8/12/2019 Kart Tune n 1986

1/10

Histopathology

1986,

10,841-850

Distribution

of

basement membrane laminin and

type IV collagen in human reactive lymph nodes

T.

K A R T T U N E N

,

M . A L A V A I K K O *

,

*Departments of Pathology, University of Oulu, Oulu and

tKeski-Pohjanmaa Central Hospital, Kokkola, Finland

M . A P A J A - S A R K K I N E N t H . A U T I 0 - H A R M A I N E N *

Accepted for publication

3

December

1985

KARTWNEN

T. , ALAVAIKKO

. ,

APAIA-SARKKINEN. AUTIO-HARMAINEN.

1986)

Histopathology 10, 841-850

Distribution

of

basement m embrane laminin

and type IV collagen in

human reactive

lymph nodes

Th e location of two basement mem brane components, laminin and t he

7-S

domain of

type

IV

collagen, was studied in human lymph nodes using the peroxidase-

antiperoxidase method. Basement membrane antigens were present on the walls of

blood vessels and of marginal, trabecular and medullary sinuses. Thin, fragmented

fibre-like staining was present also in parenchyma outside the germinal centres, in a

pattern overlapping with reticular fibres as seen on conventional reticulin stains. This

finding suggests that basement membrane components are a part of the reticular

fibres of lymph nodes, or are closely associated with them.

Keywords: lymph no de, laminin, type 1V collagen

Introduction

Despite extensive studies of the structure and function

of

lymph node cells

participating directly in immune responses, relatively little attention has been paid

to th e connective tissue comp onen ts of lymph nodes. E xtracellular matrix probably

plays an important role in the traffic of lymphocytes across the lymphatic

interstitium Fossum Ford 1985). Structurally characteristic alterations of cellular

connective tissue and extracellular matrix occur both in reactive and neoplastic

conditions. These include characteristic fibrosis in some types of non-Hodgkins

lymphomas and Hodgkins disease Re e, Leone Crowley

1982

and basement

mem brane changes in immunoblastic lymphadenopathy Knecht Lennert

1981,

Ka rttunen , Ala vaikk o Autio-Ha rmainen, unpublished observations).

Kajaanintie 52D, 90220 Oulu 22, Finland.

Address for correspondence: Dr T.Karttunen, Department of Pathology, University of Oulu,

6

841

8/12/2019 Kart Tune n 1986

2/10

042

T

Karttunen et al.

Basement membranes are a special ized form of extracellular matrix. They

separate endothelial, epithelial, muscle and fat cells from interstitial connective

tissues. They surround the endothelium of the blood vessels, but a re thoug ht

to

be

absen t fro m the lymphatic capillaries Barsky et al. 1983). The basement

membranes provide physical support to the tissues, participate in attachment

between cells and tissues and play an impqrtant part in ultrafiltration. Chemically

they are composed of a specialized form of collagen, type IV collagen, and many

non-collagenous proteins for review see Martinez-Hernandez A m en ta 1983),

o n e

of

which is a glycop rotein, laminin Timpl ef

al. 1979). It

has been suggested

that cells interact with laminin through a specific receptor molecule and that

laminin receptor may aid tumour cells to attach to basement membrane in the

me tastatic process L iotta, R ao Barsky 1983).

Except for a recent report about the distribution of laminin in hum an lymphoid

tissue Reilly

ef

al. 1985) the distribution of basement membrane antigens in

human lymph nodes has not been reported previously. We have studied the

occurrence and location

of

laminin and type IV collagen in common types of

reactive changes in human lymph nodes using antibodies against these two

components . The staining pattern

of

these components has been compared with

that

of

Go mo ris classical reticulin stain.

