SLIT2 PREVENTS RENAL ISCHEMIA REPERFUSION INJURY IN MICE

Swasti Chaturvedi

A thesis submitted in conformity with the requirements for

the degree of MSc.

Institute of Medical Science

University of Toronto

Copyright by Chaturvedi Swasti 2011

ii

Slit2 Prevents Renal Ischemia Reperfusion Injury in Mice

Swasti Chaturvedi

Master of Medical Science (MSc)

Institute of Medical Science University of Toronto

2011

ABSTRACT

The Slit family of secreted proteins act as axonal repellents during embryogenesis. Slit2 via its

receptor, Roundabout-1, also inhibits chemotaxis of multiple leukocyte subsets. Using static and

microfluidic shear assays, we found that Slit2 inhibited multiple steps required to recruit

circulating neutrophils. Slit2 blocked capture and firm adhesion of human neutrophils to and

transmigration across inflamed primary vascular endothelial cells. To determine the response of

Slit2 in renal ischemia reperfsuion injury, Slit2 was administered prior to bilateral renal pedicle

clamping in mice. This led to significant decreases in both renal tubular necrosis score and

neutrophil infiltration. Administration of Slit2 also prevented elevation of plasma creatinine

following injury in a dose-dependent manner. Furthermore, administration of Slit2 did not

increase hepatic bacterial load in mice infected with L.monocytogenes infection. Collectively,

these data demonstrate Slit2 as an exciting therapeutic molecule to combat renal ischemia

reperfusion injury without compromising protective host innate immune functions.

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ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to my supervisor and mentor, Dr. Lisa Robinson, for

her constant encouragement and guidance. I also owe my gratitude to my program advisory

committee, Dr. James Scholey and Dr Philip Marsden for their encouragement, and constructive

criticism. I am very thankful to all the members of the Robinson lab, particularly Sajeda Patel,

Yi-wei Huang, Guang Ying Liu, Ilya Mukovozov and Harikesh Wong for their constant

assistance, feedback and engaging discussions. I would also like to thank Michael Woodside and

Paul Paroutis for their technical expertise and suggestions in optimization of the neutrophil flow

assays. I would further like to acknowledge the friendly and supportive individuals of the 4th

floor. My appreciation and gratitude also extends to all the volunteers who have donated blood

for this study. Most importantly, I would like to thank my Husband and my family for their

unconditional love and support.

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TABLE OF CONTENTS

DATA ATTRIBUTION ............................................................................................................................................. vi TABLE OF FIGURES ............................................................................................................................................... vii LIST OF TABLES ..................................................................................................................................................... viii LIST OF ABBREVIATIONS ...................................................................................................................................ix CHAPTER 1 .....................................................................................................................................................................1 INTRODUCTION ..........................................................................................................................................................1

1.1 Inflammation ........................................................................................................................................................1 1.1.1 Acute kidney injury ..................................................................................................................................1 1.1.2 The leukocyte migratory cascade ........................................................................................................9 1.1.3 Chemoattractants .................................................................................................................................... 12

1.2 The Neutrophil ................................................................................................................................................. 14 1.2.1 Capture and Rolling ............................................................................................................................... 14 1.2.2 Adhesion .................................................................................................................................................... 17 1.2.3 Transmigration ........................................................................................................................................ 21

1.3 Slit2: a guidance cue for migrating cells ................................................................................................ 31 1.3.1 Slit and Robo Structure ........................................................................................................................ 31 1.3.2 Slit and Robo expression ..................................................................................................................... 34 1.3.3 Slit and Robo Function ......................................................................................................................... 35 1.3.4 Slit2/Robo1 intracellular signal transduction............................................................................... 36 1.3.5 Slit/Robo in leukocyte trafficking .................................................................................................... 39

1.4 Rho GTPases: Rac and Cdc42.................................................................................................................... 40 1.4.1 Structure and Regulation ..................................................................................................................... 40 1.4.2 The role of Rho-family GTPases in regulation of the actin cytoskeleton ......................... 45 1.4.3 The role of Rho-family GTPases in regulation of innate immune function ..................... 46

