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Alterations of Polymorphonuclear Neutrophil (PMN)
Recruitment in a Murine Mode1 of Peritonitis and a
Secondary Injury
Daniel E. Swartz, M.D.
L.D. MacLean S urgical Research Laboratories
Division of General Surgery
Department of Surzery
McGill University, Montreal
March, 1999
A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment
of the requirements for the degree of Master of Science in Ekperimental Szcrgery
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TABLE OF CONTENTS
Page
Abstract
Resumé
Acknowledgements
List of Abbreviations
Chapter 1 : Review of the Literature
1.1 Overview of the host response to uifection
1.1.1 SIRS and MODS
1.1.2 RoIe of the PMN
1.2 PMN recruitment
l.Zl Selectins
1 -2.2 Chernoattractants
1.2.3 P2 Integrins
1 -2.4 Kinetics of PMN-endothelial ce11 interactions
1.3 PMN transendothelial migration
1.3.1 Morphologie changes of activation, diapedesis and
vascular emigration
1 -3 -2 Chernotactic migration
2 -4 The PMN in host defense
1 -4.1 Phagocytosis of pathogens
vii
1.4.2 Release of degredative enzymes and toxic oxygen
Metabolites 32
1.4.3 Apoptosis 33
1.5 Consequences of altered PMN effector function 34
1 S. 1 Endo thelial ce11 destruction by PMNs 35
1.5.2 PMN recruitment to a remote site in the presence
of infiammation
1.6 Justification of a murine mode1 of secondary peritonitis
and a secondary injury
1.7 Hypotheses
1.8 Objectives
Chapter 2: Materials and Methods
2-1 Anirnals and housing
2.2 Reagants
2.3 Animal procedures
2.3.1 Cecal ligation and puncture
2.3.2 Polyvinyl sponge placement
2.3 -3 Optimal E. coli concentration
2.3 -4 Cremasteric dissection for intravital microscopy
2.4 Measurement of PMN-EC interactions using intravital
microscopy
2-5 Measurement of vesse1 kinetics
2.6
2.7
Chapter 3:
3.1
3 -2
3 -3
PMN isolation and counting
2.6-1 P M . pUnty
S tatistical anafysis
Results 53
Animal mortality 53
PMN regïonalization following cecal ligation and puncture 53
Circulating PMN counts followinp orchitis and cecal
Ligation and puncture 56
3.4 Intravital microscopy 57
3 -4. i Circulatory parameters 57
3-42 Kuietics of PMN and endothefial ce11 interactions 58
Chapter 4: Discussion 66
4.1 PMN defivery to polyvinyl sponge discs 67
4.2 Intravital microsocpic analysis of PMN fluxes, rolling and
firm adhesion 70
4.3 Future directions: a potential role for shed L-selectin 76
4.4 Conclusion 79
Chapter 5: Original Contributions to Knowledge
References
ABSTRACT
Secondary peritonitis is a significant cause of morbidity and mortality in the ICU
and ICU patients as a group have the highest rate of nosocomial infections. Once
recmited to the site of injury, the PMN interacts with endothelial cells (ECs) via rolling
adhesion, fm adhesion, and transendothelial migration. Using a murine cecal ligation
and puncture pentonitis mode1 and either skin or cremaster injury as the secondary site in
a two-front injury modei, we examined the role of injury severity on the triage of PMNs
to competing sites of injury. We demonstrated that a finite pool of PMNs was recruited to
tissues in numbers correlating to the severity major injury. Wiîh intravital microscopy we
demonstrated that numbers of PMNs invoived in rolling adhesion, rolling velocity and
stationary adhesion in the presence of one or more sites of injury were predictable,
consistent and likely mediated by changes in surface adhesion molecule expression.
La péritonite secondaire est une cause importante de morbidité et de mortalité
chez les patients à l'unité des soins intensifs, et le taux des infections nosocmiales est très
élévé chez ces patients. Une fois mobilisé au site de la blessure, le PMN interagit avec les
cellules endothéliales par l'adhérence roulant, l'adhérence ferme ainsi que la migration
transendothéliale. Dans le modèle du système de blessure à fionts chez la souris, le site de
lésion primaire consiste en une ligature caecale et en une péritonite perforée, tandis que le
site de lésion secondaire consiste en une lésion cutanée ou crémastérienne. À l'aide de ce
modèle nous avons évalué le r6le de la gravité de la blessure dans la mobilisation des
PMNs à des sites compétitifs. Nos travaux ont démontré que le nombre des PMNs
mobilisés est proportio~el à la gravité de la blessure. Nos résultats ont montré, à l'aide
de la microscopie intravitale, que le nombre des PMN impliqués dans l'adhérence
roulant, la vitesse roulante ainsi que dans l'adhérence fermement peut être prédit et qu'il
est uniforme en présence d'un ou de plusieurs sites de blessure. De plus, le nombre des
PMNs mobilisés est probablement lié à des changements de l'expression des molécules
adhésives à Ia- surface des cellules.
Many people have been instrumental in guiding me during the formulation of this
thesis which serves as the culmination of my experience in the Surgical Scientist
Program. 1 am particularly grateful to Dr. Nicolas Christou without whose support and
assistance none of this would have been possible. In addition to helping me create the
hypotheses and design the studies, Dr. Christou was there to make sense of results that 1
fomd nonsensical, to redirect me down the path when al1 roads hit a dead end, and to
refocus my thinking each tirne 1 contemp lated strangling the technician out of sheer
hstration.
Dr. Eleanor Minshall has been an endless fountain of encouragement without
whom 1 would still be revising this thesis for years to corne. Her editorial skills, artistic
illustrations and assistance with the many versions of the tables and charts were integral
in creating the polished product. Her patience and support dwing the many nights and
weekends 1 spent writing and rewriting were a tremendous sacrifice for which 1 will
always be gratefùl.
1 am gratefbl to Dr. Andrew Seely, who collaborated with me and assisted on
many of the experiments, for al1 his input and insight whether or not it was solicited.
Long hours and very late nights were spent in good Company operating on animals,
counting neutrophils, and engaging in fierce philosophical and metaphysical debates.
A special mention to some of the others whose involvement and assistance were
irreplaceable to this effort: Ms. Betty Giannias, Dr. Xuwu Chen, Ms. Mary Bouldadakis,
Dr. Felicia Huang and Dr. Teruo Sakamoto.
LIST OF ABBREVIATIONS
APACHE II
Dv
EC
EGF
ELAM- 1
£MLP
G-CSF
GlyCAM- 1
GM-CSF
GMP-140
E V
ICAM
ICU
1gSF
IL-1 p
LAD I
LAM- 1
LECAM
LTB3
MAdCAM- 1
MODS
Acute p hysiology and chronic health evaluation score II
Mean vesse1 diameter
Endo thelia1 cells
Epidermal growth factor @art of the se lech molecule)
Endothelial-leukocyte adhesion molecule-1
N-formyl-methionine-leucine-phenylalanine
Granulocyte colony-stimulahg factor
Glycosylation-dependent cellular adhesion molecule-i
Granulocyte-monocyte colony-stimulating factor
Granulocyte membrane protein- 140
High endothefial venules
Intercellular adhesion molecules
Intensive care unit
Immunoglobulin super family
Interleukin 1 p
Leucocyte adhesion deficiency type 1
Leucocyte adhesion molecule- 1
Leucocyte-endothelia1 ce11 adhesion rnolecule- 1
Leukotriene Bq
Mucosal addressin cellular adhesion molecule-1
Multiple organ dys fünction syndrome
vii
NK
PAF
PECAM
PMA
PMN
PSGL-Z
SCR
SIRS
TNF-a
VCAM-1
VBF
Vmean
Vrbc
VSR
VSS
Natural killer
Platelet activating factor
Platelet-endotheiial cell adhesion molecule
Phorbol myrktate acetate
Polymorphonuclear granulocyte
P-selectin glycoprotein ligand4
Short consensus repeats
Systemic inflarnrnatory response syndrome
Tumor necrosis factor cc
Vascular ce11 adhesion molecule- 1
Venular blood flow
Mean red blood ce11 velocity
Centerline red blood ce11 velocity
Venular shear rate
Venular shear stress
Chapter 1: REVZEW OF THE LITERATURE
1-1 Overview of the host response to infection
A successfid outcome of any injury, including infection, depends on the host's
ability to mount an appropriate immune response. The initial entry of infectious
pathogens into the host's tissues usually results fiom a failure of epithelial bamier
hc t i ons such as the gut mucosa (leading to peritonitis), lungs (leading to bronchitis and
pneumonia), and skin (leading to cutaneous and soft tissue uifections). Once entry into
host tissues has occurred, a complex interplay of cells and molecules of the immune
system attempts to contain the infection and recruit the necessary effector cells to
eradicate the rnicrobial invaders. Although the severity of infection, the type and
resistance of the pathogens and the anatomic location of the injury contribute to the
outcome, multivariate regression studies of patients with peritonitis 1 and animal studies
2 have dernonstrated that the predominant factor in detennining survival is the ability to
rnount an appropnzte immune response.
1.1.1 SIRSandMODS
The systemic inflammatory response is a double-edged sword. While an
appropriate response is essential for a successful outcorne foliowing infectious injury, a
maladaptive and dysregulated response may incite more widespread and darnaging
systemic injury leading to vascular and other tissue injury, the systemic uinarnmatoiy
response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS). The latter
entities have corne to light over recent decades as improvements in antimicrobial
treatments and care of cntically ill patients have demonstrated groups of patients who
survive their initial injury only to succomb weeks to months later of MODS.
Infection, SIRS and sepsis are distinct entities d e h e d by the 1992 Consensus
Conference of the American College of Chest Physicians and the Society of Critical Care
Medicine 3. Infection is defied as the documentation of microbial invasion of normally
sterile host tissues. SIRS, the host response to infection, is defined as two or more of the
followùig: temperature higher than 38°C or lower than 36°C; hem rate greater than 90
beats per minute; respiratory rate geater than 20 breaths per minute or artenal carbon
dioxide tension less than 32 mn Hg; and white blood ce11 count greater than 12,000 or
less than 4000 per mm' or greater than 10% bands on differential. This definition is valid
only in the absence of any other cause such as the immediate post-operative state. Sepsis
is defined as the presence of SIRS with docurnented infection.
A sustained immune response giving rise to a widespread inflammatory state
following a significant injury or insult can lead to dire systemic events such as MODS.
This syndrome is defined as a persistent maladaptive and dysregulated state of the
inflammatory and immune systems 4. As the acronym implies, multiple organs can be
involved and it is their progressive reIentIess destruction that is associated with
significant morbidity and morîality in K U patients. Multiple etiologic entities, both
infectious and non-infectious, employ the same mediators and effector moIecules in the
development of MODS. The pathophysio logy of this disease state involves both an
uncontrolled immunomediator response to injury as well as tissue hypoxia 4. Whether
MODS results purely f?om excessive or prolonged cytokinemia, uncontrolled
macrophage and PMN activation, gut epithelial breakdown with resident bacterial and
endotoxin translocation, microcircuiatory changes leading to tissue ischemia, or a
combination of one or more of these factors is still debated- Some authors favor a two-hit
phenomenon in which one of these events serves to "prime" the inflammatory system for
dramatic and excessive activation following a subsequent event 47 5.
1.1.2 Role of the PMN
The PMN is the predominant granulocyte in both the circulation as well as at the
site of acute infection in the initial 48 hours. PMNs are produced by the myeloid cell h e
in the bone rnarrow at a rate of about 101° cells per day, which may be increased by as
much as 10-fold in tirnes of injury 6 , and have a half-life of six to eight hours 7. Once
recruited to the site of injury, the PMN interacts with ECs via the specific regulation and
expression of surface receptors and ligands. The eventual goal is activation of the PMN to
become an effector ceIl capable of extravasating into the tissues and destroying the
foreign micro-organisms. Under normal circurnstances following the successfûl
elirnination of rnicrobial invaders, the PMN undergo es apop tosis, or prograrnmed ce11
death 8.
1.2 P M . recruitment
Initial interaction .s between ECs and PMNs involve dynarnic forces in which
erythrocytes moving rapidly in the center of the vesse1 exert a shear force on the larger
and less deformable PMNs pushing them outward toward the vesse1 walls 9. This
margination is thought to allow the PMN to keep in close proximity to the EC layer and
thus respond to the release of local factors. To maintain these interactions, shear forces in
the direction of blood flow must be sficiently low to permit cell-ce11 communication.
This effect can be reproduced in vitro where rolling adhesion between PMNs and ECs
varies inversely with perfusion pressure Io. The anatomic site where shear forces appear
to most favor PMN-EC interaction and transendothelial migration is the post-capillary
venule 1 1.
Under the appropriate circurnstances, specific receptors and their ligands direct a
sequence of events leading to extravasation by the P m : rolling adhesion, fim adhesion
and PMN activation. These events are mediated by a sequence of families of adhesion
molecules and chernoattractants: selectins, chernoattractants and integrins in a multi-step
mode1 as described by Springer 12. The specific leukocyte-endothelia1 interaction is
postulated to be determined by the combination of individual receptors corn each farnily
analagous to a telephone area code detemrined by the specific combination of three digits
(Figure 1.1).
