jo H.M. Berden*, Jacqueline F.H.M. Hagemann*, Wim P.M. Tamboer*,
M.José J.Th. Bogman , Robert A.P. Koene*
Departments of Medicine, Division of Nephrology and Pathology,
Sint Radboud Ziekenhuis, University of Nijmegen, Nijmegen
119
INTRODUCTION
Two experimental systems have been described in which skin
grafts carried by mice can be acutely destroyed by the administra
tion of antisera directed against the graft antigens (1,2). One
of these consisted of allogeneic skin grafted onto mice, that
were treated with anti mouse lymphocyte serum to suppress cellular
immunity (2). On day 7 after transplantation, when the grafts
showed no signs of rejection the recipients received an injection
of alloantiserum along with fresh rabbit serum as a source of
complement. Acute graft destruction occurred within 24-48 h.
Administration of exogenous complement was essential, since in
jection of antiserum alone did not destroy the grafts. This system
enabled us to study the mechanism of humorally mediated graft
destruction. Histologically, the grafts showed the characteristics
of an Arthus reaction with dense infiltration of polymorphonuclear
leukocytes (PMN) as early as 30 minutes after the antiserum in
jection, followed by extravasation of erythrocytes and necrosis.
It was subsequently shown that activation of the complete comple
ment pathway was necessary for destruction to occur (3).
Our experimental system was based on an earlier study, in
which rat skin grafted onto immunosuppressed mice was destroyed
by injection of anti rat serum (1). Histological changes in these
grafts were also of the Arthus type. Apparently the mouse endoge
nous complement was activated in this xenogeneic system of wide
antigenic disparity, since administration of exogenous complement
was not necessary to cause destruction. Further work of Winn in
this system showed that acute destruction could be induced in C5
deficient mice, while on the other hand, C3 depletion by cobra
venom factor, or PMN depletion with anti granulocyte serum, in
hibited the graft destruction (4). This suggested that activation
of C3 with consequent PMN attraction sufficed to induce tissue
damage in this xenogeneic graft model. Complement mediated cyto-
lysis of the grafts by the late acting complement components did
probably contribute to the damage because the rejection in mice
with a normal complement status had a more violent course, but
120
it played no essential role. Although these experiments show that
complement is a very important factor, it was not completely ex
cluded that graft destruction by antibody in this system also
occurred via complement independent mechanisms. The finding that,
in C3 depleted mice, grafts that were not rejected all showed
signs of inflammation might serve as an argument for such a com
plement independent destruction.
We have used a comparable xenogeneic system, to study antibody
mediated rejection and now present evidence that another, comple
ment independent, pathway of graft damage exists. This complement
dependency of graft rejection in this system was studied by using
complement deficient or complement depleted recipients, or alter
natively, by injecting non-complement fixing antibodies. Histo
logic studies were done in addition to macroscopic inspection of
the grafts, to obtain information on the mediators that were res
ponsible for the destruction.
MATERIALS AND METHODS
Animals. Inbred C57BL6/RÌJ mice were originally obtained from the
Radiobiological Institute TNO, Rijswijk (The Netherlands), and
DBA/2 (C5 deficient) mice from the N.I.H. Bethesda (Maryland,
U.S.A.). Inbred PVG/c rats came originally from the Institute of
Psychiatry, Bethlem Royal Hospital, Beckingham (Kent, U.K.).
In our laboratory these strains were kept by continuous brother-
sister matings.
Transplantation procedure. Male PVG/c tail skin was grafted onto
the flank of male C5 7BL6 or DBA/2 recipients by a modified fitted
graft technique (3). The fate of the grafts was followed by daily
macroscopic examination. Grafts were considered to be rejected
when no viable epithelium remained. Median survival times (MST)
and standard deviations (SD) were calculated according to the
method of Litchfield (5). To postpone cellular rejection of the
grafts all recipients were treated with rabbit anti mouse lympho
cyte serum (RAMLS) or the Ig fraction thereof RAMLG) on days -2,
121
-1, О, 2 and 4 after grafting in a dose of 0.25 ml i.p. This
immunosuppressive regimen prolonged the MST of PVG/c grafts to
23.0 + 1.2 days in C57BL6 recipients and to 24.5 + 1.3 days in
DBA/2 mice.
Acute reiection studies. On day 7 after grafting, when all grafts
were well healed m and showed no signs of rejection, the recip
ients received mouse anti rat serum (MARS) or its Ig fraction
(MARG) via an intravenous (i.V.) injection in the tail vein. In
mice that are not complement deficient this results in a rapid
and violent graft destruction. In this study we define this acute
antibody mediated rejection (AAR) as the complete necrosis of the
graft within 24-72 h after the i.v. injection of anti rat serum.
Complement depletion. Attempts to induce complement depletion in
vivo were first made with four agents that are known to have anti
complementary effects in vitro. These agents were: suramin
(Germanin ) provided by Bayer, Mijdrecht, the Netherlands;
flufenamic acid (Arlef ) provided by Parke-Davis, Mijdrecht, the
Netherlands; pentamidine (Lomidine ) provided by Specia, Amstelveen,
the Netherlands, and carrageenin commercially obtained from Sigma
St. Louis, U.S.A. In vivo dosages were as high as possible, but
such that there were no toxic effects to the recipients. In a
second series of experiments cobra venom factor (CoVF), 100 U/ml
(Cordis Ltd, Miami, U.S.A.) was used as an anticomplementary agent.
Antisera. RAMLS was prepared according to the method of Gray et
al (6). MARS was prepared in C57BL6 mice by weekly i.p. injections
of 5 χ 10 PVG/c lymphoid cells suspended m complete Freund's
adjuvant. After five injections the animals developed ascites,
that was tapped weekly. The lymphocytotoxic titer of the ascites
fluid was the same as the titer in the serum of these mice. The
antisera, if not used for preparation of Ig fractions, were pooled
and heated at 56 С for 45 min to destroy complement activity, and
sterilized by passage through a sterile 20 mp filter (Scheicher
and Schüll, Dassel, West Germany).