1.5 Rationale, Hypothesis and Objectives ..................................................................................................... 46 1.5.1 Rationale .................................................................................................................................................... 46 1.5.2 Hypotheses ................................................................................................................................................ 47 1.5.3 Objectives .................................................................................................................................................. 47

CHAPTER 2 ............................................................................................................................................................. 49 MATERIAL AND METHODS ........................................................................................................................ 49 2.1 Reagents and antibodies ............................................................................................................................... 49 2.2 Slit2 expression and purification ............................................................................................................... 49 2.3 Immunohistochemistry .................................................................................................................................. 49 2.4 Isolation of primary human neutrophils ................................................................................................. 50 2.5 Neutrophil endothelial adhesion Assays ................................................................................................ 50 2.6 Hypoxia-reoxygenation (H/R) of endothelial cells ............................................................................ 50 2.7 Reverse transcriptase-polymerase chain reaction (RT-PCR) ......................................................... 51 2.8 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide (MTT) assays ................. 52 2.9 Neutrophil adhesion under hydrodynamic shear flow conditions ................................................ 53 2.10 Neutrophil transmigration assay ............................................................................................................. 53 2.11 Mouse model of renal ischemia-reperfusion injury ......................................................................... 54 2.12 Histologic Scores ......................................................................................................................................... 54

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2.13 Flow cytometry analysis ......................................................................................................................... 55 2.14 Murine infection with Listeria Monocytogenes ................................................................................ 56

CHAPTER 3 .................................................................................................................................................................. 57 RESULTS ....................................................................................................................................................................... 57 CHAPTER 4 .................................................................................................................................................................. 80 DISCUSSION & CONCLUSIONS ...................................................................................................................... 80 REFERENCES ............................................................................................................................................................. 86

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DATA ATTRIBUTION

The work presented here was performed in collaboration with a number of individuals. The

purification of Slit2 was conducted in the laboratory of Dr. Yves Durocher (National Research

Council Canada). Mouse acute kidney injury experiments were done in collaboration with Dr

Mark Okusa’s lab and his lab members Aman Bajwa and Liping. Yi-wei Huang did the RT-PCR

and washed Slit experiments. Grace Lam and Yi-Wei Huang jointly did the mouse L.

Monocytogenes experiment.

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TABLE OF FIGURES Figure 1.1: Schematic overview of early immune response occurring in renal ischemia

reperfusion injury. ................................................................................................................................................7 Figure 1.2: Leukocyte Adhesion Cascade ......................................................................................................... 12 Figure 1.3: Schematic of “inside out” and “outside in” signaling ........................................................... 20 Figure 1.4: Schematic diagram of leukocyte paracellular transendothelial migration ..................... 28 Figure 1.5: Schematic diagram of signaling events initiated downstream of ICAM-1 ligation ... 29 Figure 1.6: Schematic diagram of leukocyte transcellular migration ..................................................... 30 Figure 1.7: Structure of Mammalian Slit2 and Robo-1 proteins .............................................................. 33 Figure 1.8: Slit/Robo intracellular signal transduction .................................................................................. 38 Figure 1.9: Structure of Cdc42/Rac1 .................................................................................................................... 41 Figure 1.10: Regulation of Rho GTPases ........................................................................................................... 44 Figure 3.1: Slit2 expression increases in kidney tissue in renal IRI ......................................................... 65 Figure 3.2: Slit2 inhibits neutrophil adhesion to activated endothelial cells......................................... 67 Figure 3.3: HUVEC’s express Robo-1, 2 and 4 and Slit2 inhibits neutrophil-endothelial

adhesion by its action on neutrophils ......................................................................................................... 69 Figure 3.4: Slit2 inhibits neutrophil capture and adhesion to stimulated endothelium under flow

conditions .............................................................................................................................................................. 71 Figure 3.5: Slit2 reduced neutrophil transendothelial migration ............................................................... 73 Figure 3.6: Slit2 prevents renal dysfunction after IRI .................................................................................. 75 Figure 3.7: Slit2 improves acute tubular necrosis and reduces neutrophil infiltration in renal IRI