1-21 Selectins
Rolling adhesion of the PMN along the vascular endothelium, first described by
Wagner in 1839 13, is defined as a low affuiity interaction between the leukocyte and EC
where the hydrodynarnic shear forces of the blood flow exerts a rotational motion of the
leucocyte. This event is mediated by the selectin family of adhesion molecules which
share a comrnon structure of an MIz-terminal calcium-dependent C-type lectin domairi,
an epidermal growth factor (EGF)-like region, several short conserisus
repeats (SCR) and a trammembrane region with a cytoplasmic tail 14. Multiple
molecules with this sequence homology have been identified (Table 1.1).
p Adhesion
Diapedesis
(integrin) Cliemokine
aD ICAM- 1
0 y
Figure 1.1. The sequence of receptor families involved in PMN-EC interactions:
selectins, chemokuies and integrins.
These include endothelid leukocyte adhesion molecule-l (ELAM-1), granulocyte
membrane protein-140 (GMP-140 or PADGEM) and lymphocyte homing receptor
(gp90Me1, Leukocyte Adhesion Molecule -1 (LAM-1) or leukocyte-endothelid ce11
adhesion molecule (LECAM)). As a result of the 1989 consensus conference these have
been reco*gnized as E, P and L-selectin, respectively. The role of the selectins in the acute
idammatory response is to mediate capture (or tethering) and rolling of PMNs to ECs.
Each selectin molecule is capable of recognizing specific carbohydrate ligands by the N-
temiinal lectin domain however the EGF and SCR regions may affect ligand binding 147
15. S tmctural differences between the selectin molecules include the number of SCR
uaits whereby L-selectin has 2, E-selectin has 6 and P-selectin has 10 (Figure 1.2).
Selectin Location PMN Predorninant Ligand Other Ligands
PMNs, most CD 34, PSGL-1 lymphocytes, al1 other leucocytes
ECs, phtelets PSGL- 1
ECs sLeX , unknown
E-selectin (via sLeS), GlyCPuM- 1, MAdCAM- 1
Table 1.1: Selectins involved in PMN-EC interactions (based on Springer 12).
L-selectin is constitutively expressed on al1 circulating leukocytes except a subset
of memory lymphocytes. Ct functions predominantly in interactions of PMNs and ECs in
acutely inflarned tissues as well as lymphocytes and their ligands on penpheral lymphatic
high endothelial venules (HEVs) 16. Upon PMN activation by chernoattractants, the
extracellular domains of L-selectin are rapidly shed from the ce11 surface by proteolytic
cleavage 17. AU three selectin molecules bind three types of carbohydrate moiteies via
the N-terminal lectin domain: sialylated tetrasaccharides (of which sialyl-Lewisx (sLeX)
and its steroisomer, sialyl-Lewisa (sLea) found on epithelial and cancerous cells, are the
protowes), phosphoqdated mono- and polysaccharides and sulfated polysaccharides and
lipids 14 18 19-
C Lectin domain
EGFdomain
8 Concensus repeat
rqY Mucin domain
Immunoglobulin-Iike domain
Figure 1.2. Structure and ce11 of expression of the selectins involved in PMN-EC
interactions.
The ligands to which the selectin receptors bind have not been fully elucidated.
Ligands for lymphocyte L-selectin on HEVs are constitutively expressed 20 and include
CD34 2y 2l, the soluble ligand Glycosylation-dependent Cell-Adhesion Molecule- 1
(GlyCAM-1) 22 and Mucosal Addressin Cell Adhesion Molecule (MAdCAM-1) 23.
Ligands for PMN L-selectin appear not to be constitutively expressed but rather are
upregulated in cytokine-activated endothelium 24. These ligands include CD34 25, P-
selectin Glycoprotein Ligand-l (PSGL-l) on PMNs which allows binding between PMNs
15, and although not uniformly accepted, considerable evidence exists that L-selectin can
bind directly to E-selectin via sLeX 207 26727.
P-seiectin is expressed on both ECs where they are stored in intracellular Wiebel-
Palade bodies and on platelets where they are stored in alpha granules. Upon cellular
stimulation by leukotriene Bq , histamine 28, C5a 29, thrombin, bradykuun or fiee
oxygen radicals, these storage granules are transported to and rapidly fuse with the cell
surface leading to P-selectin expression within minutes. P-selectin expression appears to
be regulated by motifs within its intracytoplasmic tail which can cause rapid
intemalization with either lysosornal degredation or recycling into storage granules 30.
The principal ligand of P-selectin located on the PMN is PSGL- 1 3 1, a mucin-like
disulfide-linked homodimer which can also bind at lower affinity to L-selectin 1% 32 and
E-selectin 33. P-selectin can also bind to sLeX 34.
E-selectin, expressed only on ECs, requires de-novo synthesis following
stimulation of the endothelium with IL1 P, TNF-u and endotoxin. Monocional antibody
studies have deterrnined that it requires approximately three to six hours for E-selectin
expression following cytokine activation 9 3 3 5 3 3G and expression can be completely -
blocked by inhibitors of mRNA transcription (Actinomycin D) or translation
(cyclohexamide) 37. Expression of E-selectin is usually transient with a duration of 10-12
hours in cytokine-stimulated human umbiLical vein endothelid cells (HUVECs) in vitro
and longer in skin and oîher sites 35. Loss of expressed E-selectin occurs b y both
internalization and lysosomal degradation 35 as well as downregulation of DNA
transcription 39. Ligands for E-selectin include HECA-452 on T lymphocytes 14, L-
selectin presenting sLeX on PMNs 2 0 ~ 41 and PSGL-1 339 429 43-
As previously mentioned the role of the selectins with respect to PMN-EC
interactions in inflammation are in the capture and rolling of PMNs along the
endothelium; a prerequisite for stationary adhesion, activation and transendothelial
migration 4. Using soluble r-arbohydrate Ligands or monoclonal antibodies to the selectin
lectin domain, Tu and CO-authors were able to abolish rolling as weLl as finn adhesion of
PMNs on ECs 15. Furthemore, at physiologie shear stresses, PMNs attach and roll along
phospholipid bilayers expressing P-selectin or P-selectin with ICAM-1 (ligand for P2-
integrin, see below), but not ICAM-1 alone 45. Ail three selectins have been shown to
mediate rolling independently both i i z vivo using knockout mice and in vitro using genetic
transfectants or g l a s surfaces coated with a particular selectin or its ligand 9. Expression
of each selectin varies according to the type of injury (ischemia/reperfusion, infection,
trauma), the effector ce11 and organ involved and the temporal relationship to the time
since stimulus for its expression. These compIexities have hindered the development of a
clear understanding of selectin involvement in leukocyte trafficking 14.
In the acute inflammatory response, initia1 tethering and rolling of PMNs is
mediated predominantly through P-selectin while L-selectin-dependent rolling is present
after 30 minutes and E-selectin is not present until after two to four hours. Studies
perforrned with P-selectin knockout mÏce undergoing intravitai microscopy of mesenteric
venules exhibited a marked 1eucocytosis with complete ablation of rolling initially that
retunied to normal levels after 2-4 hours; a duration that the authors postulated to
coincide with E-selectin expression 46. This data was corroborated with monoclonal
antibodies againçt P-selectin in dogs 47 and in mice 28. The role of L-selectin in PMN
recruitment to inflamed endothclium was demonstrated by Ley and CO-authors who
compared r o l h g adhesion of PMNs (L-selectin (+), sLex (+)) with HL-60 promyelocytes
which express sLex but no t L-selectin (L-selectin (-), sLeX (+)) and 3 00.19 cells
transfected with L-selectin (L-selectin (+), sLex (-)) in rat mesenteric venules under
intravital microscopy. The 300.19 c e h showed almost no rolling initially but increased
markedly after 20-30 minutes while the HL-60 cells demonstrated moderate rolling
initially but this was reduced to practicaily zero after 20 to 30 minutes. The wild-type
PMNs revealed consistently greater rolling t h a n the other ceIls throughout the
experiment. This data supports that early rolling at least in part involves sLex, probably
binding to P-selectin, while L-selectin-dependent rolling does not occur until after 20 to
30 minutes 48. In an ex-vivo murine study, L-selectin knockout mice whose cremaster
muscles were exteriorized and examined under intravital microscopy were compared to
normal control mice. The authors demonstrated normal rolling initially followed by up to
a 65% reduction in rolling PMNs after 30 minutes 4? E-selectin does not appear to play a
role in PMN recruitment until2-4 hours after cytokine stimulation and
monoclonal antibodies directed against E-selectin, such as EL-246, had no effect on PMN
tethering and rolling for the fkst three hours 52.
The kinetics of selectin-rnediated PMN rolling is hypothesized by Lawrence and
Splinger to involve labile bond formations between the selectins and their ligands. Upon
dissociation of one bond, the PMN is propelled downstream by the hydrodynamic forces
of the blood in a rotational motion until the ce11 is tethered by the most upstream bond.
The velocity of the rolling PMN is determined by the rates of bond association and
dissociation which are unique to each selectin-ligand pair. Applying pharmacokinetic rate
constants where b, refers to bond association and bff refers to dissociation, the
equilibrium constant, k, kn/ kr), must remain constant in order for rolling to continue
45- Puri and colleagues measured the PMN tethering (capture), rolling velocity and
rolling adhesion strength of al1 three selectins in a laminar flow in vitro assay with either
P-selectin, E-selectin or CD34 (L-selectin ligand) adsorbed to glass slides. Tethering at
physiologie shear stresses (1.5 - 2 dyn/crn2) correlated with receptor site density and was
sirnilar for al1 three selectins but slightly higher on L-selectin followed by P-selectin and
somewhat fewer numbers of PMNs tethered on E-selectin. Rolling velocity at these shear
stresses was eight-fold greater on L-selectin than E- or P-selectin. By incremeotally
increasing the shear stress, rolling adhesion strength was calculated by recording the
shear stress at which 50% of the PMNs remained bound. The rolling velocity was almost
twice as great for L-selectin as E- or P-selectin 53. While both L-selectin and PSGL-1 are
concentrated on the microvilli tips of the PMN plasma membrane 54, L-selectin is
present in two to three-fold greater concentration on the PMN ce11 surface 55. These
hd ings support an increase in L-selectin exposure to its ligand on the EC and may
account for its greater rolling adherence and velocity.
It is unclear what significance is attributed to the faster rolling velocity of L-
selectin-mediated P M ' rolling as opposed to P- or E-selectin, however it is likely that
slower velocities permit greater exPosud of the PMN to chemoattractants on the
endothelial surface leading to increased interactions with the integrin receptors 53.
Because L-selectin is expressed consititutiveiy whereas P- and E-selectin require some
fonn of stimulation for expression, L-selectin may be relegated to a role of systemic
endothelial surface sampling in the non-inflamed state characterized by greater tethering
(ability to sarnple) and faster rolling velocity (greater endothelial suiface area samp led) .
Other studies are needed to fürther delineate the individual roles of the selectins,
1.2.2 Chemoattractants
The second group of rnolecules involved in PMN recmitment in in£laxmnation are
the chemoattractants. They serve as the second of the three-digit area code analogy
advanced by Spnnger 12 (see Figure 1 -2). Chernoattractants refer to a diverse group of
rnolecules that serve several fûrictions: they can activate and alter integrin adhesiveness;
stimulate PMN changes in morphology, actin polymerization and r e sp i r a to~ burst; and
they direct migration of the leukocyte across the endotheiial basement membrane to the
site of injury. Activated leukocytes migrate along a concentration graaient of soluble
chemoattractant molecules which dif ise from their point of production. PMNs have been
demonstrated to respond to concentration differences across 1% of their diameter and
migrate progessively in the direction of higher chemoattractant concentration 56.
Chemotactic stimuli have to date been classified into two groups: classical
chernoattractants and chemokines (chemoattractant cytokines). Classical chemoattractants
have counter-receptors on monocytes and PMNs and include N-formyl peptides, C5a,
leukotriene Bq (LTB4) and platelet-activating factor (PAF), The chernokines have been
recently subdivided into four families: C-X-C, C-C, C and C-X3-C. C-X-C (a)
chemokines, which rnap to chromosome 4, and C-C (P) chemokines, which map to
chromosome 17, were the first two families identified and were described with respect to
whether or not an arnÏno acid, cX", separates the f i t two cysteine residues 12. Very
recently two fiirther goups have been added: C (y) chemokines which map to
chromosome 1 and contain a single cysteine residue 57 and CX3C (6) chemokines which
map to chromosome 16 58 (Table 1.2). C-X-C chemokines act primarily on PMNs as
well as non-hematopoietic cells involved in wound healing and include IL-8, CTAP-III,
gro/MGSA and ENA-78. C-C chemokines interact with monocytes, eosinophils and
lymphocytes and include MCP- 1, MIP-1 a, M W I P, RANTES and 1-3 09. Only the
classical chemoattractants and C-X-C chemokines pertain to PMN recruitrnent in acute
inflammation and hence the other groups will not be considered herein.