122
P r e p a r a t i o n of Ig f r a c t i o n s from a n t i s e r a . From RAMLS and MARS
Ig f r a c t i o n s (RAMLG and MARG) were p r e p a r e d i n o r d e r t o o b t a i n
p r e p a r a t i o n s f ree of complement f a c t o r s . The Ig f r a c t i o n s were
p r e c i p i t a t e d in 50% s a t u r a t e d ammoniurnsulphate, and s u b s e q u e n t l y
d i a l y s e d a g a i n s t phospha te b u f f e r e d s a l i n e (PBS). The d i a l y s a t e
was p u t on a Sepharose 4B-CL coupled P r o t e i n A column (Pharmacia ,
Uppsa la , Sweden). A f t e r s u f f i c i e n t washing wi th PBS, immuno
g l o b u l i n s were e l u t e d from t h i s column w i t h 0.05 M s o d i u m c i t r a t e
b u f f e r pH 2 . 8 , c o n t a i n i n g 1 M NaCl. The e l u a t e was immedia te ly
n e u t r a l i z e d and d i a l y s e d a g a i n s t PBS. The MARG-dialysate was
c o n c e n t r a t e d t o a volume t h a t had the same c y t o t o x i c i t y i n v i t r o
towards PVG/c lymphoid c e l l s as the MARS p r e p a r a t i o n from which
i t o r i g i n a t e d . RAMLG was c o n c e n t r a t e d and used in dosages t h a t
gave s i m i l a r p r o l o n g a t i o n of g r a f t s u r v i v a l as the o r i g i n a l
RAMLS p o o l .
P r e p a r a t i o n of IgGl and IgG2 s u b c l a s s an t ibody from MARG. Non
complement f i x i n g (IgGl) and complement f i x i n g (IgG2) a n t i b o d i e s
were p r e p a r e d from MARG by a f f i n i t y chromatography acco rd ing t o
an e a r l i e r d e s c r i b e d method ( 7 ) . Contamina t ing immunoglobulins
were removed from the IgGl and IgG2 p r e p a r a t i o n s by r e p e a t e d runs
on Sepharose columns coa ted wi th a p p r o p r i a t e immunoglobulin a n t i
b o d i e s . The p u r i t y of t he f i n a l p r e p a r a t i o n s was t e s t e d by r a d i a l
immunodiffusion. Contamina t ing IgA o r IgM could no t be demon
s t r a t e d (lower l i m i t of d e t e c t i o n < 0.05%). The f i n a l IgGl p r e
p a r a t i o n t h a t c o n t a i n e d 13,000 yg I g G l / m l , was con tamina ted wi th
0.0 5% IgG2, and the IgG2 p r e p a r a t i o n (8,700 pg IgG2/ml) w i th 0.1%
IgGl .
Se ro logy . The measurement of h e m o l y t i c complement a c t i v i t y of
t he mouse was performed wi th a C r - r e l e a s e assay t h a t has been
d e s c r i b e d e l sewhere i n d e t a i l ( 8 ) . Lymphocyto tox ic i ty of the a n t i -
s e r a was a s s e s s e d in a t r y p a n b lue e x c l u s i o n a s s a y , w i th PVG/c
s p l e e n c e l l s and r a b b i t complement ( 9 ) .
H i s t o l o g i c examina t ion of t he g r a f t s . For t h i s purpose s k i n g r a f t s
123
and surrounding recipient skin were removed at different times
after the i.V. injection of MARG and fixed in 4% buffered forma
lin. From paraffin embedded tissue blocks 4 ym sections were cut,
and stained with hematoxylin and eosin, with elastic van Gieson
stain to visualize the vessel walls, and with Mallory's phospho-
tungistic acid hematoxylin stain to detect fibrin. The histologi
cal sections were studied by the pathologist (M.J. B.) who had
no knowledge of the experimental protocol used. At each tirae-
interval three grafts were studied, and all experiments were per
formed twice.
RESULTS
Complement consumption du r ing g r a f t r e j e c t i o n . Mice were b l e d
from the r e t r o - o r b i t a l ve in a t v a r i o u s i n t e r v a l s a f t e r the i n -
time after ¡.v. injection
0 1 2 8 24 48 72 (hr) I 1 1 1 — I — ¡ — г - " — ι — " — ι — " — ι 1 1 1
'U c h a n g e CH50 titer T
.20
О
,20-
-40
.60-
-80-
-100-
Fig. 1. Changes of CHSO titer in CS7BL6 -n.ale mice during the resection of a
P'/G/a skin graft induced by the i.V. administration of 0.25 ml mouse
anti rat serum (ш). Control experiments: CHSO titers in C57BL6 mice
carrying a FVG/a skin graft (o), and in CS7B16 nice without a skin
graft that received 0.2S ml mouse anti rat serum (·). Each point is
the mean + S.E.M. of determinations in 8 mice. All determinations were
done in triplicate.
124
jection of MARS, and hemolytic complement activity was measured
in these sera. The results are given in Fig. 1. Two control ex
periments were performed. One group of mice that received MARS
but did not carry a graft, showed no changes in complement levels,
The second group was grafted and treated with RAMLS but did not
receive MARS. This treatment induced a 40% decrease of the CH50
titer on day 7, with a gradual rise to normal values on day 9.
The injection of MARS in mice carrying a PVG/c graft resulted in
an immediate decrease of the CH50 titer. After 24 h when the
graft was completely rejected, the CH50 titer returned towards
normal values. These results show that there was consumption of
complement during AAR.