................................................................................................................................................................................... 77 Figure 3.8: Slit2 does affect hepatic bacterial load of L.monocytogenes. .............................................. 79

viii

LIST OF TABLES

Table 1 Acute kidney injury (AKI) classification in adults and children ..................................................4 Table 2 Summary of the primers used for RT-PCR ....................................................................................... 52

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LIST OF ABBREVIATIONS

AKI acute kidney injury ATP adenosine triphosphate CNS central nervous system DAG diacyl glycerol EGF epidermal growth factor EMT epithelial mesenchymal transition fMLP FormylMethionylLeucylPhenylalanine GDI GDP dissociation inhibitor GDNF glial derived neurotrophic factor GPCR G protein coupled receptor GAP GTP activating proteins GEF guanine nucleotide exchange factor HGF hepatocyte growth factor HUVEC human umbilical venous endothelial cells IL-1 interleukin-1 IL-8 interleukin-8 IGF-1 insulin like growth factor-1 IP3 inositol (1,4,5)-triphosphate IRI ischemia reperfusion Injury JAM junctional adhesion molecule LFA-1 lymphocyte function-associated Antigen-1 LRR leucine rich repeats Mac-1 macrophage-1 antigen MAPK mitogen activated protein kinase MCP-1 monocyte chemotactic protein-1 MLCK myosin light chain kinase MIP-1α macrophage inflammamtory protein- 1α MIP-1β macrophage inflammatory protein-1β MYPT-1 myosin-specific phosphatase-1 ICAM-1 intercellular adhesion molecule-1 PAI-1 plasminogen activator inhibitor-1 PAK P21-activated kinase PDGF platelet derived growth factor PECAM1 platelet /endothelial cell adhesion molecule 1 PLC phospholipase C ArfGAP ADP ribosylating factor GTPase activating protein PIX PAK interacting exchange factor PSGL-1 P selectin glycoprotein ligand-1 RANTES Regulated and normal T cell expressed and secreted TGF-β transforming growth factor-β TNF-α tumor necrosis factor-α VCAM-1 vascular cell adhesion molecule-1 VLA-4 vascular leukocyte adhesion molecule-4 VVOs vesiculo-vacuolor organelle

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CHAPTER 1

INTRODUCTION

1.1 Inflammation

1.1.1 Acute kidney injury

Acute kidney injury (AKI) is a complex, sometimes life threatening illness defined by the

presence of reduced glomerular filtration rate and azotemia (Star 1998). AKI develops in ~5%

of hospitalized patients and leads to significant morbidity, mortality and financial costs (Brady

and Singer 1995; Korkeila, Ruokonen et al. 2000; Bagshaw 2006). Despite significant

advances in understanding the cellular and molecular events that cause AKI, specific therapy

remains elusive and management is mainly supportive (Jo, Rosner et al. 2007).

For a long time, the lack of universal definition of AKI acted as a major barrier in the

ability to compare studies, predict clinical course, test therapeutic strategies and improve

outcome. To overcome this limitation, nephrology and critical care groups proposed empiric

working AKI definition in 2004 (Bellomo, Ronco et al. 2004), Table 1. The RIFLE (risk, injury,

failure, loss, and end stage renal disease) has been extensively studied and demonstrate that AKI

is an independent predictor of survival after correcting for co-morbities, complications and

severity of illness (Chertow, Soroko et al. 2006; Hoste and Kellum 2006). A modified pediatric

RIFLE (pRIFLE) has been proposed for use in children (Akcan-Arikan, Zappitelli et al. 2007),

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Table 1. In 2007, the Acute Kidney Injury Network (AKIN) developed a new streamlined

scoring system, based in part on the RIFLE criteria (Mehta, Kellum et al. 2007), Table 1.