Chemoattractant Ce11 or Process of Origin Target CeiI
CIassical chemoattractants N-formyl peptides CSa L m 4 PAF
C-X-C chemokines IL-8
C-C chemokines MCP- 1
Lymp htoac tin
Bacterial protein processing Complement activation Arachidonate metaboIism Phosphatidylcholine rnetabolism
T cell, monocyte, EC, fïbroblast, kentinocyte, chondrocyte, rnesothelial ceil Platelets Fibroblast, melanomas, EC, monocyte Epithelïum
T cell, monocyte, fibroblast, EC, smooth muscle Monocyte, T cell, basophil T ceIl, platelets T cell, rnast ceii
Cytotoxic T celIs, thymocytes
Granulocyte Granuiocyte PMN, monocyte Granulocyte
PIvfN, basophil
PMN, basophil, Fibroblast PMN, melanoma, fibroblast
PMN
Monocyte, basophil
GranuIocyte, T ce11 Monocyte, eosinophil, T ceiI monocyte
T cell, monocyte
Table 1.2: chernoattractants @ased on Springer l2 and Ward 59).
Chemokines, produced by virtually al1 cells involved in inflammation, consist of
approxirnately 7-8 kd proteins which diffuse across the endothelium. Over forty distinct
chemokines have been charactenzed to date 59. Once in the bloodstream, it is thought
that chemoattractants are rapidly diluted and can no longer effect PMN-EC binding.
Administration of IL-8 intradermally in rabbits led to marked extravasation of PiMNs
whereas this did not occur in intravenous administration 60. It is generally accepted that
PMN tethering and rolling enhances ce11 exposure to chemoattractants. Chemokines bind
to a G-protein-coupled receptor on the PMN which transduces the chemokine signal into
integrin adhesiveness 61.
The presence of chemokines have been demonstrated in most inflamed tissues
including the skin, brain, joints, lungs, vessels, kidney and gut as well as virtually al1 cells
involved in the inflammatory response. Stimuli for chemokine secretion are IL-1 P, TNF-
a, IFN-y, interleukin 4 (IL-4) as well as bacterial products such as lipopolysaccharide 62.
The specific chemokine involved is determined by the particular tissue or cell involved,
the type of infiammatory infiltrate and the nature of the invading organisrn. When the EC
is stimulated by pro-idiammatory cytokines, such as TNF-a and IL-10, it produces IL-8
and other chemoattractants which become bound to the luminal surface of the activated
cell.
The role of chemokines in PMN attachent and emigration has been borne out in
many in vitro and in vivo investigations. Using transwell chambers separated by a human
umbilical vein endothelial cell (HUVEC)-lined filter (a common endothelial ce11
monolayer culture grown in vitro), cytokine-stirnulated ECs synthesized and secreted IL-
8 into the basal chamber. PMNs added to the apical charnber were subsequently
identified in the basal charnber. IL-8 added to the apical charnber inhibited PMN
emigration 63. In a rabbit mode1 of ischemia-reperfusion injury to the lung, monocIona1
antibody against IL-8 inhibited PMN emigration into the lung and markedly reduced
tissue injury 64. Oda and CO-authors topically adrninistered the chemoattractants N-
formyl-Met-Leu-Phe ( W P ) and LTB4 to hamster cheek pouch post-capillary venules
and examined the effect on PMN recruitment using intravital microscopy. They noted a
marked increase in adherence peaking within 10 minutes followed by the disappearance
of PMNs fi-om the bloodstream and a reappearance 30 minutes later in the interstitiun 64.
1.2.3 p21ntegrins
PMN rolling on endothelium is characterized by low-affinity selectin-receptor
binding. It is a necessary prerequisite to firm adhesion under conditions of flow as well as
activation and sub s equent emigration uito extravascular tissues 9. Extensive information
on the role of integrins in PMN recruitment has been obtained fkom studying patients
with the congenitally acquired syndrome of Leucocyte Adhesion Deficiency type 1 (LAD
I), where the affected individual is devoid of P2 integrins. Examination of the PMN-EC
interactions in these patients reveals a chronic neutrophilia, normal rolling but absence of
firm adhesion, a marked reduction in PMN emigration and an increased susceptibility to
severe infections despite high circulating PMN counts 65.
The integrin family of receptors and their Iigands mediate stationary, or "çm"
adhesion of the PMN to the EC. Integrins are leukocyte-associated heterodimeric
molecules composed of an a and a P subunit of which the susbset involved in PMN-EC
binding utilize the P2 subuaits and are known as "P2 integrins". There are three P2
integrins: Leucocyte Function-associated Antigen- l (LFA- 1) (aLP2, CD 1 1 dCD la),
Mac-1 (ccMP2, CD 1 WCD 18) and p l5OY% (aXB2, CD 1 WCD 18). CD 1 1 aKD18 is
expressed on lymphocytes, monocytes and PMNs wlule CD 1 1 b/CD 18 and CD I 1 c/CD 1 8
are expressed only on monocytes and PMNs 12 (Table 1.3). LFA-1 and Mac4 are
constitutively expressed on PMNs (and other leucocytes) and when activated by
infiammatory mediators, undergo a rapid conformational change to a high avidity state
allowing stationary (b) adhesion 9.
In tegrins Names Location Ligands
P2 integtrins d B 2 LFA-1, CD 1 1aICD 18 PlMN, monocyte, B and
T cells
CM32 Mac- 1, CD 1 1 b/ CD 18 PMN, monocyte
P150,95, CD 1 lc/CD 18 PMN, monocyte
VLA-4, CD49dCD29 B and T celis, monocyte, neural crest cells, fibroblast, muscle
LPAM-1, CD49dfCD- B and T ceiis
ICAM-1, ICAM-2, ICAM-3 ICAM-1, iC3b, fibrinogen, factor X iC3b, fibrinogen
VCAM- 1, fibronectin
MAdCAM- 1, VCAM- 1, fibronectîn
Table 1.3: Integrins in Leukocyte-Endothelia1 Interactions (fiom Springer 12).
Adhesiveness of integrin molecules is activated and altered by chemoattractants.
With respect to PMN-EC interactions in the inflarnmatory response, fMLP and IL-8
moderate the P2 integrins LFA-1 and Mac-l 66. Yuan and CO-authors demonstrated
increased PMN adhesiveness using intravital microsocpy of porcine coronary venules
under flow conditions by pretreatment of the PMNs with CSa without effect on PMN
rolling 10.
PMN activation by chemoattractants induces shedding of L-selectin and rapid up-
regulation of Mac4 expression and adhesiveness 67. The increased adhesiveness is likely
due to the concentrated clustering of P2 integrins on the PMN surface as demonstrated in
reçponse to phorbol ester stimulation 68. However chernoattractants in the absence or
inhibition of the P2 integrins are insufîïcient to promote firm adherence and emigration of
PMNs. Arfors and CO-authors administered murine monoclonal antibody directed against
CD18 with intradermal injection of fMLP, LTB4, CSa and histamine to rabbits and
examined the tenuissimus muscle under intravitai microscopy. They noted that rolling
was not altered however PMN adherence and emigration in post-capillary venules was
abolished 69.
The ligands for the B2 integrins, located on ECs, belong to the Immwoglobulin
Superfamily (IgSF) of adhesion molecules composed of an immunogiobulin domain of
90- 100 amino acids. They include intercellular adhesion molecule- 1 (ICAM- 1), ICAM-2,
and ICAM-3. Other IgSF members which bind to a4 integrins are involved in mainiy
lymphocyte adhesion and include vascular ce11 adhesion moIecule-1 (VCAM-1) and
mucosal addressin ce11 adhesion molecule (MAdCAM-1). MAdCAM-1 is unique in
lymphocyte binding properties in that it has homology to immunoglobu1in (and thus
binds to integrins) as well as a carbohydrate domain (to bind to L-selectin) 23. While
ICAM-1, ICAM-2 and ICAM-3 can bind to LFA- 1, only ICAM-1 can bkd to Mac-1.
Thus in order to inhibit LFA-1 interactions with the endothelium, ail three ICAMs need
to be bIocked 70.
1.2.4 Kinetics of PMN-endothefial cell interncdions
Ln surnrnary, the three step mode1 for PMN recnritment prior to transendothelial
migration involves the adhesion molecu1e:ligand sequence of selectin-chemoattractant-
integrin as advanced by Springer 12. The selectins (L on the PMN and P and E present on
the endothefium) with their ligands mediate PMN capture (tethering) and rolling along
the endothehm via association and dissociation of l ow-awty bonds. Frorn a
physiological viewpoint this is thought to reduce the shear forces present in venular blood
flow as it passes the lruninal wall to permit adhesion and exposure to chemoattractants.
Chemoattractants, produced and released by the EC in response to IL4 and TNF-cc,
induce activation of P M . with shedding of L-selectin and up-regulation of high-avidity
P2 integrins such as LFA-1 and Mac-1. Leucocyte integrin binding to their respective
cellular adhesion molecule counter-receptors on the EC lead to stationary, or £km,
adhesion of the PMN. Now in its activated form, the neutrophil undergoes ernigration
from the vascular space between endothelid junctions and across the basement
membrane where it will migrate along a chernotactic gradient to the site of rnicrobial
invasion,
1.3 PMN transendothelial mi.gration
Transendothelial migration of the PMN generally requires al1 of the
aforementioned recruitment steps. The evidence for the requirement of selectins has been
demonstrated by studies in which anti-l-seiectin monoclonal antibody 717 72 and
knockout mice deficient in both E and P-selectin 73 are associated with a significant
reduction in ernigration. Likewise the importance of the R2 integrins in transendothelial
migration are supported by its ablation in the presence of anti-CD 18 monoclonal antibody
T4 and anti-ICAM-1 monoclonal antibody 7 5 9 76. Fuaher evidence for the requirernent of
P1 integrins in transendothelial migration cornes ~ o m studies of patients with leucocyte
adhesion deficiency syndrome (LAD) type 1 who are deficient in PMN expression of
LFA-1 and Mac-1. PMNs fiom these patients demonstrate a virtual ablation of vascular
emigration 65. The impairment of emigration following the administration of Pertussis
toxin, which irreversibly inhibits the G-pro tein-coupled receptor, to marnmalian
leukocytes in vitro demonstrates a necessary role for chemoattractants 77.
In vitro studies have also demonstrated that the presence of a chernotactic gradient
involving IL-8 63, PAF 783 79, £MLP or LTB4 807 gL can induce transendothelid
migration. Migration involves dynamic changes of PMN receptor expression. From
studies in which PMN receptor expression was examined following PMN migration in a
skin window preparation 82 or fiom pustules 83, it has been shown that PMN exudation
is associated with up-regdation of fMLP and C3bi receptors as well as £MLP-induced
chernotaxis, hydrogen peroxide and fiee oxygen radical production 87. In addition PMN
transmigration across TNF-a-stimulated endothelium in vitro is dependent on IL-8- TNF-
a-stimulated HUVEC monolayers was associated with three-fold increase in PMN
transmigration and high concentrations of IL-8 in the supernatant compared with
unstirnulated endothelium and CO-incubation with anti-IL-8 monoclonal antibody or
actinomycin D-inhibition of protein synthesis almost completely inhibited transmigration
84- Studies in vitro have demonstrated that LPS from E. coli up-regulates DL-8R
expression on PMNs to peak around 30 minutes before down-regulation to baseline
within 2 hours 85. Changes in IL-8 receptor (IL-8R) expression correlated with
intracellular Ca++ levels and low Ca* levels prevented IL-8R down-regdation 86.
A recent in vivo human study usin% skin windows compared IL-SR and CSaR
expression on PMNs before and after transmigration in ICU patients with SIRS and
healthy controls. The process of transmigration correlated with a reduction in IL,-8R but
not CSaR in both patients and controls. Furtherrnore patients with SIRS had reduced
CSaR but not IL-8R in their circulation. TNF-a levels were increased in the exudate
environment over the circulation and in vitro incubation of PMNs with TNF-a was
associated with a reduction in IL-SR expression suggesting that TNF-a may contribute to
akered PMN IL-8R expression before and after transendothelial migration. This study
provides fiuther evidence for the dynarnic effect of PMN ce11 surface receptor expression
by the process of transendothelial migration. This data also suggests a potential for
different roles for specific chemoattractants such that sorne, such as IL-8, may contribute
to vascular emigration while others, such as C5a, may mediate post-migration chernotaxis
87
1.3.1 MovhoZogic changes of activation. diapedesis and vascular ernigration
The rnorphology of PMN emigration hüs been documented via elecîron
rnicroscopic studies. Initial contact of the EC by the PMN involved rnicrovilli-like
extensions where L-selectin and PSGL-1 are concentrated 54 while the nucleus,
organelles and majority of the cytoplasm rernained in a relatively spherical cellular
envelope. Once contact is made, the PMN acquire a flattened appearance which increases
the apposition between the cells. The mechanisms to account for these changes have not
been well-defhed but are likely mediated by chemoattractants. Pseudopodia are then
extended by the PMN between two adjacent ECs and the cytoplasm and granules
followed by the organelles and finally the nucleus flow into the pseudopodia 88. The
PMN having migrated rapidly across the EC then remains superficial to the basement
membrane in close association with the EC 88 for up to 30 minutes before migration
across this layer is observed 81.