Materials

and methods

The material consisted

of

five reactive lymph nodes removed for diagnostic

purposes Table 1). Sections 5p m in thickness were stained with haem atoxylin and

eosin for routine histology and with Gomoris reticulin stain to evaluate the

distribution

of

reticulin. Antibodies used in immunohistochemical stainings were a

kind gift from Dr Leila Risteli and Dr Juha Risteli, Collagen Research Unit,

Depar tment of Medical Biochemistry, University

of

Oulu. Antibodies against the

7-S domain of type IV collagen from hum an kidney Risteli et al. 1980) and laminin

from hum an placenta Risteli Timpl 198l) , were raised in rabbits and purified by

Table 1. Lymph nodes studied

Case no . A geh ex Location Histopathological diagnosis

1 39/M Cervical Non-specific reactive lymphaden itis

follicular hyperplasia, slight

dilatation

of

sinuses, slight

paracortical reaction)

follicular hyperplasia, sinus

histiocytosis)

paracortical hyperp lasia)

2 13/F Cervical Non-specific reactive lymphaden itis

3 20/M Axillary Non-specific reactive lymphaden itis

4

18/M

Axillary Dermatopathic lymphadenopathy

5

55/M Cervical Dermatopathic lymphadenopathy

6

60/M Inguinal Fibrosis

8/12/2019 Kart Tune n 1986

3/10

Basement membrane in

lymph

nodes

843

immunoabsorption, tested radioimmunologically to be non-cross-reactive and used

in the peroxidase-antiperoxidase procedure on sections cut from formalin fixed,

paraff in .embedded lymph node biopsies Karttunen et

a f . 1984).

Normal rabbit

serum and phosphate buffered saline PBS) were used instead

of

the primary

antibody

as

controls.

Results

Th e staining pattern of both antigens was similar in all cases. In the lymph node

capsule and trabeculae there were single cells or small groups of cells, where

Figure

1. Ca psul e and m arginal sinus of a fibrotic lymph node Case 6) stained for type IV collagen.

Small blood vessels in the cortex and in the capsule have strongly staining basem ent mem branes. T he

afferent lymphatic

L)

has strong staining on its walls, which continues on the outer wall

of

the

marginal sinus

S).

In the inner wall

of

the marginal sinus the staining pattern is faint

or

attenuated

arrows). In the capsule some cells, probably smooth muscle cells, are surrounded by faint staining

arrowheads).

~ 6 6 0 .

8/12/2019 Kart Tune n 1986

4/10

844 T . Karttunen et al.

individual cells were surrounded by strong and more or less continuous staining.

The staining pattern around the cells was similar

to

that seen around smooth

muscle cells in oth er organs Figure

1).

Afferent and efferent lymphatics had a

continuou s linear staining on their walls Figures

1

and

5).

A continuous line

of

staining outlined the outer wall

of

the marginal

subcapsular) sinus and sinuses along connective tissue trabeculae extending from

the capsule. In the inner side of marginal and trabecular sinuses staining

of

both

antigens was present, but its intensity was more variable and generally less than

tha t on the capsu lar side, and th e staining was distinctly discontinuous Figures 2

and

3).

The amount and length

of

the discontinuities varied in different regions,

but no constant pattern with relation to deeper structures such as germinal centres

was detectable. In cortical and medullary sinuses the staining pattern was similar to

that of the inner side

of

th e marginal sinus Figures 2 an d 5 ) . Thin strips

of

staining

we re see n ne ar t o a few reticular cells in all types of sinuses Figures 1, 2, 3 and 5 ) .

The parenchyma outside sinuses contained thin, fibre-like staining, which

formed frag me nted , irregular nets around

3-10

cells Figures 3 and 6 . This net was

absent from the germinal centres and the compact corona

of

small lymphocytes

around them. However, small fragments

of

staining, not thought

to

represent

blood vessel basem ent m em bra ne were present in germinal centres Figure 6 . In

Figure

2.

Capsule and cortex of a reactive lymph node Case 1 stained

for

type

IV

collagen. Almost

continuous staining

is

present on the walls

of

both trabecular sinus

T)

and cortical sinuses

C ) . X 120.