Despite the use of standardised classifications, the diagnosis of AKI remains

problembatic due to reliance on two functional abnormalities: changes in serum creatinine (a

marker of glomerular filtration rate or GFR) and oliguria. Both of these changes appear late in

the course of injury and have several significant shortcomings (Al-Ismaili, Palijan et al. 2011).

For example: serum creatinine may not change until 25-50% of kidney function is already lost;

serum creatinine concentarion varies with hydration status, muscle mass, age and gender; at

lower GFR, serum creatinine will overestimate renal function due to tubular secretion of

creatinine; different methods used to measure creatinine (enzymatic vs. Jaffe reaction) give

different values and serum creatinine cannot be used for assessment of kidney function in

patients on dialysis as serum creatinine is easily dialyzed (Al-Ismaili, Palijan et al. 2011).

Ideally a biomarker for AKI should be up-regulated shortly after injury and be independent of

the GFR besides being highly sensitive and specific.

There are several novel AKI biomarkers under evaluation in humans (Devarajan 2011).

The most promising of these biomarkers are neutrophil gelatinase-associated lipocalcin (NGAL),

kidney injury molecule-1 (KIM-1), interleukin 18 (IL-18), and serum cystatin C (Mak 2008).

Human NGAL is a 25kDa protein covalently bound to neutrophil gelatinase and is normally

expressed at very low levels in kidney, lungs, stomach and colon. Its expression increases in

epithelial injury and NGAL is a useful early biomarker of AKI in a wide range of clinical

settings (Haase, Bellomo et al. 2009). KIM-1 is transmembrane glycoprotein expressed in low

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levels by normal kidney. It is highly up-regulated in proximal tubule cells after an ischemic or

nephrotoxic AKI and its proteolytically cleaved extracellular domain is detected in urine

(Devarajan 2011). IL-18 is a pro-inflammatory chemokine produced systemically and in renal

tubular epithelial cells in AKI (Leslie and Meldrum 2008). Cystatin C is a cysteine protease

inhibitor synthesized by nucleated cells and released into the blood at a relatively constant rate.

It is freely filtered by the glomerulus, completely absorbed by the proximal tubule, and not

secreted. Its levels are not significantly affected by age, gender, race or muscle mass, thus

making it a better predictor of glomerular function than serum creatinine (Dharnidharka, Kwon

et al. 2002).

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Table 1 Acute kidney injury (AKI) classification in adults and children Adult Pediatric

AKIN AKIN/RIFLE RIFLE pRIFLE

Stage S.Cr Urine output Class Serum Cr or

GFR

Class eCCl by

Schwartz

Urine

output

I ↑ S.Cr > 0.3

mg/dl or ↑S.Cr

> 150-200%

< 0.5ml kg per h x

6 h

Risk ↑S.Cr >150% or

GFR ↓ by 25%

Risk eCCl ↓ by

25%

< 0.5ml kg

per h x 8 h

II ↑ S.Cr by

200-300%

< 0.5ml kg per h

> 12 h

Injury ↑S.Cr >200% or

GFR ↓ by 50%

Injury eCCl ↓ by

25%

< 0.5ml kg

per h x 16 h

III ↑ S.Cr > 300%

or S.Cr > 4.0

mg/dl with

acute rise of

atleast 0.5

mg/dl

< 0.3ml kg per h

>24h or anuria for

> 12h

Failure ↑S.Cr >300% or

S.Cr > 4.0 mg/dl

with acute rise of

atleast 0.5 mg/dl

or GFR ↓ by

>75%

Failure eCCl ↓ by

25%

< 0.3ml kg

per h for24h

or anuria for

> 12h

Loss Failure> 4 weeks Loss Failure> 4

weeks

ESRD Failure > 3

months

ESRD Failure > 3

months

Cr- creatinine; GFR- Glomerular filtration rate; eCCl- estimated creatinine clearance; ESRD-end

stage renal disease

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There are many causes of AKI including ischemia reperfusion injury (IRI), glomerulonephritis,

interstitial nephritis, nephrotoxins, vascular lesions, renal developmental dysplasia, hypoplasia,

obstructive uropathy, infection and sepsis (Lameire, Van Biesen et al. 2005). Ischemia

reperfusion injury (IRI) is the leading cause of AKI in both native and transplanted kidneys (Star