Burns and CO-authors using astrocyte-conditioned HLTVEC monolayers (which
create interendothelid tight junctions as found in the in vivo endothelium 89)
demonstrated that PMN emigration occurs around ti&t junctions at tricellular comers of
ECs 90- Platelet-endothelid cellular adhesion molecule (PECAM, CD3 l), a member of
the irnmmoglobulin superfamily (IgSF) located on monocytes, granulocytes, natural
killer (NK) cells, some T cells and concentrated at tricellular comers of ECs, is required
for emigration across EC junctions- Monoclonal anti-PECAM antibodies administered to
cytokine-stimulated HUVECs prevented Ieukocyte migration across ECs but not PMN
binding to EC junctions gl . PECAM appears to be the sole adhesion molecule to mediate
transendothelial migration and has no other known fûnction and thus, in contrat to the
redundancy of the other adhesion molecules, appears to serve as the final common
mediator of leukocyte recruitment to extravascular tissue 92. This factor has Ied to
theoretically attractive anti-idammatory therapy directed at PECAM 937 94.
1-32 Chernotactic migration
Neutrophil cytokinesis, the process of ce11 migration, has not been fully elucidated
however it is clear that migration is directly irnplicated in the capacity of the PMN to
undergo respiratory burst and oxygen-fiee radical formation 95. The cytoskeleton,
particularly F-actin and microtubules, are intunately involved in this activity 96. 97.
PMNs were previously thought to migrate by cyclical endocytosis, a process in which
cells such as fibroblasts and lymphocytes maneuver. Here, coated pits at various areas of
the plasma membrane containing specific receptors and other proteins, but no lipids, are
internalized by endocytosis and are returned to the membrane at the front of the ce11 98.
This creates a rearward flow of membrane Iipids toward the back of the ce11 and which
directs movement in an anterograde fashion. Lee and CO-authors elegantly refiited this
theory by photobleaching a line ont0 membrane lipids perpendlcular to the axis of ce11
movement and dernonstrating that PMN membrane lipids actually move toward the fiont
of the ce11 during anterograde motion 99.
It is likely that PMNs migate by F-actin filament rearrangement, similar to the
process of neuronal growth, such that portions of the plasma membrane in the front of the
ce11 undergo contraction and internalization. This induces gradients of tension which pull
the ce11 forwards while the internalized portion of membrane are passed to the back of the
ce11 where they fuse with the membrane 96.
1.4 The PMN in host defense
1.4.1 Phagocytosis of pathogens
Following recruitment of the PMN to sites of injury and inflammation, the PMNs
ingest and destro y microbial invaders. P hagocytosis, the process of intracy-top lasmic
ingestion, involves surrounding opsonized microbial pathogens with pseudopodia that
fuse to create the enclosed intracytoplasmic vesicle known as a phagosome 100.
Phagocytosis is mediated by two classes of receptors: immunoglobulin (Fcy) and
cornplement (C3b/C3bi) receptors. The immunoglobulin Fcy receptors, namely FcyRI,
FcyRII and FcyRm, recognize the Fc domain of imrnunoglobulin G (1gG)-opsonized
particles. Cytokine-stimulated and unstimulated PMNs have different mediators of
phagocytosis: stimulated PMNs require protein kinase C activation while unstimulated
PMNs require phospholipase D activation - These are not the only signalling
pathways involving Fcy receptors since fMLP-stimulated PMNs, for instance, use neither
protein kinase C nor phospholipase D activation but rather involve increased
intracytoplasmic calcium ions and iiiositol L,4,5-trisphosphate 102- Complement
receptors for C3bK3bi are alternately involved in the signalling pathway of phagocytosis
and have been previously demonstrated to be up-regulated following transendothelial
migration 82. These interactions induce a signal transduction event involving the PMN
membrane and cytoskeleton which is required for phagocytosis. A variety of molecular
stimuli at infiammatory sites can induce pha~ocytosis which is locally-confined thus
preventing widespread proinflammatory and tissue-destructive processes 103-
1.4.2 ReZeme of degredative en,ymes and toxic O-xygen nzetubolites
Once enplfed by the PMN, the foreign material remains bound within
phagosomes where lysosomal enzymes contained in cytoplasmic granules fuse and
" degranulate" to destroy the pathogens O3 - Primary (azurophilic) granules contain
rnyeolperoxidase and lysosomal enzymes such as cathepsin G and elastase Io4. Many of
these proteases are positvely-charged and thus bind with more aanity to negatively-
charged ce11 membranes and extracellular rnatrix proteins enhancing their destructive
capacity 105. Secondary (specific or peroxidase-negative) granules contain lactoferrin,
lysozyme and other enzymes without myeloperoxidase 106-
The PMN membrane-associated NADPH-induced respiratory burst pathway
represents the other primary mechanism by which PMNs kill bacteria. Activation of this
NADPH oxidase system is associated wîth a respiratory burst in which oxygen
consumption is increased and superoxide anions are generated. Although hydrogen
peroxide is produced by the respiratory burst, rnyeloperoxidase released by azurophilic
granules catalyzes a reaction between hydrogen peroxide and chloride radicals to yield
hypochlorous acid, a powerful oxidant that is considered the predominant agent of PMN
oxygen-fiee radical injury 107-
Althou& the oxidative and non-oxidative mechanisms of microbial killing may
function independently, the destruction of many pathogens rquire both systems
functioning together. Gram-negative bacteria have been show to be resistant to lysozyme
alone but are killed when exposed to both lysozyme and oxidants 108. Turnor ce11 lysis in
vitro has also been demonstrated to require both defensin and oxygen species 109. The
serine protease, elastase, also acts synergistically with oxygen-fiee radicals. Among the
PMN degradative enzymes, elastase is most commonly associated with tissue injury 110
given its ability to hydro lyze many extracellular matrix (elastin, fibronectin and CO llagen
types DI and IV) and plasma proteins (complement proteins and clotting factors) 1 11.
Tissue destruction by elastase is compounded by either the inactivation of plasma anti-
proteinases, such as ai-proteinase inhibitor, or by the presence of hypochlorous acid 1 12.
1.4.3 Apoptosis
Apoptosis, or prograrnmed ce11 death, is the standard by which PMNs are cleared
fkom the site of inflammation. The activated PMN having engulfed and destroyed the
microbial pathogen in normal circurnstaiices will undergo apoptosis and the rernnants
phagoc ytosed by surrounding mononuclear phagocytes 13. The process of apoptosis is a
tightly regulated process in which the ce11 undergoes shrinkage, fragmentation
(karyorrhexis), dissolution (karyolysis), internucleosomal DNA cleavage with
partitioning into membrane-bound apoptotic bodies L4. Apoptosis rates have been
enhanced upon PMN ingestion in vitro of E- coli in an oxygen-dependent rnanner 1 15.
Mediators of the in£larnmatory process, such as C5a, fM3LP, LPS and GM-CSF have been
demonstrated to delay apoptosis rates of PMNs 87 l3 . Seely and CO-authors
demonstrated 50% of transmigrated PMNs remained present d e r 8 hours and were
unresponsive to TNF-a-induced apoptosis when compared to PMNs in circulation l6.
Teleologically the point in time at which apoptosis occurs during the recruitment
and effector activities of the PMN determines which side of the "double-edged sword" is
at work This paradox of PMN function refers to both their essential role in host
defense to eradicate rnicrobial invasion and ensuing infection as well as their capacity to
effectuate host tissue injury, a maladaptive systernic inflamrnatory response and MODS.
Resolution of an inffammatory process relies on the inactivation and clearance of the
extravasated PMNs and their products; a process performed by inflammatory and
monocyte-derived macrophages 1 18 within a period of minutes 19.
1.5 Consequences of altered PMN effector fùnction
The paradox of PMN recruitment lies in its necessity for an individual's suMval
in the face of injury as well as its capacity to eIicit inflamrnatory injury in the host leading
to cellular, tissue and organ destruction (or MODS), the leading cause of death in the K U
120. Chronic uinammatory conditions such as rheurnatoid arthritis 121 and ischemia-
reperfusion injury 1073 122y 123 are also mediated prirnarily by PMNs. There are several
theones regarding the meçhaïiisms of maladaptive PMN responses. Some microbes (e-g.
Candida) rnay be too large to be fully engdfed by the PMN such that phagocytosis rnay
be partial or absent with release of Iysosomal enzymes and oxygen-fi-ee radicals into the
irnmediate vicinity 124- Alternatively, extensive release or backflow of inflammatory
mediators into the circulation rnay induce premature PMN activation and release of
cytotoxic agents by the PMN overwhelm host antioxidant protease inhibitors which lead
to EC injury and destruction 125. Oxidant injury rnay trigger a vicious cycle since
superoxide production also induces fùrther PMN recruitment to the site of inflammation
126-
The host defense mechanisrns against PMN-induced injury include the
antiproteases and antioxidants. When deficient, such as in ai-antitrypsin deficiency in
pulmonary ernphysema 12', or ovenvhelrned, as occurs in a dysregulated inflammatory
response 1 17, hydrolytic tissue injury c m occur. Despite adequate concentrations in the
circulation of most individuals, antiproteases are effectively excluded fiom the sites of
PMN-EC interaction by the dense adherence of these two ceIl types. Antiproteases are
also excluded fiom sites of interstitial inflammation by the high oxidative stress which
destroys the molecules by binding to exposed thiol groups 125. Sorne antiproteases, such
as ai-protease inhibitor and a2-macroglobulin, are inactivated by hydrogen peroxide and
chlorinated oxidants 2 17.
PMN activation and oxidative burst rnay be triggered by the sarne mediator at
different concentrations or two mediators which induce activation rnay act synergistically
to cause premature burst. N-formyl peptides, such as fMLP, induce chernotaxis and
migration when present in the nanomolar range and respiratory burst in the micromolar
range 128- Phorbol esters, such as phorbol myristate acetate (PMA), and fMLP at levels
which induce activation independently may act together to trigger oxidative burst and
release of toxic oxygen species 129.
1.5.1 Endothelial cell destruction by PMNs
Sepsis in ICU patients is invariably associated with a loss of vascular integrity
proportional to the degree of the inflamrnatory response manifesthg as interstitial edema
and hypotension despite extensive volume resuscitation. This endo thelial destruction has
been attributed to Pm-dependent mechanisms since Sacks and CO-authors demonstrated
that activated PMNs in vitro, when incubated with cultured ECs, induced EC lysis which
was inhibited by the addition of catalase and superoxide dismutase l30- Other studies
have supported that proteolytic enzymes induce EC detachment 2 132 while reactive
oxygen species are responsible for EC destruction l33-l35- Agonists of PMN activation
irnplicated in Pm-dependent EC injwy and detachrnent include PIVIA, W P , CSa, PAF
136. Phospholipase C, p hospholipase A2 and streptolysin S are membrane-active agents
which have been demonstrated to enhance susceptibility of ECs to injury by hydrogen
peroxide 137. Finally unstimulated PMNs incubated with cytokine-activated endothelium
can also induce EC detachrnent in vitro, an effect which can be inhibited with the
addition of IL-8 136. This latter effect rnay be due to the stimulatory effect of vascular
emigration by IL-8 which would lessen the duration of contact between PMNs and the
endo theliurn.
Endothelial damage in ischemia-reperfision injury aIso implicates PMN
involvement since hypoxic ECs demonstrate an increased PMN adherence 138 and
activation 139- Anti-CD18 monoclonal antibodies administered to a rabbit hemorrhagic
shock mode1 resulted in impaired PMN-EC adherence, enhanced survival and marked
reduction in end-organ injury 140- PMN activation results in cytoskeletal alterations that
render the celIs less deformable and can lead to microvascular plugging during penods of
ischemia with enhanced exposure to ECs and thus increasing the risk for damage to the
endothelium 107-
Most in vitro studies of PMN-induced EC injury taise place in plasma-fi-ee
systems using a variety of inert balanced salt solutions 135-1389 141. When human
plasma was used for the medium in which EC cytotoxicity was assayed, there was almost
a complete ablation of endothelial cytolysis suggesting the presence of an unidentified
protective factor in plasma 142. Little is known about this hypothesized factor, other than
that it is likely a protein since its effect is decreased following pronase digestion of
denaturation of plasma proteins 143, and fùrther investigations are required to elucidate
its structure and mechanism.