Influence of treatment with anticomplementary agents on hemolytic
complement activity and AAR. C57BL6 male mice received three i.p.
injections of 0.5 mg pentamidine, 2 mg carrageenin, 2.5 mg flu-
-4Θ -Z4 О
t ime after i.p. injection anticomplementary agent
72 9 6 (hr) - i
% change CH50 t i t e r
* 2 0 1
- 2 0
- 4 0
-60
- 8 0
-ίσο
Fig, 2. Changes of the CHSO titer in CS7BL6 mice after three i, p. administra
tions (-48 h, -24 h, and 0 h) of various antiaomplementary agents.
• 0, S mg pentamidine
m 2 mg aarrageenin
a 2. 6 mg flufenamia aaid
о 5 mg suramin
Each point represents the mean + S.E.M. of determinations in four
different miae. Each determination was performed in dupliaate.
125
ТЛВЬЕ I. Influenae of treatment with anti eomplenentary agents
on AAR of PVG/a skin grafted onto CS7BL6 recipients
Group number Anticomplementary agent Occurrence of AAR
1 none 19/19
2 pentamidine (0.5 mg) Β/θ
3 carrageenin (2 mg) 9/9
4 flufenamic acid (2.5 mg) 8/8
5 suramin (5 ng) 5/7
a) injected i.p. 48, 24, and 2 h before the i.v. injection of HARG, on day 7
(daily dose listed in ng)
b) AAR was induced by i.v. injection of 0.1 ml MARG on day 7 after grafting
c) number of mice with complete graft necrosis within 24-72 h after IIARG
injection/number of mice tested
fenamic acid, or 5 mg suramin at 24 h intervals. The CH50 titers
measured 24, 48, 72 and 96 h after the third gift are presented
in Fig. 2. Only with suramin a substantial decrease in CH50 titer
was found. C57BL6 mice carrying a PVG/c graft received a similar
treatment with these anticomplementary agents at 48, 24, and 2 h
before the injection of 0.1 ml MARG on day 7.
Table I shows that AAR was not prevented by pretreatment with pen
tamidine, flufenamic acid, or carrageenin, which were the drugs
that scarcely influenced the CH5Û titer. Suramin that induced a
substantial depletion of hemolytic activity of complement pre
vented AAR in 2 out of 7 mice.
Influence of CoVF treatment on hemolytic complement activity.
Prior to the experiments in which it was tried to inhibit AAR with
CoVF we studied the effects of increasing CoVF dosages on comple
ment activity in otherwise untreated C57BL6 mice. Fig. 3 shows
that, 24 h after a dose of 20 U, hemolytic activity had almost
time after i.p. injection of CoVF 72 96 120 (hr)
-100
Fig. 3. Influence of CoVF treatment on the CH50 titer of С57БЬв mice.
• single dose of 2.S U i.p.
a single dose of 5 U i.p.
m single dose of 20 U i.p.
о daily dose of 20 U i.p.
Each point represents the mean * S.E.M. of determinations in four
different mice. Each determination uas performed in triplioate.
127
00
TABLE I I . Influenae of CoVF treatnent on AAR of P/G/o
skin grafted onto СБ7вІв геаг^гепЬв
Group number Dose MARC (nl) CoVF treatment a)
Occurrence of AAR
0.1 ml
0.1 ml
0.25 ml
0.5 ml
0/15 (MST: 23.0)
19/19
0/8
6/11
10/10
d)
a) 20 U CoVF daily i.p., started on day 5 after grafting and continued for 7 days or
until rejection of the graft was complete
b) AAR was induced by i.v. injection of listed volumes of MARG on day 7 after grafting
c) number of mice with complete graft necrosis within 24-72 h after MARG injection/
number of mice tested
d) MST = median survival time
dropped to zero. Daily injections of this dose ensured a complete
depletion of hemolytic complement activity during at least 5 days.
Influence of CoVF treatment on AAR in normal and C5 deficient mice.
C5 7BL6 mice carrying PVG/c skin grafts were treated with daily
injections of 20 U CoVF from day 5 after transplantation. Injection
of 0.1 ml MARG at day 7 did not induce AAR, while such a dose
induced rapid destruction in recipients with a normal complement
status (Table II). However, the grafts in CoVF treated recipients
were not completely protected against the destructive effects of
the anti rat serum since at higher doses AAR occurred. A dose of
0.25 ml destroyed the grafts in about half of the recipients,
while 0.5 ml MARG induced AAR in 100% of the cases. Since complete
and sustained loss of hemolytic complement activity had been in
duced by CoVF treatment, these results suggested that graft des
truction could be mediated through complement independent mecha
nisms. More arguments for this assumption were sought by performing
similar experiments in C5 deficient mice. The results are shown
in Table III. Similarly to the findings in CoVF treated mice, 0.1
ml MARG did not induce AAR in these animals. With increasing
doses of MARG the grafts again became sensitive to the antibody.
That AAR in these animals only occurred at higher antibody doses
was apparently a consequence of the C5 deficiency since 0.1 ml
MARG injected along with 0.5 ml of non complement deficient nor
mal mouse serum induced AAR in all recipients (group 5). The
results in these C5 deficient mice confirm the findings of Winn
et al (4). He presumed that in these animals activation of C3
was sufficient to induce destruction and that, although activa
tion of lytic components of complement contributed to the damage,
these were not essential. If this were the case, one would expect
that C3 depletion in C5 deficient mice would further decrease the
sensitivity of the grafts to AAR, or would even protect them
completely against destruction. Therefore we treated DBA/2 mice
with a protocol identical to that used in the mice that had no
genetic complement deficiency. The results given in Table III
(groups 6 and 7) show that this CoVF treatment caused no sub
stantial change in the sensitivity to MARG.