1998; Devarajan 2006). Blood flow to the human adult kidney comprises ~ 25% of the cardiac

output with the renal cortex receiving the majority of the renal blood supply (Janssen, Beekhuis

et al. 1995). Blood flow to the renal medulla is via efferent arterioles of juxtamedullary

glomeruli which give rise to vasa recta. During the ischemic phase of IRI, there is regional

reduction in blood flow with the outer renal medulla being the worst affected (Okusa 2002).

Reduced blood flow and oxygen delivery leads to ATP depletion, impaird oxidative metabolism,

generation of reactive oxygen species and inhibition of Na/K ATPase pump which in turn

results in epithelial and endothelial cell dysfunction, swelling and death (Okusa 2002; Legrand,

Mik et al. 2008). There is increased renal synthesis of pro-inflammatory cytokines and

chemokines, most notably tumor necrosis factor-α (TNF-α) and CXCL1 (interleukin-8; IL-8)

(Baggiolini, Dewald et al. 1994; Daemen, van de Ven et al. 1999; Donnahoo, Meng et al. 1999;

Donnahoo, Meldrum et al. 2000; Furuichi, Wada et al. 2002; Furuichi, Wada et al. 2003).

Ischemia also activates the transcription factors including NF-Kb, heat shock factor-1, and

hypoxia-inducible factor-1 (HIF-1) (Eickelberg, Seebach et al. 2002; Cao, Ding et al. 2004).

HIF-1 in turn regulates critical biological processes important for survival of hypoxic cells such

as anaerobic glycolysis, oxygen delivery through increased angiogenic growth factor production

and erythropoiesis, as well as cellular proliferation and apoptosis (Gunaratnam and Bonventre

2009).

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The reperfusion phase is characterised by leukocyte recruitment, endothelial cell

activation and generation of inflammatory and vasoactive mediators which perpuate the tissue

injury. These chemoattractants both attract and activate leukocytes and blocking these individual

inflammatory cytokines leads to partial protection in mice subjected to renal IRI (Figure 1.1).

For example, administration of anti-TNF-α antibodies improves renal injury following IRI

(Daemen, van de Ven et al. 1999). Similarly neutralisation of IL-8 ameliorates renal damage

after IRI (Miura, Fu et al. 2001). The early reperfusion phase is characterised by a massive

influx of circulating neutrophils to the injured kidney (Figure 1.1) (Linas, Shanley et al. 1988;

Hellberg and Kallskog 1989; Klausner, Paterson et al. 1989; Willinger, Schramek et al. 1992;

Bonventre and Weinberg 2003). Indeed, neutrophil infiltration has been demonstrated in animal

models of ischemic acute kidney injury and in renal biopsies from patients with acute kidney

injury (Solez, Morel-Maroger et al. 1979; Linas, Shanley et al. 1988; Hellberg and Kallskog

1989). The recruited neutrophils then becomes activated and releases cytokines, chemokines,

eicosanoids, proteases, and reactive oxygen species that perpetuate and promote the

inflammatory damage (Bonventre and Weinberg 2003; Hayama, Matsuyama et al. 2006). Not

surprisingly, neutrophil depletion and therapies targeting neutrophil migration cues have been

found to be protective in renal IRI (Klausner, Paterson et al. 1989; Chiao, Kohda et al. 1997).