8/12/2019 Kart Tune n 1986

5/10

Basement membrane in

lymph nodes

845

Figure

3. Detail

of

a junction of marginal and trabecular sinuses stained

for

type

IV

collagen Case 1 .

Staining is present on the walls of both the sinuses and basement membrane material is also seen as

fine fibres

in

the sinus arrow s) and cortical stroma arrow heads). ~ 9 0 0 .

dermatopathic lymphadenopathy, fibre-like staining was largely absent from

paracortical areas with increase in reticulum cells.

The distribution

of

reticular fibres as seen in the reticulin stain showed some

overlap with the staining pattern of basement membrane components . However,

the nets formed by reticular fibres were more continuous and regular. Sinus walls

stained more distinctly on immunostaining than with Gomoris reticulin stain

Figure 4 .

The blood vessels had a strong continuous line of staining visible on the

endothelial basement membrane. The walls of larger vessels also showed a

reticulated staining reaction , corresponding

to

the basement m em brane of pericytes

or smooth muscle cells of the vessel wall. In germinal centres, one to four

capillaries with ordinary staining basement me mb ranes were present Figure 6).

8/12/2019 Kart Tune n 1986

6/10

Figure 4.

Gom oris reticulin stain Case

1).

There is no definite silver-positive wall on the subcapsular

sinus S). When co mp ared to the immunostaining results Figures 2 and

5 )

there are more positively

stained fibres both in the sinus and in the cortical strorna. x380.

Figure 5. Hilus of a reactive lymph node stained

for

type JV collagen Ca se 1 . Thin and fragmented

staining is present on the parenchymal side while co ntinuous strong staining is seen on the hilar side of

the medullary sinuses

M).

Efferent lymphatics E) have continuous staining on their walls. Veins

V )

and arteries A) have strongly stained basement membranes. X

120.

8/12/2019 Kart Tune n 1986

7/10

Basement membrane

in

lymph nodes

847

Figure

6

Lymph n ode cortex stained for laminin Case 1). A germinal centre G) with the surrounding

corona of small lymphocytes is practically devoid of staining except for the basement membrane of

small capillaries. Th in fibre-like staining is present in the paracortical strom a arrows). Post-capillary

venules P) are strongly stained. ~ 3 8 0 .

Discussion

This study shows that all types of sinuses in human lymph nodes have a basement

membrane of varying thickness and degree of continuity. This finding is in

agreem ent with th e results of an electron microscopic study by Forkert , Thliveris

Bertalanffy 1977) o n hum an lymph nodes. Ho wev er, M ori Len nert 1969)

comm ented o n the ab sence of a definite basement m embrane from the inner s ide

of

the subcapsular sinus in human lymph nodes. Electron microscopic studies in

expe rimen tal animals suggest either that a definitive mem brane is lacking, although

basement membrane-like material is present on the sinus walls

Moe

1963,

Nopajaroonsri , Luk Simon 1971, Luk , Nopajaroonsri Simon 1973),

or

comm ent on the total absence of basement mem branes Yam ada Yamagishi

1961, Farr , Cho De Bruyn 1980). An immunofluorescence study on bovine

lymph nodes using antisera against type IV collagen suggested the presence of

basem ent m em bra ne on the walls of the sinuses Konom i, Sano Nagai 1981).

Laminin has be en shown to be present o n the walls of the m arginal sinus

of

human

lymph node s Reilly

et

al. 1985).

Reticular fibres form th e supporting meshwork of the lymph node stroma. They

are characteristically identified by silver impregnation, and have been shown to

contain collagen types I and

I11

Konomi

et

af 1981), and fibronectin Stenman

8/12/2019 Kart Tune n 1986

8/10

848

T Karttunen et al.

Vaheri 1978). The staining pattern of basement membrane antigens in our study

resembled that

of

reticular fibres as seen in silver impregnation, although the net

formed by the components was not

so

dense and regular. Our results suggest that

basement membrane material is an inconstant component, or close associate of

reticular fibres. This is in agreement with the electron microscopic study by

Tykocinski, Schinella an d Gr ec o 1983), who saw focal accumulations of basement

membrane material between fibroblastic reticulum cells and reticular fibres.