129
TABLE III. Influence of Ci deficiency and CoVF treatment on AAR
of PVG/c skin grafted onto DBA/I reaipients
Group number Dose MARG (nl) CoVF treatment Occurrence of AAR
1 - - 0/15C) (HST: 24.5)
2 0.1 ni - 0/16
3 0.25 ml - 10/17
4 0.5 ml - 11/11
5 0.1 ml + 0.5 nl NMSd) - 10/10
6 0.25 ml + 2/7
7 0.5 nl + 9/9
a) 20 U CoVF daily i.p., started on day 5 after grafting, and continued for 7 days or
until rejection was complete
b) AAR was induced by i.v. infection of the listed volumes of MARG on day 7 after grafting
c) number of mice with complete graft necrosis within 24-72 h after MARG injection/
number of mice tested
d) NHS = normal mouse serum not deficient in C5# injected along with MARG
Destructive activity of non-complement fixing IgGl subclass anti
bodies . We surmised that non-complament fixing antibodies could
also cause AAR if complement independent mechanisms indeed played
a role in our system. For this purpose we tested the activity of
purified IgGl antibodies prepared from MARG. As stated in the
methods the preparation was slightly contaminated with IgG2. If
tested in a cytotoxic assay against PVG/c lymphocytes residual
complement dependent cytotoxicity was found resulting in 50% cell
lysis up to a dilution of 1:8. This was attributed to the conta
minating IgG2, present in a concentration of 6.5 ug/ml. The ori
ginal MARG pool contained 6 ,900 pg IgG2/ml and had a cytotoxic
titer of 1:20,480. The destructive effects of increasing doses
of IgGl were studied in C57BL6 recipients with a normal complement
status (Table IV). The limiting dose for the induction of AAR was
approximately 800 pg IgGl. This dose was contaminated with 0.4
yg of IgG2. From the results of the dose response study for IgG2,
also presented in Table IV, it can be seen that the amount of
IgG2 contamination of the IgGl preparation cannot be held res
ponsible for the destruction, since the limiting dose for IgG2 is
between 65 and 131 yg and thus at least 160 times higher.
Histology of AAR in mice with a normal complement status and in
complement depleted animals. Induction of AAR in complement de
ficient animals did not only require higher amounts of antibodies
but the grafts also showed a different rejection pattern. In mice
with a normal complement status there was rapid necrosis of the
graft that was complete in 8 to 24 h. In C3 or C5 deficient
animals the process occurred more slowly. It started with hyper-
aemia of the graft followed by cyanosis and necrosis that was
complete after 48-72 h. The histological findings reflected these
macroscopic differences. Grafts, that were carried by mice with
a normal complement status, showed intravascular coagulation with
damage of vessel walls and extravasation of erythrocytes at 30
min after the injection of 0.25 ml of MARG. There was no intra
vascular increase of PMN. Necrosis was found after 2 h and was
complete after 8 h. In grafts of mice in which C3-depletion was
induced by CoVF, the first event was a dense intravascular accu-
131
ы
TABLE IV. AAR of FVG/o skin grafted onto CS7BL6 recipients induced by the administration of
IgGl and IgG?, subclass antibodies specifically directed against the donor antigens
Preparation IgGl (pg) IgG2 (pg) Occurrence of AAR a)
IgGl
1625
812
406
0.Э
0.4
0.2
5/5"
2/6
0/5
IgG2
0.3
0.2
0.1
261
131
65
5/5
5/5
0/5
a) AAR was induced by i.v. injection of the listed amounts of IgGl or IgG2 on day 7 after
grafting
b) nunber of nice with complete graft necrosis within 24-72 h after the IgGl or IgG2
injection/nunber of mice tested
mulation of PMN at one half and two hours after the injection of
0.25 ml of MARG. There were no signs of intravascular coagulation
at that time. Slight vessel damage with incipient extravasation
of erythrocytes and intravascular coagulation started at 8 h.
Partial or complete necrosis was present at 36 and 48 h after the
injection. The accumulation of PMN was not caused by the CoVF
treatment since sections of normal skin of the same recipient,
and grafts in mice that were treated with CoVF but not with MARG
showed no increase of PMN. These results show that dependent on
the complement status of the recipients two distinct types of
antibody mediated graft destruction occur.
DISCUSSION
It is clear from this and previous studies (2-4,9) of anti
body mediated rejection, that in recipients with a normal comple
ment status, activation of complement is an important effector
system that leads to graft damage. The decrease of the CH50 titers
during AAR of PVG/c skin grafts indicates that complement con
sumption took place. The CH50 titer remained depressed until re
jection was complete. Similar results have been reported during
rejection of xenogeneic kidneys in dogs (10).
An in vitro anticomplementary effect has been described for
pentamidine (11-15), flufenamic acid (16) and carrageenin (17,18),
but we were not able to induce in vivo complement depletion with
these drugs. With respect to carrageenin it has been reported
earlier that its anticomplementary activity in vivo is not very
impressive (19) . Only treatment with suramin induced a substantial
decrease of the CH50 titer in vivo that was in line with its
reported anticomplementary activity in vitro (20,21). The residual
complement activity was obviously sufficient to induce graft des
truction, since only 2 out of 7 grafts were protected against the
destructive effect of a relatively low dose (0.1 ml) of MARG.
Only with long lasting depletion of complement activity by CoVF
protection against 0.1 ml of MARG was obtained. However, when the
dose was raised to 0.5 ml, destruction of all grafts again occurred,
although the reaction was not as abrupt as seen in animals with
133
a normal compleirent status. It is possible that traces of comple
ment activity not measurable in the hemolytic complement assay
in vitro, mediated the destruction at higher doses of antibody.
Two findings argue against this explanation. First, in C5 defi
cient animals the decrease in sensitivity was similar to that in
C3 depleted animals and there was no further drop when these ani
mals were also treated with CoVF. One would anticipate a decrease
to occur if C3 activation, without activation of terminal comple
ment components, played an important role in the destruction.
A second and stronger argument for the involvement of complement
independent mechanisms, was our finding that non-complement
fixing IgGl antibodies also caused destruction. This destructive
effect of non complement fixing IgGl is not present in all systems.