Macrophages, T lymphocytes and dendritic cells are recruited in the later phases of

inflammation (1-5 days after reperfusion) and exacerbate inflammation by activating adaptive

immune responses (Figure 1.1) (Rabb, Daniels et al. 2000; De Greef, Ysebaert et al. 2001; Day,

Huang et al. 2005; Jo, Sung et al. 2006; Schlichting, Schareck et al. 2006; Loverre, Capobianco

et al. 2007). Individually blocking the recruitment of monocyte/macrophages or T cells partially

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protects against IRI. Systemic macrophage depletion results in reduced inflammation and less

tubular damage and apoptosis (Jo, Sung et al. 2006). Mice lacking CD4+/CD8+ T lymphocytes,

or cell adhesion receptors on T lymphocytes that allow them to adhere to injured endothelium,

are partially protected from IRI (Rabb, Daniels et al. 2000). Adherent leukocytes, platelets and

red blood cells also cause capillary plugging and further compromise the microvascular flow in

the vasa recta of the outer medulla (Okusa 2002).

Figure 1.1: Schematic overview of early immune response occurring in renal ischemia

reperfusion injury.

In experimental ischemic acute kidney injury models, an ischemic insult precedes the reperfusion

phase. During the reperfusion phase, leukocytes, including neutrophils, macrophages,

lymphocytes, and dendritic cells are recruited to the injured kidney. Once recruited, these

leukocytes then exacerbate the inflammatory damage.

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After IRI, repair is initiated by dedifferentiated epithelial cells that express vimentin, an

embryonic marker for multipotent renal mesenchymal cells (Devarajan 2006). Besides vimentin,

several other embryonic genes are markedly induced during recovery phase. These include Wnt-

4, transcription factor Ets-1 and leukemia inhibitory factor (Yoshino, Monkawa et al. 2003;

Terada, Tanaka et al. 2005). The change from differentiated phenotype to less differentiated

phenotype recapitulates renal development (Hammerman 2000). The source of these cells is not

entirely clear and is an area of intense research. A small subset may be bone-marrow derived

cells (Duffield, Park et al. 2005; Krause and Cantley 2005). Another potential source of these

cells could be resident stem cells with tubulogenic potential (Maeshima, Sakurai et al. 2006).

However, the dedifferentiated resident tubular epithelial cells are perhaps the most important

source of these reparative cells (Lin, Moran et al. 2005; Stokman, Leemans et al. 2005).

In the next phase of recovery, there is upregulation of genes encoding growth factors,

such as hepatocyte growth factor (HGF), insulin like growth factor-1 (IGF-1) and fibroblast

growth factor on these dedifferentiated cells which migrate and rapidly proliferate to replace the

irreversibly damaged tubular epithelial cells (Nigam and Lieberthal 2000). The final phase of

the repair process is redifferentiation of the epithelial cells where most tubules regain the

essential functions and re-establish cell polarity (Bonventre and Weinberg 2003). However,

depending on the severity of the initial damage, recovery is frequently incomplete and may lead

to progressive kidney dysfunction due to progressive interstitial fibrosis, peritubular capillary

loss causing tubular damage, and loss of functioning nephrons (Azuma, Nadeau et al. 1997;

Okusa, Chertow et al. 2009; Waikar and Winkelmayer 2009). Platelet-derived growth factor

(PDGF) and transforming growth factor-β (TGF-β)-dependent cellular signaling cascades are the

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key mediators of fibroblast proliferation, epithelial-to-mesenchymal-transition (EMT), enhanced

matrix synthesis and reduced matrix turnover leading to enhanced inter-cellular matrix

deposition (Tang, Ulich et al. 1996; Chai, Krag et al. 2003; Kalluri and Neilson 2003; Zeisberg,

Hanai et al. 2003). Epithelial-mesenchymal transition is a process by which injured tubular

epithelial cells undergo transition to a matrix producing fibroblast and myofibroblast thereby

contributing to tissue fibrogenesis (Iwano, Plieth et al. 2002; Liu 2004). Thus, therapies

inhibiting this process or perhaps even stimulating the conversion of myofibroblast back into

epithelium again (mesenchymal-to-epithelial transition) may help delay the development of

chronic kidney disease(Liu 2004; Zeisberg, Shah et al. 2005).