1.5.2 PMN recrzritment to a remote site in thepresence of inflammation
Although granulocytopoiesis is a well-recognized compensatory event in
inflammation, significantly fewer PMNs are delivered to remote sites when an active
inflammatory response to an injury elsewhere is present. Using a skin window technique
82 where plasma-filled polyethylene chambers are applied over denuded skui for eighteen
to twenty hours, Ahmed and CO-authors demonstrated a 72% reduction in PMN delivery
to ICU patients as compared to healthy controls 144. Although it is unclear why
neutrophilic states, such as a significant infection, are associated with reduced PMN
delivery to an idammatory focus, the consequences of this reduced delivery are
apparent,
As fewer PMNs are available at sites remote fkom the point of principal injury,
individuals with one insult may be at greater nsk for subsequent infections and death.
Critically il1 patients with intra-abdominal infections are more susceptible than healthy
controls or non-critically il1 hospital patients to infectious complications such as wound
infections, pneumonia, urinary tract and vascular-catheter-related infections l. In a recent
multi-center evaluation of over 10,000 critically iII patients, there was a 45% rate of ICU-
acquired infections and risk factors for rnortality were identified as pneumonia,
bacteremia and sepsis 145. These cntically il1 patients with sepsis also demonstrate an
increased rate of non-uifectious complications, such as acalculous cholecystitis,
pancreatitis and ARDS, which are recognized as predisposing factors for the development
of MODS 4. The hi& rnortality (50-80% of ICU deaths 4) and lack of an effective
treatment for MODS serves as the impetus for investigating t5e mechanisms of PMN
recruitment in sepsis; how PMNs are recruited to one site over another.
1.6 Justification of a murine mode1 of peritonitis and a secondary injury
Secondary peritonitis is an injury to the pentoneal space, often uifectious in origin
secondary to breakdown of the gut epithelial lining with release of enteric bactena.
Mortality rates in the literature Vary from O to 70% with a recent senes of 239 patients
with severe peritonitis (APACHE II scores greater than 10) demonstrating a 32%
rnortality 1. In animal models, pentonitis has been most reproducible and clinically
analagous following cecal ligation and puncture first reported by Chaudry l46 and
exhaustively used elsewhere 147-150 as well as in studies pertaining to this thesis. In this
procedure, animals are subjected to laparatomy, ligation of the cecum without causing
proximal bowel obstruction, and one or more punctures of the cecum with a needIe.
In the following experiments, we used a murine cecal ligation and puncture
peritonitis mode1 \ . th intra-abdominal infection as the primary site of injury and either
skin or cremaster injury as the secondary site.
1.7 Hypotheses
This aim of this thesis is to investigate the PMN-endothelial interactions in sepsis
with respect to differences of a primary and second site of injury. The following
hypoîheses will be tested.
Hypothesis 1. Prior to a cornpensatory response of increased neutrophil
production by the marrow, a regionalization phenomenon exists such that PMN delivery
to a pnmary site is proportional to the d e p e of injury with fewer cells available to sites
of secondary injury.
Hypothesis 2. Following an event such as secondary peritonitis, the PMN
activation results in decreased PMN adherence to sites remote fiom the prirnary injury.
Hypothesis 3. Following an event such as secondary peritonitis, shedding of L-
selectin results in decreased rolling and adhesion of PMNs at remote sites.
1.8 Objectives
The soal of this thesis is to both quanti@ and gain insight into the mechansims of
PMN delivery to secondary sites of inflammation. In the first part, polyvinyl sponge discs
placed in the abdomen and/or the dorsal subcutaneous tissue of mice served as both
irritants (recruïters ofPMNs) as well as a vehicle for collecting the ceils. In the second
part, intravital microscopy of the cremaster muscle permitted ex vivo analysis of numbers
of rolling and stationary PMNs as well as rolling velocity. Using the cremaster muscIe as
a point of reference, three scenarios of PMN-EC interactions were studied: a site remote
fiom a prirnary injury (pentonitis), a local primary injury (orchitis) and a local secondary
injury in the presence of a primary injury (orchitis plus peritonitis). Unlike previous
studies which use in vitro conditions with transfected cells, monoclonal antibodies and
flow chambers of adhesion molecule-coated polyrners, the methodo l o g in this thesis
parailels clinical conditions of peritonitis and secondary infections which involve
multiple adhesion molecules and idammatory mediators in an unrnodified fashion.
Chapter 2: MATERIALS AND METHODS
2.1 m a l s and housing
The protocols for these studies were approved by the McGill University Animal
Care Cornmittee. CD1 male mice (25-35 gram; Charles River, St. Constant, Quebec)
were used &er a 3-5 day acclimitization period to the Royal Victoria Hospital Animal
Facility. Al1 procedures, care and housing took pIace in this facility and conformed to the
Canadian Coucil on Animal Care.
2.2 Reagants
Dulbecco's Phosphate Buffered Saline
Ficoll-Paque (research grade)
Heparin (Heapalean;
1000 USP unitshl)
Iso fluorane
Ketamine (Ketaset)
Phorbol 12-Myrisatate 13-Acetate
Turk's stain
Xylazine (Rompurn)
Gibco, Grand Island, NY
Pharmacia, Uppsala, Sweden
Organon Teknika, Toronto, ON
Ayerst Laboratories, Montreal, QC
Sigma Chernicals, Oakville, ON
O.Olg Gentian violet, 3% Glacial acetic acid
Bayer hc., Etobicoke, ON
Bicarbonate-buffered saline was prepared as follows: NaCI, 13 1.9 rnmol/L; NaHC03, 20
mmoI/L; KC1,1.7 mmoYL; MgC12, 1.2 mmol/L.
2.3 Animal procedures
2.3-1 Cecal ligation and puncttrre
Anesthesia was administered by isoflouorane induction and nose-cone ventilation
with 2-4% isofluorane titrated to optimal anesthesia and analgesia. The abdomen was
shaved with electric clippers and the skin prepped with 1% proviodine solution. A 1 cm
rnidline Iaparotomy incision was made and the cecum was carefully delivered into the
operative field. Peritonitis was created in CLP mice according to the technique of
Chaudry et al 146 in which stool was gently milked into the cecum prior to Ligahon with
3-0 silk proximal to the iliocecal valve so as not to cause intestinal obstruction. Two
punctures with a 21 guage needle were made I cm apart and manual compression
extnided feces fkom the puncture sites. The cecum was replaced into the peritoneum and
the abdomen was closed in two layers withr 3-0 Dermalon suture: a continuous layer for
the abdominal fascia and intempted for the skin. The anirnals were allowed to awake
under heating lamps and received buprenorphene analgesia (0.05-0.1 mgkg
subcutaneously) immediately post-operatively and then every eight to tweIve hours as
needed.
2.3.2 Polyvinyl sponge placement
Polyvinyl sponge discs (Ni-PACT, Eudora, Kansas) 5rnm in diameter and 3mrn
thick were prepared according to the technique of Brozna and Ward 151. After washing
in tap water for 90 minutes, they were soaked in distilled water at room temperature for
30 minutes and then boiled in distilled water for an additional 30 minutes. The sponges
were stored in sterile distilfed water until use.
Four groups of 20 mice were assigned as follows: CLP (cecal ligation and
puncture), CM (cecal manipulation), SP (sponge placement) and CON (control) (Table
2.1).
Table 2.1: PMN Regionalization Study Protocol
Group CLP CM SP CON
L-O laparotomy L- CLP X
t=6h laparotomy and abdominal sponge insertion X
t=6h back incision and sponge insertion X
t=3Oh sacrifice and sponge removal X
Table 2.1: Animal were assigned to the following groups: CLP (cecal Sgation and
puncture), CM (cecal manipulation or, sham laparotomy), SP (sponge placement only),
CON (control with sponges placed in back oniy).
At time = O, CLP mice undenvent cecal ligation and puncture as previously
descrïbed. CM mice had their cecums replaced into the abdomen without ligatioo and
puncture and the laparotomy closed in two layers with 3-0 Dermalon suture. Peritonitis
was created in CLP mice according to the technique of Chaudry et aï 146 in which stool
was gently rnilked into the cecum prior to Iigation with 3-0 s i k proximal tu the iliocecal
valve so as not to cause intestinal obstruction- Two punctures with a 21 guage needle
were made 1 c m apart and manual compression extnided feces f?om the puncture sites.
The cecum was replaced into the peritoneum and the abdomen was closed as previousIy
mentioned. The animds were allowed to awake under heating Iamps and received
buprenorphene analgesia (0.05-0- 1 mgkg subcutaneo us ly) imrnediately post-operatively
and then every eight to twelve hours as needed.
Six hours foiIowing the initial laparotomy, ( t h e = 6h) the CLP and CM groups
were re-anesthetized and prepped in the same fashion and their laparotomy wounds
reopened. The SP group also undenvent laparotomy at this time. The CLP mice had their
necrotic cecums resected and al1 three groups had their peritoneal cavities imgated with
30cc of w m e d 0.9% saline. One polyvinyl sponge was placed in each of the four
abdominal quadrants in each group and the Iaparotomy wounds were closed in the same
manner. AI1 anirnals were then placed in a prone position, the rnidline dorsum was shaved
and prepped and a 1-5 cm transverse incision was made through the skin and dennis- Four
subcutaneous pockets were created in each animal by blunt dissection. One sponge was
placed in each of the four pockets. The dorsal incision was closed as a single layer with
3-0 nylon intermpted suture. Each animal received 3.5 ml of warm 0.9% saline and
buprenorphene 0.05 mg/kg subcutaneously, and gentamycin 3mgkg and metronidazole
7.5 mgkg intrarnuscularly. The CON group underwent placement of dorsal sponges only.
Twenty-four hours after sponge placement, the animals were sacrificed using a
CO2 chamber. The sponges were carefully rernoved, placed in 5 ml syringes and the fluid
was squeezed into pre-weighed test tubes. The tubes were weighed with the fluÏd and
then 2 ml of s tede 0.9% saline were drawn through the syringe bearing sponges and the
fluid was expressed into the test-tube. This was repeated twice for a total of 6 cc. Each
tube was centrifuged for 8 minutes at 1500 rpm. The supernatant was discarded and the
pellet reconstituted with 300 pl of PBS of which 50 pl were added to 450 pl of Turks
stain. PMNs were counted with a hemocytometer and light microscope.
23 .3 Optimal E-coli concentration
To determine the optimal concentration of E. coli to create a local inflarnmatory
response in the cremaster muscle sheath for intravital microscopy, serial dilutions o f 1 o',
10~, 1 o7 and 10%rganisms in l5Opl of sterile 0.9% saline were generated using E. coli
NTCBOO 1. These organisms were graciously provided by the Department of
Microbiology and Immunology at McGill University and the material was handled using
aseptic technique and standard laboratory precautions. Each concentration was
administered to three mice by intracremasteric injection and the rnice were observed for
18 hours. Animals receiving 1 o8 and 1 o9 oorganisms becarne rnarkedly systernically toxic
and succumbed upon induction with anesthesia. Anirnals receiwig 107 and 10' organisms
appeared moderately il1 but tolerated the anesthesia well. Upon cremasteric dissection,
anirnals which had received 107 organisms demonstrated both gross and microscopie
inflarnmatory changes while those with 106 organisms had no visible evidence of such
changes. Thus 1 o7 organisms in 150 pl 0.9% saline was selected as the optimal
concentration for use in this study.
2.3.4 Cremastenc dissection for intravital rnicrascopy
Animals were subjected initially to intraperitoneal anesthesia with ketamine (200
mgkg) and xylazine (10 mgkg). Under a heat lamp, a longitudinal midline neck incision
was made and the right interna1 jugula vein was cannulated, During the course of the
expebent , ketamine and xylazine were administered intravenously in 50-1 00 pl boluses
titrated to anesthesia. Cremasteric muscle dissection for intravital microscopy was
perfomied according to the technique of Granger 9. In the supine position with the
assistance of a dissection light microscope (Nikon SMZ-IB Stereoscopic Dissecting
Microscope, Nikon Canada Inc., Montreal, QC), a 3 mm transverse skin snip of the
scrotum was made and the cremasteric sheath was exteriorized and cleared of its fascia1
attachments. The muscle was splayed open by an anterior longitudinal incision with
electrocautery and secured by a five-point suture fixation at the penphery. The tissue was
kept moist with warmed bicarbonate buffer pefision (Peristaltic Microperfusion Purnp,
Instecli Laboratories hc., Plymouth Meeting, PA). The testis, epididymus and vas
deferens were gently reduced into the abdominal cavity. The animal was then turned
prone and resecwed ont0 the intravital microsopy specimen board and placed ont0 the
microscope under a heat larnp.
2.4 Measurement of PMN-EC interactions using intravital microscopy
Mice were assigned to three groups: CLP (peritonitis via cecal ligation and puncture),
ORC (orchitis via E. coli injection into the crernaster muscle sheath), O+C (orchitis +
cecd ligation and puncture) or CON (control: neither peritonitis nor orchitis). At time =
0, animals assigned to CLP underwent creation of peritonitis as descnbed in section 2.3.1.
ORC mice underwent anaesthesia and 3 mm transverse skin snip of the scrotum. The
crernaster muscle was extenorïzed and injected with 107 E. coZi cells in 100 pl of 0.9%
saline. The muscle was replaced in the scrotum and the skin closed with a single 3-0
Dermalon suture. Animals in the O+C group underwent both cecal ligation and puncture
as well as E. coli orchtitis at the same time.