However, type IV collagen had an entirely different distribution from that

of

reticular fibres as seen in silver impregnated bovine lymph nodes Ko nom i et al.

1981). On the inner wall of sinuses, basement membrane material was not

completely impregnated by the reticulin staining. This finding emphasizes the lack

of

chemical specificity of the reticulin stain Puch tler W aldr op 1978). T h e

distribution of basem ent m em brane antigens in the white pulp and B illroths cords

of spleen Apaja-Sarkkinen et

af

1986) seems to be comparable to that

of

lymph

node parenchyma.

No specific ultrastructural features have been seen in the basement membrane

of the blood vessels of lymph node s Mori et at 1969, No pajaroo nsri, Luk Simon

1971). Neither did ou r study reveal any notable differences in th e staining pa ttern

in

lymph n od e vessels compared with tha t

of

extranodal vessels. Our observation of

the presence

of

basement membrane components on afferent and efferent

lymphatics is in agreement with the results of Barsky

et al.

1983).

Sm ooth m uscle cells are constantly p resent in the capsules and trab eculae

of

the

lymph nodes

of

man and cattle Folse, Beath ard Granholm 1975) and we re

demonstrated here by the staining of basement membrane antigens around them.

This method offers a new possible approach to estimate the number

of

smooth

muscle cells in the lymph nodes.

It is not known whether basement membranes have any special role in the

lymph nodes. Guidance of the cells arriving

in

lymph nodes has

so

far been

attributed to th e cellular microenvironmen t see Fossum Ford 1985), but recent

findings suggest that the extracellular matrix may also regulate the function of both

monocytes Huard

et al.

1984) and lymphocy tes Shields, Ha ston Wilkinson

1984).

Acknowledgements

We thank Miss Eija Hiltula for skilful technical assistance. This work was

supported in part by Sigrid Juselius Foundation.

References

APAJA-SARKKINEN. , ALAVAI KKO. , KARTTUNEN. AUTIO-HARMAINEN. 1986) Basement

membran e proteins in the spleen: immu nohistochemical demonstration and relation to reticulin.

BARSKY

. H . ,

BAKER . , SIEGAL

.P., TOGO.

LIOTTA .A . 1983) Use of anti-basement membrane

antibodies

to

distinguish blood

vessel

capillaries from lymphatic capillaries.

Amer ican Journal

of

Surgical Pathology 7 667-677

Histopathology

10,

295-302

8/12/2019 Kart Tune n 1986

9/10

Basement membrane in

lymph

nodes

849

FARRA . G . , C HO

Y . DE

B RUYN .P .H.

1980) T he structure of the sinus wall of t he lymph node

relative to its endocytic properties and transmural cell passage. American Journal of Anatomy

FORKERT.G., THLIVERIS.A. BERTALANFFY

.D.

1977) Structure of sinuses in the human lymph

node. Cell and Tissue Research

183,

115-130

FOLSED.S., BEATHARD.A. GRANHOL M. A . 1975) Smooth muscle

in

lymph node capsule and

trabeculae. Anatomical Record 183, 517-522

FOSSUM . FORDW . L . 1985) Th e organization of cell populations within lymph nodes: their origin,

life history a nd functional relationships. Histopathology

9,

4 6 9 4 9 9

HUARD . ,

WOOD . ,

MALINOFF . , MAHON EY

. ,

HEPPNER

.

WICHA

M . 1984) Laminin promotes

macrophage tumor cell binding by specific plasma membrane proteins.

Proceedings

of

the

American Association of Cancer Research

25,

196

K A R I T U N E N

T.,

AUTIO-HARMAINEN. , RASANEN

O.