We have previously shown that mouse skin allografts that can be
destroyed by administration of specific alloantiserum together
with rabbit complement, are completely unaffected if IgGl anti
bodies are used (7). These studies were performed in a congenie
model with limited antigenic differences and this suggests that
the occurrence of destruction by IgGl depends on the antigenic
disparity of the donor-recipient combination. Λ further argument
for the fact that complement-independent mechanisms can only
become operative in graft models with a wide antigenic disparity,
is our previous finding that AAR of mouse allografts only occurred
in the presence of a completely intact complement system (3).
Histologic studies can give insight into the spectrum of
reactions that may lead to antibody mediated vascular damage. The
results in animals with a normal complement status show that acute
intravascular coagulation can occur, without involvement of PMN.
It is likely that under these circumstances there is direct cyto-
lysis of vascular endothelium by antibody and complement with
consequent rapid platelet aggregation and coagulation. This has
happened before PMN, attracted by activated chemotactic factors,
can reach the graft. It, thus, seems that PMN are not absolutely
necessary to cause graft destruction initiated by antibody. The
destruction can, however, also take place via a classic Arthus
type reaction with activation of chemotactic complement compo
nents and secondary attraction of PMN. We have shown previously
134
that this type of reaction occurs in an allogeneic system, where
destruction is seen only in the presence of exogenous complement
(2). After accumulation of PMN in the graft vessels, there is
vascular damage, extravasation of erythrocytes and necrosis. Sim
ilar histologic changes have been found in another model of anti
body mediated destruction of rat skin grafts (1,4). On the basis
of our findings we propose that a third pathway of vascular damage
should be added to this spectrum. Although this pathway is comple
ment independent it is mediated by PMN as shown by the histologic
studies in animals depleted of complement by treatment with CoVF.
It is not clear which factors are responsible for the attraction
of PMN under these circumstances. It might be the result of immune
adherence of PMN to the Fc part of the immunoglobulins bound to
the vascular endothelium (22). Since there is no amplification of
this process by the generation of chemotactic factors of the com
plement system, the reaction develops more slowly and requires a
relatively high amount of antibodies. This does not exclude that
the process is amplified by other mechanisms. It has been shown
that mouse IgG antibodies can induce a complement independent
release of histamin and serotonin from leukocytes (23), and this
could obviously contribute to the rejection process. Furthermore,
immune adherence of PMN can cause release of lysosomal enzymes
(22), that can enhance further accumulation of PMN, causing damage
of the vascular endothelium (24). Elucidation of the mechanism in
this type of rejection clearly requires further study. Our findings
are in apparent contrast to earlier observations in reversed pas
sive Arthus reactions and in experimental nephrotoxic nephritis,
where depletion of the complement system could prevent PMN accu
mulation (25-29). This stresses the fact that in graft rejection
other mechanisms can become operative, whose activation is proba
bly influenced by the extent of antigenic disparity that exists
between donor and recipient.
The existence of this mechanism might have practical implica
tions for clinical transplantation. Attempts to influence humoral
graft rejection by complement depletion might fail because this
approach does not provide absolute protection against antibody
mediated graft destruction.
135
SUMMARY
Complement dependency of acute antibody mediated rejection
(AAR) was studied in a xenogeneic skin graft model in the mouse.
PVG/c rat skin grafted to immunosuppressed mice is quickly des
troyed by intravenous administered mouse anti rat serum on day 7
after grafting. We demonstrated depression of the CH50 titer in
the recipients, indicative for complement consumption during the
rejection process. Of four anticomplementary agents: pentamidine,
carrageemn, flufenamic acid and suramin, only treatment with
suramin led to a substantial complement depletion in vivo.
Administration of suramin prior to anti rat serum protected 30%
of the grafts against AAR, while the three other drugs were in
effective in this regard. Complete and long lasting complement
depletion induced by treatment with cobra venom factor (CoVF)
decreased the sensitivity of the grafts for AAR. However, complete
protection was not achieved, since high doses of anti donor serum
again induced destruction. Similar results were obtained in C5
deficient recipients, and m CoVF treated C5 deficient recipients.
These results indicated that complement independent rejection
mechanisms were operative. This was further substantiated by the
finding that pure non-complement fixing IgGl subclass antibodies
were able to elicite AAR. Histologic studies showed that AAR of
grafts in mice with a normal complement status was caused by
intravascular coagulation without primary involvement of poly
morphonuclear leukocytes. In complement depleted animals an
Arthus-like reaction was seen, with dense intravascular accumula
tion of PMN, that, apparently, were attracted to the graft through
complement independent mechanisms.
ACKNOWLEDGMENTS
The skillfull technical assistance of Miss Cathy N. Maass and the
staff of the animal laboratory (head: Dr. W.J.I, van der Gulden)
is gratefully acknowledged.
136
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tation. 24:175.
10. Gewürz, H., D. Scott Clarck, J. Finstad, W.D. Kelly, R.L.
Varco, R.A. Good, and A.E. Gabrielsen. 1966. Role of the com
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11. Coats, Ε.Α. 1973. Comparative inhibition of thrombin, plasmin,
trypsin, and complement by benzamidines using substituent
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12. Asghar, S.S., K.W. Pondiran, and R.H. Cormane. 1973. Inhibition
of Clr, Cls and generation of Cls by amidino compounds.
Biochem. Biophys. Acta. 317:539.
13. Hausch, С , and M. Yoshimoto. 1974. Structure-activity rela
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benzamidines. J. Med. Chem. 17:1160.
14. Glovsky, M.M., M. Cory, and A. Alenty. 1974. Inhibition of
guinea pig complement by dérivâtes of benzamidine. Immunology.
20:819.
15. Hauptman, J., and F. Markwardt. 1977. Inhibition of the hemo
lytic complement activity by derivatives of benzamidine.
Biochem. Pharmacol. 2 5:325.