Eighteen to 24 hours after creation of peritonitis or orchitis, animals underwent
cremasteric muscle dissection for intravital rnicroscopy as described in section 2.3.4. The
entire cremaster was visually scanned under light microscopy (Nikon Eclipse TE 2000
Inverted Microscope with Epifluorescence, Nikon Canada Inc., PvIontreaI, QC) for
adequate visualization of blood flow in post-capillary veinules. A straight, unbranched
segment approximately 150 pm in Iength and 30-50 prn in diameter was located and
centered. The animal was observed via video rnicroscopy (Cohu 49 15-20 20 CCD
Monochrome Video Camera, Scion Corporation, Fredenck, MD) for 20 minutes to aliow
PMN kinetics to return to baseline values pnor to taking measurement. The animal was
excluded if centerline red blood ce11 velociv, measured continuously by Optical Doppler
Velocimeter (Microcirculation Research Institute, College Station, TX), fell below 3
rnm/second. Vessels were also excluded if mean velocity was greater than 6 mrn/second
at which shear stresses would significantly impact PMN-EC interactions. Vesse1 blood
flow was then recorded for ten minutes by video cassette recorder (RCA Video Cassette
Recorder VR4564, Thomson Consumer Electronics, Indianapolis, IN) with a time-date
generator (Panasonic WJ-8 10, Secaucus, NJ). One to 3 venules per animal were recorded
depending upon adequate vesse1 quality. At the end of the data acquisition, the rnice were
sacnficed in a COz chamber and then underwent percutaneous cardiac punchire and the
blood was stored in a heparinized tube on ice for PMN counting.
2.5 Measurement of vessel kinetics
Off-he video playback analysis of the recorded PMN-EC interactions were
performed using a stage micrometer to calibrate on-screen measurements of vessel
diameter and length. Centerline red bIood ce11 velocity (Vrbc) was deterrnined as
previously mentioned using an Opticai Doppler Velocimeter with the sensors placed in
the center of the vessel. Mean red cell velocity (Vmean) was determined by the formula
Vrbdl.6 and venular blood flow W F ) cdculated by Vmean*cross-sectional area
assuming cylindrical geometry. Venular shear rate (VSR) was calculated by 8*Vmean/Dv
(vessel diameter) and venular wall shear stress by VSRçq where tl is 0.25 poise, the
viscosity of blood (Table 2.2).
PMN rolling fluxes were calculated by averaghg the nurnber of PMNs which
crossed a h e drawn perpendicular to the axis of the vessel per minute over two minutes.
Rolling PMNs were defined by those cells which moved at a constant rate slower than
that of the red blood cells. PMN rolling velocity was calculated by timing twenty PMNs
which moved at a constant rate over a distance of 100 pm and reporting the average
velocity in pn per second. Adherent PMNs were defined as PMNs that become
stationary for at least 30 seconds in a 100 p m segrnent over 5 minutes.
Table 2.2: Formulae for the calculation of vesse1 kinetics
Parameter Formula
VSR
rneasured directly fiom the Optical Doppler Velocimeter
Table 2.2: centerline red ce11 velocity (Vrb,), mean red ce11 velocity Or,,,,), venular blood
flow (VBF), vesse1 diameter (Dv), venular shear rate (VSR), venular wall shear stress
(VWSS) and viscosity (q).
2.6 PMN isolation and counting
Al1 mice undergoing intravital rnicroscopy underwent cardiac puncture and blood
withdrawal after they were sacrificed. The volume of the blood was recorded and the
blood gently layered over 3 ml of Ficoll-Paque and centrifuged (Centra-8R Refi-igerated
Centrifuge, International Equipement Co., Needham, MA) for 25 minutes at 400g (1600
rpm). The supernatant was discarded and the pellet was resusbended in 6 ml of sterile
water for 10 seconds to induce erythrocyte lysis after which the osrnolarity was rapidly
restored with the addition o f 2 ml of 3.6% sodium chloride. M e r centrifugation for 5
minutes at 400g, the supernatant was again discarded and the pellet resuspended in 8 ml
of phosphate buffered saline (FBS). After a final centrifigation at 4003 for 5 minutes, the
pellet was resuspended in 100 pl of PBS and the PMNs were counted as previously
descnied.
2.6- I Pniinpuri~,
PMNs were isolated fi-om the blood using a standardized technique described in
the previous section. Purity of the PMNs were assessed in six rnice; three controls and
three with pentonitis by isolation, smear and hematoxylin and eosin staining. The
differential was counted in three hi&-power fields per slide and PMNs were reported as
botti raw number and percentage of total leucocytes. Figure 2.1 dernonstrates a
differential smear of animals with pentonitis and figure 2.2 depicts that of controls. The
number and percent purity of PMNs was 94% for controls and 68% for animals with
pentonitis (CLP).
2.7 Statistical analysis
Al1 analyses of data were performed using Systat 8.0 (SPSS, Chicago, IL). Means
and standard errors of two groups were compared by Student's t-test. Analysis of
variance was used for cornparisons of three or more groups of data with Bonferroni
correction for multiple comparisons. A p value c 0.05 was considered statistically
sigmtlcant.
Figure 2.1. Giemsa-stained smear of PMNs following isolation fiom whole blood fiom a
control animal. PMNs have a characteristic multi-lobed nucleus while lymphocytes have
a prominently stained, large unilobed nucleus.
Figure 2.2. Giernsa-stained smear of PMNs following isolation fiom whole blood fiom a
cecal ligation and puncture (CLP) animal. PMNs have a characteristic multti-lobed
nucleus while lymphocytes have a prominently stained, large unilobed nucleus.
Chapter 3: RESULTS
3.1 A n b a l mortality
Animals which did not survive the penoperative period were excluded fiom the
study and subsequent animals were added to the study until adequate goup size was
achieved. Approximately 170 animals excluding mortalities were used in these studies,
80 in the assessrnent of PMN regionalization and 90 in the intravital microscopy
experïment, ~Mortality rates were on the order of 10%.
3.2 PMN regionalization between peritoneal and skin in jury
The results of the PMNs recovered ffom abdominally placed sponges are
demonstrated in Figure 3. la. In the abdomen, both the mice receiving the cecal Ligation
and puncture and cecal manipulation had significantly greater numbers of PMNs
recovered fiom the sponges wlien compared to the control animals.
CLP CM S P
Figure 3.la. Recruitment of P M . to the peritoneal cavity (x 105) in mice undergoing
cecal ligation and puncture (CLP), mice with cecal manipuIation (CM) and mice
undergoing sponge placement alone (SP). Data are represented as rnedian (horizontal
h e ) , 25 and 75% (box) and 10 and 90% (error bars) confidence intervals with outlying
points. PMN exudation in mice with either CLP or CM were sipnificant-y greater than
those mice undergoing sponge placement alone.
In the sponges placed in the dorsal subcutaneous tissue, there were the greatest
numbers of PMNs recovered in the control group which undenvent no abdominal
intervention (Figure 3.1 b).
CLP CM SP Control
Figure 3.1 b. Recruitment of PMN to the dorsal skin (x 10') in mice undergoing cecal
ligation and punct-are (CLP), rnice with cecal manipulation (CM) and mice undergoing
sponge placement alone (SP). Data are represented as median (horizontal line), 25 and
75% (box) and 10 and 90% (error bars) confidence intervals with outlying points. Mice
with CLP were significantly different fiom those undergoing sharn laparotomy (CM), SP
or contro 1s.
3.3 Circulating PMN counts folIowing orchitis and cecal ligation and puncture
The numbers of circulating PMNs arnong the intravital microscopy study groups
are demonstrated in Figure 3 -2.
Control CLP Orchitis CLP + Orchitis
Figure 3.2. Circulating PMN counts (1 03/ml) in control mice, mice undergoing cecal
ligation and puncture (CLP), mice with orchitis and mice undergoing both CLP and
orchitis. Data are represented as median (horizontal line), 25 and 75% (box) and 1 0 and
90% (error bars) confidence intervals with outlying points. CLP mice with or without
concomitant orctiitis were significantly different fi-om both orchitis alone and controls.
Mice with orchitis alone were also si,onificantly different kom those with concomitant
CLP.
Within the blood, there was no signincant difference in the numbers of circulating
PMNs between the control group and those mice which undenver~t E. di-induced
cremasteric muscle infection (orchitis). In contrast, when compared to the control mice,
there was a significant reduction in the nurnbers of circulating PMNs in the animds
which undenvent cecal ligation and puncture. In mice receiving both cecal ligation and
puncture and a cremasteric muscle infection, thire was no M e r reduction in the
numbers of circulating PMNs compared to cecal ligation and puncture alone.
3 -4 fntravital microscopy
3.4.1 Circulatory parameters
The circulatory parameters for the groups of mice in the intravital microscopy
study are shown in Table 3.1. Mean vesse1 diarneter and venular blood flow did not differ
signifrcantly between the four groups of anirnals. However mean red blood ce11 velocity
was significantly reduced fiorn 2.9 to 2.3 mm/sec in those mice undergoing cecal ligation
and puncture whether or not this was accompanied by cremasteric muscle id5ection.
Fwthermore, venular wall shear stress was si,onificantly reduced in those animais which
undenvent orchtitis compared to controls and this was observed both in the presence and
absence of cecal ligation and puncture.
Table 3.1. Baseline circulatory parameters (expressed as mean i- SEM) in mice
undergoing cremaster muscle infection (orchitis, ORC), cecal ligation and puncture
(CLP) or both (ORC + CLP). * p < 0.05 compared to control mice (CON).
Group n Mean rbc Vesse1 Venular Venutar wall velocity diameter blood flow s hear stress ( d s e c ) ( ~ m ) (1 0' ml/sec) ( 1 o5 dyn/cm2)
Control 24 2.9 k 0.1 35.9 t 1.2 29.6 + 2-1 16-8 t 1.1
ORC 18 2.6 t 0.1 38.7 t 1.3 31.1 -t- 2-0 13.9 t 0.7*
CLP 21 2,S-tO-l* 35.1-i-1.1 24.3t1.9 14.4 t 0.8
ORC+CLP 10 2,3+0.1* 38.7I1.7 16.6t2.0 12.0 +_ O.L*
3 - 4 2 Kinetics of PMN-endothelid ceZZ interactions
The results for PMN rolling flux, which was defïned as the number of PMNs
rolling past a stationary point per minute, are represented in Figure 3.3a Alth~ugh the
greatest PMN rolling flux was observed in the control group of mice, these values did not
sipificantly differ from those animals undergoins cremasteric E. coli infection. In
contrast, the mice which undenvent cecal ligation and puncture showed a significant
reduction in rate of PMN rollin,o compared to the control mice. Moreover, this rolling rate
was M e r reduced in those animals which had a concomitant muscle infection.
20
O
Control C LP Orchitis CLP + Orchitis
Figure 3.3a. Rolling PMN flux (ceilshin) in control mice, mice undergoing cecd
ligation and puncture (CLP), mice with orchitis and mice undergoing both CLP and
orchitis. Data are represented as median (horizontal line), 25 and 75% (box) and 10 and
90% (error bars) confidence intervals with outlying points. CLP mice with or without
concomitant orchitis were significantly different fiom both orchitis alone and controls.
Mice with orchitis alone were also significantly different from those with concomitant
CLP.
Since circulating PMN concentrations was demonstrated to be different among
the groups, this rnight serve as a confounding variable in the determination of rolling
flux. In order to account for any effect of PMN concentration, an adjustment factor for
each data point was created by the ratio of circulahng PMN concentration to group mean
PMN circulating concentration. The adjusted rolling count was determined by the product
ofthe rolling nurnber and the adjustment factor (Figure 3 -3b).
Con trol CLP Orchitis CLP + Orchitis
Figure 3.3 b. Adjusted roliing flux in which values are adjusted for circulahng PMN
concentration in control mice, mice undergoing cecai Ligation and puncture (CLP), mice
with orchitis and mice undergoing both CLP and orchitis. Data are represented as rnedian
(horizontal line), 25 and 75% (box) and 10 and 90% (error bars) confidence intervals with
outlying points. CLP rnice with or without concomitant orchitis were significantly
diifferent fi-orn both orchitis alone and controls. Mice with orchitis alone were also
sigdicantly different from those with concomitant CLP.
Adjushg the rolling numbers of PMNs for the effect of circulating PMN
concentration did not signihcantly alter the results.
Mean PMN rolling velocities for the four groups of rnice are represented in Figure
L I
Controi CLP Orchitis CLP + Orchitis
Figure 3.4. Rolling PMN velocity (prnkec) in control mice, mice undergoing cecal
Ligation and puncture (CLP), mice with orchitis and mice undergoing both CLP and
orchitis. Data are represented as median (horizontal line), 25 and 75% (box) and 10 and
90% (error bars) confidence intervals with outlying points. Orchitis mice with or without
concomitant CLP were significantly different f?om controls. Mice with CLP alone were
also significantly different fkom those with concomitant orchitis.