RISTELI. RISTELI. 1984) Immunohistoche-

mica1 localization of epidermal basement membrane laminin and type

IV

collagen in bullous

lesions of dermatit is herpetiformis. British Journal of Dermatofogy

11

1, 389-394

KNECHT

H.

LENNERT

. 1981) Ultrastuctural findings

in

lymphogranulomatosis

X

angio-)im-

munoblastic lymphadenopathy). Virchows Archiv Cell Pathology)

37,

29-47

K O N O M IH . , S AN O

.

NAGAI

Y. 1981) Immunohistological localization of types 1 , 111 and

1V

basem ent m em bran e) collagens in the lymph node: co-distribution of types

I

and 111 collagens in

the reticular fibers. Biomedicat Research

2,

5 3 6 5 4 5

L I O T ~ A .C . , RAO C.N . B ARSKY . H . 1983) Tumor invasion and the extracellular matrix.

Laboratory Investigation

49,

6 3 6 6 4 9

LUK S .C . , NOPAJAROONSRI. SIMON

.T . 1973) Th e architecture of the normal lymph node and

hemolymph no de. A scanning and transmission electron microscopic study. Laboratory Investiga-

tion

29,

2 5 g 2 6 5

M ART I NE Z - HE RNANDE Z. AM E NT A

.S. 1983) The basement me mbra ne in pathology. Laboratory

Investigation

48,

656-677

M OE R.E . 1963) Fine structure of the reticulum and sinuses

of

lymph nodes. American Journal of

Anatomy

112,

311-335

MORI Y .

LENNERT

. 1969) The so-called normal lymph node. In Electron Microscopic Atlas of

Lymph Node Cytology and Pathology, pp . 4-19, eds

Y.Mori

K.Lennert. Springer, Berlin

NOPAIAROONSRI., L U K S . C . SIMONG.T . 1971) Ultrastructure of the normal lymph node.

American Journal

of

Pathology

65,

1-11

PUCHTLER. WALDROP

.S. 1978) Silver impregnation m etho ds for reticulum fibers and reticulin.

Histochemistry

57,

177-187

RE E H.J . , L E ONEL.A. CROWLEY.P . 1982) Sclerosis in diffuse histiocytic lym phom a:

A

clinicopathologic study of 25 cases. Cancer

49,

1636-1648

REILLY. T . , NAS H .R . , M ACKI E

. J .

MCVERRY. A . 1985) Distribution of fibronectin an d laminin

in normal and pathological lymphoid tissue. Journal of Clinical Pathology 38, 849-854

RISTELI . ,

BACHINGER

.P.,

E NGE L . , FURTHMAYR. T I M P L

. 1980) 7-S collagen: characteriza-

tion of unusual basement m embrane structure. European Journal of Biochemistry 108, 239-250

RISTELI .

TIMPL

. 1981) Isolation and characterization of pepsin fragments

of

laminin from

human placental and renal basement membranes. Biochemical Journal

193,

749-755

S HI E L DS. M . , HASTONW . W I L K I N S O N.C. 1984) Invasion of collagen gels by mouse lymphoid

cells. Immunology

51,

259-268

STENMAN. V A H E R I . 1978) Distribution of a major connective tissue protein, fibronectin in

normal human tissues. Journal

of

Experimental Medicine

147,

1054-1064

T I MP L

. , ROHDE

. ,

ROBE Y .G. , RE NNARD

. I . ,

FOIDART.M. MARTIN .R .

1979) Laminin. A

glycoprotein from basement membranes. Journal of Biological Chemistry

254,

9933-9937

TYKOCINSKI

. ,

S CHI NE L L A. A . G R EC O .

1983) Fibroblastic reticulum cells

in

human lymph

nodes. An ultrastructural study. Archives of Pathology and Laboratory Medicine

107,

418-422

YAMADA . YAMAGISHI. 1961) Light and electron microscopical studies

on

the structure of

lymphatic sinus in lymph nodes. Nagoya Medical Journal 7 7-16

157,

265-284

8/12/2019 Kart Tune n 1986

10/10