16. Harnty, Th.W., and M.B. Goldlust. 1974. Anti-complement
effects of two anti-inflammatory agents: Niflumie and flu-
fenamic acids. Biochem. Pharmacol. 2 3: 3107.
17. Davies, G.E. 1963. Inhibition of guinea pig complement in
vitro and in vivo by carrageenm. Immunology. 6:561.
18. Davies, G.E. 1965. Inhibition of complement by carrageenm:
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19. Borsos, T., H.J. Rapp, and C. Crisler. 1965. The interaction
between carrageenm and the first component of complement.
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20. Fong, J.S.C., and R.A. Good. 1972. Suramin, a potent reversible
and competative inhibitor of complement system. Clin. Exp.
Immunol. 10:121.
21. Eisen, V., and C. Loveday. 1973. Effects of suramin on comple
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Br. J. Pharmac. 43:678.
21. Henson, P.M. 1971. Interaction of cells with immune complexes:
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23. Casey, F.B., and S. Tokuda. 1973. A comparative study of the
mechanisms of passive cutaneous anaphylaxis induced by mouse
138
IgG, rabbit IgG, and rabbit F(ab,)2 antibodies. Int. Arch.
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24. Janoff, A. 1970. Mediators of tissue damage in human poly-
norphonuclear neutrophils. Ser. Haemat. 3:96.
25. Ward, P.A., and C.G. Cochrane. 1965. Bound complement and
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polymorphonuclear leukocytes and complement in nephrotoxic
nephritis. J. Exp. Med. .222:99.
27. Cochrane, C.G. 1967. Mediators of the Arthus and related
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28. Cochrane, C.G. 196 8. The role of immune complexes and comple
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29. Cochrane, C.G., H.J. Müller-Eberhard, and B.S. Aiken.
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139
Chapter Vili
SUMMARY
141
Rejection mediated by antibodies accounts for a considerable
amount of graft losses in renal transplantation. The detrimental
effects of antibodies that are already circulating at the time of
grafting or that are formed after transplantation might be di
minished by interfering with the effector systems triggered by
these antibodies. Of these effector systems the complement system
is one of the most important, and an understanding of its precise
role in antibody mediated rejection (AAR) might provide additional
tools for the management of the rejection process. The aim of
this study was to investigate the role of the complement system
in AAR in an experimental animal model.
A protocol was used in which graft destruction could reproduci-
bly be elicited by the passive administration of anti donor anti
serum to skin graft recipients, whose cellular immune response
was suppressed by treatment with anti mouse lymphocyte serum.
The first set of experiments was performed in an allogeneic skin
graft model in the mouse. In this model administration of exogenous
complement together with antiserum is required for AAR, since the
recipient's endogenous complement system is inefficiently activa
ted by mouse alloantisera.
The influence of antigenic disparity on AAR was studied in nine
donor-host combinations whose histoincompatibility ranged from a
single non-H-2 difference to a disparity for the complete H-2
complex plus multiple non-H-2 differences. With the exception of
combinations with a single or few non-H-2 differences AAR could
be induced in all combinations. It was found that with increasing
histoincompatibility, the amount of antibodies required for the
induction of AAR decreased, parallel to the decrease of the median
survival time of skin grafts in untreated animals, and also paral
lel to the increase of the cytotoxic titer of the anti donor serum
in vitro. It is concluded that the results for immunogenicity of
the different histocompatibility loci as described for cell-mediated
rejection, also apply to our model of humoral graft destruction.
Complements from various species differ in their effectiveness
in in vitro lymphocytotoxicity tests with mouse alloantisera. All
antisera, when tested with rabbit complement (RC), gave rise to a
higher cytotoxic titer than with guinea pig complement (GPC) or
142
human complement (HC). These differences in effectivity were
mirrored in the in vivo studies. In some donor-host combinations
with relatively small antigenic disparity (multiple non-H-2 dif
ferences, H-2D or H-2K) , where AAR could be elicited by injection
of RC along with anti donor serum, GPC and HC were ineffective.
In the combinations with a greater histo-incompatibility (H-2D
plus non-H-2, H-2K plus non-H-2, H-2 and H-2 plus non-H-2) GPC
and HC were also effective. However, dose response studies showed
that GPC was less efficient than RC, for a greater amount of anti
serum was necessary to induce AAR with it. In none of the allo
geneic donor-host combinations were we able to induce AAR by in
jection of antiserum alone. This is in agreement with the fact
that mouse complement never caused lymphocytotoxicity of target
cells in vitro, not even with the strongest antiserum. In general
terms one can conclude that the cytotoxic titer of an antiseruir.
as tested in vitro gives a good indication about its destructive
power in vivo.
The question whether all components of the complement system
are necessary for the induction of AAR was studied in an allo
graft model in which a complete H-2 disparity existed between
donor and recipient (B10.D2 -* C57BL6). As stated above AAR can be
evoked in such a combination by injection of anti donor serum
along with RC, GPC or HC. Experiments were done in which allo-
antiserum was injected together with sera from animals deficient
in single complement factors or, alternatively, with sera that
were artificially depleted of one of the complement components
(R-sera). The in vivo effects of the various R sera were again
compared with their in vitro activity in the lymphocytotoxicity
assay. None of the R-sera (Rl, R4, R3, R6 or R9) was able to
mediate lymphocytotoxicity when tested with B10.D2 target cells
and C57BL6 anti B10.D2 serum. Reconstitution with the deficient
complement factor restored the lymphocytotoxic capacity of all
the R sera. If normal mouse serum was added to Rl and R9 serum,
these regained their cytotoxicity, demonstrating that mouse CI
and C9 could replace the deficient factor. For CI this was further
substantiated by the finding that a crude mouse CI preparation
had the same restoring effect on Rl serum as whole mouse serum.