The highest rolling velocity was observed in the control group of mice, although
this did not significantly differ f o m the animals undergoins cecal ligation and puncture.
There was however a significant reduction in rolling velocity in those mice with orchitis.
Animals which underwent cremasteric muscle infection with E, coZi and had a
concomitant cecal ligation and puncture were not sigdicantly different fiom those with
cremasteric muscle infection alone, however they did have a reduced rolling velocity
compared to the cecal ligation and puncture group.
The results of PMN adherence in the four groups of mice, defined as the number
of cells which becarne adherent to a 100 Fm section of the venule endothelium over a 5
minute period, are demonstrated in Figure 3.5. in the group of r i c e with cremasteric
muscle infection (orchitis), there was a significant increase in the numbers of adherent
PMNs compared to the control animals.
Control CLP Orchitis CLP + Orchitis
Figure 3.5a. PMN adherence (cells) in control rnice, mice undergoing cecal ligation and
puncture (CLP), mice with orchitis and mice undergoing both CLP and orchitis. Data are
represented as median @orizontal line), 25 and 75% (box) and 10 and 90% (error bars)
confidence intervals with outlying points. Orchitis rnice were significantly different fiom
those with concomitant CLP or controls,
There was no difference in the nurnbers of PMNs adhering to the venular
endothelium in the anirnals which had cecal ligation and puncture when compared to
control @ > 0.05). Those mice which underwent orchitis with concomitant cecal ligation
and puncture had significantly fewer adherent PMNs than the orchitis alone group @ c
0.001). However the concomitant cecal Ligation and puncture with orchitits rnice were not
significantly different in their numbers of adherent PMNs to the cecal ligation and
puncture alone group (p > 0.05).
In order to account for any effect of PMN concentration on adherence, the same
adjustment factor was used to calculate the adjusted adherence count. This value was
detennined by the product of the adherent PMNs and the adjustment factor (Figure 3.5b).
Control CLP Orchitis CLP + Orchitis
Figure 3.5b. Adjusted PMN adherence (cells) in which values were adjusted for
circulating PMN concentration in control mice, mice undergoing cecal ligation and
puncture (CLP), mice with orchitis and mice undergoing both CLP and orchitis. Data are
represented as median (horizontal line), 25 and 75% (box) and 10 and 90% (error bars)
confidence intervals with outlying points. Orchitis mice were significantly different fiom
those with concomitant CLP or controls.
Adjusting the adherence data for the effect of circulating PMN concentration did
not significantly alter the results.
Chapter 4: DISCUSSION
These studies were designed to examine the local recruitment of neutrophils in a
murine mode1 of pentonitis and secondary injury. PMNs delivered to polyvinyl sponge
discs in the extravascular space were studied in a manner encompassing the sequential
interactions with the endothelium of capture, rolling adhesion, £hm adhesion and
transendothelial migration. The resdts demonstrated a regionalization of neutroptiils to
the prïmary site of inflammation with a concomitant reduction in neutrophil recruitrnent
to site of secondary inflammation. With the aid of the intravital microscope, the
component parts of PMN-EC interactions prior to vascular emigration were examined.
These included capture (ro lling adhesion), ro lling velocity and adherence of PMNs.
Using the post-capillary venules of the cremaster muscle as a reference point, the
interactions between PMNs and ECs in each of the following four perspectives were
investigated: at the site of injury, at a point remote fiom the site of injury, in the absence
of any injury and at the site of a secondary injury in the face of a severe primary injury.
We demonstrated that at the site of injury, both PMN rolling and firm and adherence
were greatest and P M ? velocity was reduced corresponding to enhanced recruitment.
Conversely, at a site remote fiom significant injury, fewer numbers of rolling and
adherent PMNs were present but they rolled at a higher velocity likely corresponding to
an enhanced delivery of PMNs to a distant site of injury. At a site of an injury of Iesser
severity when a significant primary injury is present distantly, PMN rolling and fim
adherence rates were the lowest and the PMNs that were present rolled at the slowest
rates. This irnplies a triage of PMNs away fiom the secondary injury for enhanced
delivery to a distant site. Together these hdings suggest important implications
regarding PMN recruitment to injury and raise M e r questions about m e c h ~ s m s of
recruitment.
4.1 PMN delivery to polyvinyl sponge discs
In d l animais regardless of the site or degree of injury, PMNs were recruited
primarily to the peritoneum, the site of major injury. The degree of recmitment at the
prïmary site of injury corresponded to the extent of injury. A simultaneous secondary
injury to the skin of lesser severity yielded fewer PMNs to an extent inversely
proportional to the degree of injury to the primary site. These results were corroborated
by circulating PMN counts in mice with cecal ligation and punctwe-induced primary
injury and E.coli-induced cremaster muscle uifection as a secondary injury. Significantly
fewer PMNs were circulating in anirnals with peritonitis compared with cremaster muscle
infection or controls, and even fewer were counted in the presence of both injuries,
however the latter value was not statistically significant,
A consistent neutropenia was observed in mice in response to infectious injury
while similar infections in human patients dcmonstrate a consistent neutrophilia.
Furthemore, other investigators have reported a hypothermie response in rnice
undergoing cecal ligation and puncture while patients with peritonitis generally manifest
a fever. These differences either relate to either the rodent mode1 itself which may
manifest a different sephc response than that found in hurnans or that the cecal ligation
and puncture provides an extrernely severe injury which ovenvhelms the immune system
in the acute period. Our data supports the latter hypothesis since al1 measurements were
taken within 24 hours of this injury after which the animals were sacrificed since
preliminary data revealed that virtually al1 animals succurnbed in the following 12-24
hours. The presence of a neutrophilia implies that an adaptive compensatory myeloid
response to injury has occurred- Further studies are warranted to determine if a
compensatory neutrophilia occurs following a less severe injury.
Recruitment of PMNs to the sponge discs occurred in mice whose only injury was
the subcutaneous irnp lantation of the po lyvinyl sponge discs alone. The mechanisms
responsible for the induction of PMN recruitment following sponge placement have been
previouçly described elsewhere 151. It is clear that the polyvinyl sponge discç are more
than passive vehicles for the collection of PMNs since the cells rnust undergo al1 the steps
of recruitment including vascular exudation before they contact the sponges. Upon
sponge removal, there was never any evidence of bleeding or sanpinous fluid suggesting
vascular disruption tlius PMN exudation is a prerequisite to collection by the sponge. The
polyvuiyl mesh serves as an irritant leading to a consistent, reproducible recruitment of
PMNs sirnilarly documented 52.
With respect to the intraperitoneally-placed sponges there was no significant
difference between nurnbers of PMNs recruited by sponges placed in cecal ligation and
puncture anirnals or those with cecal manipulation. This suggests that either the creation
of peritonitis adds little to the degree of injury caused by sharn lapamtomy with
subsequent placement of sponges or that a maximum PMN response was achieved by the
lesser injury which has depleted a finite reserve of PMNs. Our data supports the latter
(Le. hypothesis #1) since exudated PMNs collected Gom the sponges placed in both the
abdomen and dorsal subcutaneurn demonstrate a trend of sequential increase of PMNs
delivered to the primary site of injury (the abdomen) depending on the degree of injury
with a concomitant reduction of PMNs dehered to the sponges placed at a secondary site
of injury. Because the cecal ligation and puncture arimals dernonstrated signincantly
fewer PMNs in the sponges in the back than the sharn laparatomy group (cecal
manipulation), it appears that peritonitis does add a significant "hit" above that of a sham
laparatomy. These data support hypothesis #1 in that they sugsest the likelihood for a
relatively finite reserve of PMNs available within 24-30 hours d e r inciting event for
delivery to one or more sites of injury. The mechanism by which this pool of PMNs is
triaged to one or more injuries deserves fùrther investigation.
The unifjhg concept fkom the data represented in Figures 3.1 a and 3.1 b support a
regionalization phenornenon by which a reserve pool of PMNs are triaged to sites of
injury depending on theie relative severities. The clinical analogy to this situation is the
ICU patient with peritonitis who is not only at increased risk of developing a secondary
infection, such as a pneumonia, but the morbidity and mortality associated with a
secondary pneurnonia are increased over a pneurnonia developing in othenivise healthy
patients. A recent study in murine models of bacterial-induced peritonitis demonstrated
that neutrophil recruitment is associated with intrapentoneal granulocyte and
granulocyte-rnonocyte colony stimulating factors (G-CSF and GM-CSF, respectively).
Since intrapentoneal injection of these hematopoietic factors failed to yield enhanced
PMN recruitrnent into the peritoneum, they likely senre as systemic markers of an up-
regulated response in the bone marrow of granulocyte production within three hours 153.
Such hematopoietic factors in response to injury serve to increase PMN production to up
to 10" cells per day 6. It is unclear how long before the increased production impacts on
the immediate pool of PMNs, but in the mode1 of murine peritonitis this time hime is at
least 24-30 hours.
4.2 Intravital microsocpic analysis of PMN fluxes, rolling adhesion and firm adhesion
In the intravital microscopy study we exarnined the numbers of PMNs undergoing
rolling adhesion (PM? flues), the mean velocity at which PMNs roll as well as the
numbers undergoing firm adherence in the selected groups in order to determine whether
differences were due to changes in circulatory parameters or another mechanism such as
adhesion molecule expression. With respect to baseline hemodynamic circulatory
parameters measwed for each group, we demonstrated that cecal ligation and puncture in
mice was associated with a reduction in mean red blood ce11 velocity. This effect
occurred whether or not a secondary injury, E. coli-induced cremasteric muscle iniCection
(referred to herein as "orchitis"), was present. Furthermore, orchitis, with or without cecal
Iigation and puncture, was associated with a reduction in the venular shear stress. These
changes occurred despite no differences in vessel diarneters or venular blood flow among
the four groups of animals.
To determine the extent of any relationship between mean RBC velocity and
venular wall shear stress, and the kinetics of PMN-endothelial ceIl interactions, we
performed correlational analyses. These analyses demonstrated that there were significant
relationships between both the mean RBC velocity and vessel shear stress, and PMN
rolling (2 = 0.39 and r2 = 0.56, respectively; p c 0.001). No other signincant correlations
between baseline hemodynamic parameters and kinetics of PMN-endothelid ce11
interactions were observed. This implies that only differences in numbers of rolling
PMNs between the groups, and not PMN rolling velocity or adherence, could be
attributed to differences in RBC velocity or vesse1 wall shear stress.
Our data supports that changes in mean red ce11 velocity act systernically while
changes in venular wall shear stress acts only at the site of local injury without affecthg
PMN delivery elsewhere. A reduction in mean red ceIl velocity was seen only in the
groups of animals with the most severe insults: peritonitis with or without orchitis. The
reduced velocity seen in animals with a major injury in a site rernote fiom the point of
reference implies a systemic effect has occurred that may account for differences in
numbers of rolling PMNs. The reduced venular wall shear stress seen in only in the
groups where a local injury occurred: orchitis with or without peritonitis sugsests that the
change in wall shear stress was simply a local phenornenon since the most severely
injured group (peritonitis and orchitis) did not manifest these changes. Thus while it is
unlikely that changes in venular wall shear stress occurred systemically in the face of
injury, we cannot exclude a reduction in mean red ce11 velocity as a cause of differences
found in numbers of rolling PMNs. Factors responsible for the reduction in mean RBC
velocity in our experirnentd groups rernain to be established. However, a reduction in
biood pressure which comrnonly occurs in association with SIRS, may have been
conûibuting factors. Further experiments to assess blood pressure in these animals and to
institute masures to prevent differences in baseline hemodynamic variables are
warranted.
Since mean red ce11 velociv differences may contribute to changes in numbers of
PMNs undergoing rolling adhesion, we wondered if differences that occurred were due to
the actual adherence properties themselves (Le. adhesion molecule expression and
hypotheses #2 and 3) or simply due to a change in overall numbers of PMNs in the
circulation. Simply put, did we rneasure dserences in defivery of PMNs to the segment
of venule being studied? In order to resolve this question, we adjusted each group for the
effect of circulating PMN concentration by calculathg an adjustment factor fiom the
quotient of the group mean circulating PMN count into the individual animal's number of
circulating PMNs. The product of the adjustment factor and either the rolling or the £ïrm
adherence nurnber deterrnined the respective rolling or fïrm adherent value adjusted for
the effect of circulating concentration of PMNs. When adjusted values were p lotted
against each group, no alteration in statistical significance for each group were noted
when compared to unadjusted data. Therefore any results which occurred could not be
attributed to differences in PMN concentrations in the circulation. By virhie of this fact,
the differences in hemodynamic parameters seen did not significantly alter the rolling or
firm adherence of the PMNs.