143
The R4, R3 and R6 sera could not be reconstituted with normal
mouse serum. If the different R sera were injected with anti donor
serum in C57BL6 recipients of a B10.D2 skin graft results analogous
to the in vitro findings were obtained. Rl and R9 serum induced
AAR without prior reconstitution. On the other hand C4, C3 and C6
depleted complement sources did not evoke AAR. This was only
obtained if, prior to administration, these R sera were reconsti
tuted with the appropriate complement factor. These experiments
permit the following conclusions. The close correlation between
the in vivo activity and the cytotoxicity in vitro strongly sug
gests that activation of the classical complement pathway is
necessary to induce AAR in this allogeneic model. This is corro
borated by the finding that R4 serum, which can be activated nor
mally via the alternative pathway, did not elicit AAR. Furthermore,
the complete complement sequence has to become activated in this
model. Generation of chemotactic complement factors (C3a and C5a)
alone, is not sufficient to cause AAR, since R6 serum, that can
generate normal chemotactic activity, did not induce AAR. Lastly,
the low cytotoxic properties of mouse complement do not reside
in factors CI and C9, because these factors could effectively
restore Rl and R9 serum.
The aim of the second set of experiments was to elucidate the
role of endogenous complement in AAR. For this purpose a xeno
geneic skin graft model was used in which a PVG/c rat skin graft
was placed on immunosuppressed C57BL6 or DBA/2 mice. In this model
of greater antigenic disparity administration of exogenous comple
ment is not required and the injection of mouse anti rat serum
alone causes a rapid and violent destruction. Complement consump
tion was measured during AAR, and complement dependency of the
destruction was studied by using complement depleted recipients
and by the administration of non-complement fixing antibodies.
Before this study was undertaken a sensitive assay of mouse
complement had to be developed in order to measure complement con
sumption and complement inhibition. Because under test conditions
normally used for the measurement of hemolytic activity of human
and guinea pig complement no activity is found with mouse comple
ment, several conditions of the test were investigated to increase
144
its sensitivity. It was found that 7S IgGl amboceptor activated
mouse complement better than 19S IgM amboceptor. The use of a
Cr release assay allowed the reduction of the number of target
cells. Furthermore it was found that optimal incubation conditions
were: an ionic strength of the incubation medium of 0.13 M NaCl,
an incubation time of 90 minutes, and an incubation temperature
of 30 C. With this test it was possible to measure hemolytic ac
tivity of mouse complement sensitively and reproducibly.
With the above mentioned method we found complement consumption
during AAR of a PVG/c skin graft carried by C57BL6 recipients.
Ten minutes after the i.v. injection of mouse anti rat serum there
was a sharp decrease in CH50 titer, which remained depressed during
the rejection process. After the rejection had been completed the
CH50 titer gradually rose to normal levels. Subsequently, attempts
were made to inhibit complement activity and to prevent AAR with
four agents which are anticomplementary in vitro. With pentamidine,
carrageenin and flufenamic acid no significant decrease of the
complement activity in C57BL6 mice was obtained. Treatment of
these agents did not prevent the occurrence of AAR. Although ad
ministration of suramin caused a substantial decrease in CH50 titer
(20% of normal values), it protected only 2 of 7 grafts against
AAR. Prevention of AAR was only found if a complete and prolonged
depletion of the complement system of the C57BL6 recipient was
induced by treatment of CoVF prior to the administration of anti
donor serum. Similar results were obtained in C5 deficient DBA/2
recipients. The protective effect of CoVF pretreatment or of C5
deficiency, was, however, overcome if a greater dose of mouse anti
rat globulin (MARG) was given to the recipients. This was even the
case if C5 deficient recipients were also treated with CoVF.
With 0.5 ml MARG, AAR was obtained in all recipients despite C3
depletion, C5 deficiency, or C3 depletion plus C5 deficiency.
These results indicated that complement independent mechanisms
became operative in this xenogeneic skin graft model, provided
sufficient amounts of antibody were presented to the graft.
A further argument for this was obtained by the finding that non-
complement fixing IgGl antibodies, prepared from MARG, could in
duce AAR. Although it is as yet not clear which mechanisms are
145
operative in the complement independent destruction, we have
acquired some clues from histologic examinations of the grafts.
In animals with a normal complement status the destruction of the
xenografts was brought about by acute intravascular coagulation
without primary involvement of polymorphonuclear leukocytes (PMN).
Xenografts rejected by complement depleted recipients showed
dense intravascular accumulation of PMN, with only secondary signs
of intravascular coagulation. These PMN must have been brought
into the graft by complement-independent mechanisms. This is
possibly the result of immune adherence of PMN to the Fc part of
the antibodies bound to the vascular endothelium. Elucidation of
the mechanisms in this type of rejection clearly requires further
s t udy.
When extrapolating these results to the clinical transplanta
tion situation, one may conclude that it is worthwile to continue
the search for effective anticomplementary agents, since these
might lead to attenuation of the humoral rejection process.
Agents that inhibit the complement components of the classical
activation unit are preferable, because they have the advantage
of leaving the alternative pathway intact. This is important for
the complement dependent defense mechanisms against bacteria.
Whether complement independent mechanisms can become operative in
the vascular rejection of human kidney grafts remains speculative,
until we know more about the role of immunoglobulin subclasses
and about the extent of antigenic disparity necessary to initiate
this process.
146
WOORDEN VAN DANK
Allen die hebben bijgedragen aan het tot stand komen van dit
proefschrift wil ik gaarne bedanken.
De transplantatie experimenten en de ontelbare serologische be
palingen konden worden verricht dankzij de enthousiaste hulp van
Jacqueline Hagemann. Zij werd in de loop der jaren bijgestaan door
Trees Borggreve, Ton Lensen en Peter Daamen.
Voor het verkrijgen van diverse sera en de bereiding van meerdere
antisera werd ik op deskundige wijze geholpen door de medewerkers
van afdeling IV (hoofd: dhr. J. Koedam) van het Centraal Dieren
laboratorium van de Universiteit (hoofd: Dr. W.J.I. v.d. Gulden).