Anunals with pentonitis with or without orchitis (CLP and CLP + Orchitis
groups) demonstrated a marked reduction in numbers of rolling PMNs compared to
controls. These differences correspond to similar results reported elsewhere 154. The
greatest nurnbers were associated with the orchitis and control anirnals. Surprisingly the
creation of a local injury, orchitis, did not result in increased nurnbers of rolling PMNs
over controls. These results when taken together imply that either the addition of a local
infection has no fiirtlzer stimulus above baseline PMN recruitment or that changes in
recrutirnent are not manifested in the numbers of rolling PMNs. It is unlikely that orchitis
does not add to PMN recruitment over controls since PMNs fcom animals with orchitis
roll at a slower velocity and adhere in greater numbers than controls. It appears that the
minor injury of orchitis does not lead to increased numbers of rolling PMNs over control
animals, however increased local recmitment of PMNs to injury is demonstrated in the
reduced velocity and increased firm adhesion seen in orchtitis animals. Specific selectin
rnolecule expression has been associated with changes in either rolling adherence or
velocity or both and this rnay account for increased PMN recmitment to infected
cremasteric tissue. Other investigators have demonstrated elevated systemic CDllb
expression in CLP animals compared to controls in a sirnilarly-designed study 154.
The possibility that a minor local infectious injury does not result in increased
PMN fluxes in a finite pool of PMNs must also be considered. In other words the
quiescent control state is associated with maximum numbers of PMN rolling penpherally
and the presence of an injury serves to reduce numbers of rollhg cells elsewhere without
increasing them locally. The PMNs recruited &om the peripheral maiginated pool may
appear as adherent or sIowly rolling PMNs at the site of injury. Since adherent PMNs are
not included in the rolling numbers and that the reduced velocity may result in fewer
fluxes as well, it is possible that the changes in PMN-EC interactions seen in sepsis are
manifested in PMN velocity and adherence alone.
PMN rolling velocity was reduced in the mlce with orchitis with or without
concomitant cecal ligation and puncture (Orchitis and CLP + Orchitis groups). Rolling
velocity of PMNs reflects the presence of adhesive interactions between selectins and
their ligands on the PMN and the underlying endothelial cells. In a mode1 of acute
inflammation such as orchitis, these adhesion molecules would be upregulated both on
the PMNs and vascular endothelium. The time course of Our experiments, 18 - 24 hours
following intra-cremasteric injection of the E. Coli organisms, suggested that E-selectin
wodd have been the predominant selectin expressed 353 507 51. The predominance of
this adhesion molecule may account for the reduced rolling velocity of the PMNs within
the cremasteric vasculature since in vitro studies of E-selectin have demonstrated slower
rolling than P or L selectin s3. Further studies to address this hypothesis using specific
anti-E-selectin antibodies in this mode1 are necessary.
Those animals with cecal Iigation and puncture alone did not demonstrate any
significant difference in PMN rolling velocity fiom controls. This was to be expected
since there was no locaiized inflammation occwrïng within the cremasteric muscle in
these animals and therefore no stimuli for the recruitment of PMNs in this region.
Although the specific adhesion molecule mediating PMN rollirg in the crernaster muscle
in the absence of local injury (CLP or controls) was not determined in these experiments,
evidence fiorn previous investigations suggests that rollinj in these venules is most likely
mediated by L-selectin. Although L-selectin is the only selectin to be constitutively
expressed, induction of P-selectin can occur within minutes following cytokine-induced
EC stimulation. It is possible that the trauma of general anesthesia and crernastenc
muscle dissection alone c m be a sufficient stimulus for P-selectin upregulation. Baseline
recording of PMN-EC interactions took place 20 minutes after dissection was completed
and rnounted ont0 the microscope stage in order to eliminate srnall peiturbations in
cytokines and other mediators of adhesion rnolecule expression and circulatory dynamics.
This is a standard technique employed in similar intravital microscopie investigations 52,
155-157- Using a laminar flow assay to measure rolling velocity of PMNs on g l a s slides
with P-, E-selectin or a L-selectin ligand, L-selectin-mediated rolling velocity was double
that of the other two selectins 53. Our data similarly demonstrated significantly increased
velocity of control animals (where L-selectin is likely to predomioate) as compared to
animals with orcilitis both with and without cecal Ligation and puncture (Orchitis and
CLP + Orchitis groups) where E-selectin is likely to predominate-
PMN £km adherence to the endothelium requires the sequential involvement of
selectins, chemoattractants and integins under physiologie conditions of blood flow.
Animals undergoing local injury (orc hitis) dernonstrated significantly greater numbers of
adherent PMNs in the cremasteric vascuIature than animals undergoing cecal ligation and
puncture or controls. This was to be expected since adhesion molecule up-regulation is
present locally in the orchitis group and not in the others. Tliat there was no dinerence in
CLP and contrai groups with respect to £km adherence (thus rejecting hypothesis #2) has
been demonsîrated both here as well as elsewhere 154- Althouph we expected that
pentonitis would have been associated with reduced firm adherence at remote sites
Oîypothesis #2), it is possible that the circulatins and marginating pool of PMNs buffer
such a change. Furthemore, despite the likelihood of increased adherence at the
mesenteric venules, we saw no change frorn baseiine peripherally- This stands in contrat
to rolling adhesion which is significantly reduced peripherally in the face of peritonitis.
When a primary injury is present remotely combined with a relatively minor
injury locally (CLP + Orchitis group), we observed a drarnatic reduction in PMN
adherence within the cremaster muscle. This suggests a picture of competing sites of
injury for a relatively finite pool of PMNs available within the f i s t 24 hours of acute
injury. AIthough the precise mechanism this effect was not investigated, it is likeIy due to
differences in levels of chemoattractants and clustering of P2 integrhs. Regardess of the
actual mechanisrn, this hding m e r supports hypothesis #1 that a regionalization
p henomenon exists among stimuli of cornpeting injuries.
Chemoattractants function to alter adhesiveness of integins and are produced and
released in concenûations which correlate with the severity of the inciting injury 158.
The degree of P M . delivery has also been demonstrated to correlate zh vivo with
chernokine concentration in a dose-dependent rnanner 159. Thus it is possible that the
major injury (peritonitis) caused a greater magnitude of chemokine syntiesis and release
than the minor injury (orchitis) and resulted in an increased PMN recruitment to the
peritoneum with fewer cells available for recniitment by the cremasteric vasculature.
Sirnilarly increased clustering of P2 integrins on the PMN ce11 surface has been
demonstrated in response to chemoattractants carising increased numbers of adherene
PMNs as well as increased sîrength of adherence to endothelial in vivo Io. This correlated
with our data which demonstrated a drarnatic reduction in adherence of PMNs to the
cremasteric vendes in the presence of orchitis and peritonitis.
The concentration of PMNs in the circulation of these anirnals M e r supports
this hypothesis. Circulating PMN counts in control mice or mice with orchitis were
significantly greater than cecal Ligation and punchlre mice with or without orchitis (CLP
and CLP + Orchitis groups). The concentration of circulating PMNs for each group of
mice appears inversely proportional to the nurnbers of adherent PMNs. This finding
supports the presence of a relatively finite pool of circulating PMNs available for
recruitment and fûrther suggests a possible role for adhesion molecule and
chemoattractant as regulators of these alterations in the kinetics of PMN-EC interactions.
These results were further corroborated by the PMN smears fiom CLP and control
animals used for pmity analysis. In the photographs (which were consistent findings in al1
six animals who undenvent purity analysis of PMN preparations), the raw number of
lymphocytes per high power field was maintained while a 90% reduction in raw PMN
counts were obtained between control and CLP anirnals.
4.3 Future directions: a potential role for shed L-selectin
The results fiom these investigations suppoas the existence of a relatively finite
pool of PMNs available for the immediate period following injury- In a murine mode1 of
cecal ligation and puncture-induced peritonitis, this pool of PMNs is present for at least
24-30 hours following injury. The duration and extent of compensatory hematopoietic
responses by the bone marrow warrants further investigation. The myeloid response is
likeiy mediated by endogenous colony stimulating factors. The potential benefit of
administering exogenous hemopoietic colony stimulating factors in the early period
followirg injury also merits investigation.
We have also demonstrated the presence of a regïonalization phenornenon by
which PMNs fiom the relatively f i t e pool are triaged to the site of major injury. No
attempt was made here to determine the mechanism of this effect but it is likely that
alterations in adhesion rnolecule expression play conîributory roles. Chemoattractants
such as IL-8, f M U , and LTB4 have been previously shown to increase the adherence and
exudation as well as influence chernotactic migration of PMNs to and across HLNECs in
viho 63 and rabbit demis 60 and hamster cheek pouch in vivo 64. When PMNs were
pretreated with IL-8, CSa, or MLP, adherence to HLJVECs under flow conditions was
inhibited in a dose-dependent rnanner 160. The explanation for why chemoattractants at
times can either facilitate or inhi'bit PMN adherence to ECs is that these chernoattractants
induce shedding of L-seleciin 72. Shedding of L-selectin is a concomitant step in
transition fiom r o l h g to firm adhesion and is associated with up-regulation of the P2
integrin molecules on the PMN 67. When PMNs are pretreated with IL-8 and shed their
L-selectin prior to interaction with the endothelium, adhesion does not occur 160.
Little is known of the fate or the subsequent role played by these soluble L-
selectin molecules on PMN-EC interactions downstream. It is postulated that once shed,
the soluble L-selectin may bind to its receptors peripherally and competitively block
quiescent PMN rolluig on the endothelium, This would lead to a reduced PMN flux and
fïrm adhesion at sites remote from injury (hypothesis #3) and increased numbers at sites
of injury where the soluble L-selectin would less effectively compete with up-regulated
L-selectin receptors. Using a murine thioglycollate-induced peritonitis model, Watson
and CO-authors demonstrated that the intravenous administration of a soluble
immunoglobulin chimera containing the extrallular L-selectin domains significantly
reduced PMN ernigration to the peritoneum 161. It is possible that shed L-selectin may
contribute to the regionalization effect of PMlV delivery to sites of injury. The sites of
major injury such as pentonitis would presurnably recruit a greater proportion of the pool
of available PMNs since shed L-selectin would compete less effectively with the
associated up-regulated endothelial expression of selectin receptors. Conversely,
uninjured tissue or less severe sites of injury such as an E-coli-induced crernaster muscle
infection would accumulate fewer PMNs since the relatively fewer selectin receptors
would be more efficiently blocked by soluble L-selectin.
Further studies are indicated to determine the role of soluble L-selectin on PMN
delivery to sites of injury. Tech icd and ethical bamers need to be overcome in order to
produce the necessary quantities of murine shed L-selectin as few investigators have been
able to achieve this end. Without the data to support or contradict the shedduig of L-
selectin as well as the role of soluble L-selectin on rolling and fum adherence, we can not
at this time test hypothesis #3.
4.4 Conclusion
Using polyvinyl sponge discs implanted in the peritoneum and skin of the back in
mice with or without peritonitis, a relatively f i t e pool of PMNs was demonstrated to be
present with PMNs delivered to each site in proportion with the severity of injury to the
major site. In this mode1 this pool exists for at least 24-30 hours following injury before a
compensatory response by the myeoloid organ occurs. These results support hypothesis
#l however the precise mechanism of PMN delivery, or regionalkation, remains uncIear.
With intravital microscopy we demonstrated a that numbers of PMNs involved in
rolling adhesion with the endotheliurn are significantly reduced at a site remote f?om
peritonitis and this effect was unchanged if a local injury was present as well-
Correlational analyses revealed that differences in mean red blood ceIl velocity, a rnarker
of intravascular hemodynarnic change, could account for differences in rolling PMNs by
altering number of PMNs arriving at the post-capillary venule. However, adjusting for
circulating P M . concentration and replotting the data, we found that there were no
differences significant outcomes. Since liemodynamic changes do not account for these
differences, the mechanism of altered PMN-EC interactions in the presence of a two-kont
injury mode1 is likely due to alterations in both ceU surface-expressed and soluble
adhesion molecuIes. In addition, PMNs rolled at sIower velocities in the presence of local
injury whether or not peritonitis was present. The presence of peritonitis did not result in
reduced numbers of finrily adherent PMNs to the cremasteric vasculature, thus rejecting
hypothesis #2, and this reIationship was not altered by the addition of a Iocal injury.
Further studies investigating the role that shed L-selectin plays on PMN-EC interactions
downstream fkom a primary site of injury are warranted to determine whether or not this
results in enhanced delivery of the marghating PMN popu1ation to the prirnary injury
(hypothesis #3).
Chapter 5: CONTRIBUTION TO ORIGINAL KNOWLEDGE
1, PMNs are regionalized in the acute period fdlowing a severe injury. In a murine
peritonitis model, PMN delivery to a second site when peritonitis is present is
inverseIy proportional to the degree of injury to the primary site,
2. PMN adherence is greatest in the presence of a localized injury (orchitis) than when
the injury is distant (peritonitis) or in the presence of a cornbined localized and
massive distant injury (pentonitis-orchitis).
3. Numbers of rolling PMNs were greatest in the absence of injury and reduced in the
presence of distant, massive injury (peritonitis) and virtually abolished in the
cornbined presence of a localized and a massive, distant injury (peritonitis-orchitis).
4, The PMNs rolled most rapidly in the absence of injury and at a slower velocity in the
presence localized injury and even slower in the combined presence of a localized and
massive, distant injury.
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