Bij het selecteren en verkrijgen van de diverse dierstammen deed
ik nooit vergeefs een beroep op de kennis en hulp van dhr. J. van
Gaaien en dhr. F. Janssen van het Centraal Dierenlaboratorium.
De discussies over de resultaten van het onderzoek met de mede
werkers van het laboratorium voor nefrologie (Rob de Waal,
Simon Lems en Wim Tamboer) waren steeds erg plezierig en vormden
een essentiële bijdrage aan de voortgang en de tot stand koming
van dit proefschrift. Ondanks vele tegenslagen isoleerde Wim Tam
boer met onnavolgbare vasthoudendheid de verschillende immuun-
globulinen en subklas antilichamen.
Van de adviezen van Mej. Drs. W.C.A. Buys (Ir. Reichertlaborato-
rium) bij het ontwikkelen van de Cr release assay werd dankbaar
gebruik gemaakt. Evenmin mag onvermeld blijven de vriendschappe
lijke hulp die ik kreeg van Mevr. A.C. Feiten (Ir. Reichert-
laboratorium) en dhr. G. Grutters (Isotopenlaboratorium van het
Centraal Dierenlaboratorium) bij de praktische uitvoering van de 5 ΙΟ r assay.
De tekeningen voor dit proefschrift werden met zorg gemaakt door
dhr. H. Berris van de afdeling Medische Illustratie (hoofd: dhr.
W. Maas). De medewerkers van de afdeling Medische Fotografie
(hoofd: dhr. A. Reynen) vervaardigden de foto's.
De Heer E. de Graaff (hoofd Medische Bibliotheek) en zijn staf
waren behulpzaam bij het verzamelen van de literatuur.
Het typen van de talrijke versies van de tekst werd met grote
toewijding en nauwkeurigheid verricht door Angèle Wentholt.
148
CURRICULUM VITAE
De auteur van dit proefschrift werd geboren op 26 mei 1948 te
Blenck. Hij bezocht het St. Thomascollege te Venlo en behaalde
het einddiploma HBS-B in 1965. Daarna studeerde hij geneeskunde
aan de Katholieke Universiteit te Nijmegen, waar hij in 1971 het
doctoraal- en in 19 73 het artsexamen aflegde. Vanaf 1 augustus
1973 was hij m opleiding tot internist, aanvankelijk m het
St. Joseph Ziekenhuis te Eindhoven (hoofd van de opleiding:
Dr. P.F.L. Deckers) en vanaf 1 februari 1975 aan de Universiteits
kliniek voor Inwendige Ziekten (hoofd: Prof.Dr. C.L.H. Majoor).
Op 1 augustus 19 78 werd hij ingeschreven als internist in het
specialistenregister. Vanaf 1 januari 1979 is hij werkzaam op de
afdeling nierziekten (hoofd: Prof.Dr. P.G.A.B. Wijdeveld) van de
Universiteitskliniek voor Inwendige Ziekten.
Hij is getrouwd met Tilla Crommentuyn, en heeft 2 kinderen:
Joost en Bas.
149
STELLINGEN
1
De regels voor de immunogeniciteit van de diverse loci binnen het
H-2 complex gelden niet alleen voor de cellulaire maar ook voor de
humorale afstoting van huidtransplantaten.
2
Acute antilichaam afhankelijke afstoting van allogene huidtrans
plantaten in de muis vereist een aktivatie van de volledige
klassieke route van het complement systeem.
3
Acute antilichaam afhankelijke afstoting van xenogene huidtrans
plantaten in de muis kan via complement onafhankelijke mechanismen
verlopen.
4
Transplantaatbeschadiging door antilichamen kan zowel het gevolg
zijn van een lokale Shwartzman reactie als van een klassieke
Arthus reactie. Welke van de twee tot ontwikkeling zal komen hangt
af van het antigene verschil tussen donor en ontvanger, en van de
titer en subklas specificiteit van de circulerende antilichamen.
5
In tegenstelling tot allogene niertransplantaties bij het konijn,
induceert Cyclosporine A geen blijvende tolerantie bij muizen die
een allogeen huidtransplantaat dragen.
- S.P.M. Lems en R.A.P. Koene (1979)
ter publicatie aangeboden
- C.J. Green en A.C. Allison
Lancet (1978) , 1, 1182
6
Bij een patient met een niet-purulente mono- of oligoarthritis
dient men ook een Campylobacter jejuni infectie als mogelijke
oorzaak uit te sluiten.
- J.H.H. Berden, H.L. Muytjens en L.B.A. v.d. Putte
British Medical Journal (1979), !_, 380
7
Immuuncomplexen en complement aktivatie spelen een belangrijke
rol bij de ontwikkeling van de complicaties van een Plasmodium
falciparum infectie.
- WHO Technical Report (1975) 579
- eigen waarneming
8
Bij een onbegrepen acute nierinsufficientie moet als mogelijke
oorzaak de ziekte van McArdle (niet-traumatische rhabdomyolyse)
overwogen worden.
- eigen waarneming
9
Er zijn voldoende aanwijzingen voor een immunologische Pathogenese
van multiple sclerose om goed opgezette pogingen ter behandeling
van deze ziekte met immunosuppressiva te rechtvaardigen.
- P. Delmotte, O.R. Hommes en R. Gonsette (redacteuren)
Immunosuppressive treatment in multiple sclerosis.
European Press, Gent, 1977
10
In een toekomst ige t r a n s p l a n t a t i e wetgeving v e r d i e n t t en a a n z i e n
van d o n a t i e van organen h e t "geen-bezwaar-sys teem" de voorkeur
boven h e t " toesternmingssysteem" .
- H.J.J. Leenen
Nederlands Juristen Blad (1978) , 53_, 871
11
Het onderwijs in de medische geschiedenis verdient een ruimere
plaats in de medische opleiding.
Nijmegen, 27 april 19 79 J.H.M. Berden