The recruited leukocytes not only exacerbate acute injury but also accelerate renal

fibrosis and chronic kidney disease through release of fibrogenic growth factors including TGF-

β, PDGF, plasminogen activator inhibitor-1 (PAI-1) and endothelin-1 (Isaka, Fujiwara et al.

1993; Bottinger and Bitzer 2002; Hirschberg and Wang 2005; Floege, Eitner et al. 2008).

Inhibition of the early leukocyte infiltration reduces not only the acute inflammatory damage

also ameliorates the development of renal tubulointerstitial fibrosis and preserves renal function

(Forbes, Hewitson et al. 2000; Persy, Verhulst et al. 2003; Furuichi, Gao et al. 2006).

1.1.2 The leukocyte migratory cascade

Following IRI, leukocytes are recruited to the injured tissue in a series of precisely

coordinated dynamic interactions with vascular endothelial cells. The classical leukocyte

migratory cascade involves three main steps: leukocyte capture and rolling, adhesion to the

activated endothelium and eventual leukocyte transendothelial migration (Fig. 1.2). In the first

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step, circulating leukocytes are captured by and roll along the endothelial surface, a process

mediated mainly by selectins (Chamoun, Burne et al. 2000). Selectin deficiency or blockade

reduces acute renal injury as well as late renal dysfunction and tissue damage in animal models

of renal IRI (Takada, Nadeau et al. 1997; Singbartl, Green et al. 2000).

The next step is leukocyte arrest and firm adhesion on activated endothelium. This

process is triggered by chemokines and other chemoattractants and mediated by the binding of

leukocyte integrins to the immunoglobulin superfamily members intercellular adhesion

molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), expressed on

endothelial cells (Campbell, Hedrick et al. 1998). Chemokines, including IL-8, are secreted by

activated endothelial cells (Huber, Kunkel et al. 1991; Springer 1995). Platelets also secrete

chemokines, such as CC-chemokine ligand 5 (CCL5; RANTES), CXC-chemokine ligand 4

(CXCL4) and CXCL5, thus triggering leukocyte arrest on the inflamed endothelial cells (von

Hundelshausen, Koenen et al. 2005; Weber 2005). Binding of chemokines to their specific G

protein coupled receptors (GPCR’s), expressed on the surface of leukocytes leads to a rapid

increase in integrin affinity due to a conformational change resulting in increased ligand binding

energy and decreased ligand dissociation rate. Binding of chemokines to their receptors on the

surface of leukocytes also increases the integrin valency (the density of integrins per area of

plasma membrane involved in adhesion). This is referred to as inside-out signalling (Figure 1.3)

(Shamri, Grabovsky et al. 2005). In contrast, outside-in signaling refers to downstream effects of

integrin ligand binding and contributes to adhesion stability of leukocytes (Figure 1.3) (Kinashi

2005). Adhesion is the critical step in leukocyte tissue infiltration and ICAM-1 blockade has

been shown to be protective in renal IRI (Kelly, Williams et al. 1994).

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Following firm arrest on activated endothelium, leukocytes migrate across the three

vascular barriers: endothelial cells, the underlying basement membrane, and the pericyte sheath

(Nourshargh and Marelli-Berg 2005). Leukocytes can cross the endothelium either through the

endothelial cell junctions (paracellular route) or directly through an endothelial cell (transcellular

route) (Engelhardt and Wolburg 2004). Although the leukocyte adhesion cascade has been

divided into distinct steps, these are not temporally exclusive, but instead work together to

achieve the desired effect of leukocyte arrest and transendothelial migration. In the past decade,

new insights have been gained into the signaling events that underlie integrin activation, post-

adhesion strengthening of leukocyte attachment, and the molecules involved in transendothelial

migration.

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Figure 1.2: Leukocyte Adhesion Cascade

Leukocytes are recruited to the sites of inflammation in a series of coordinated interactions with

endothelial cells (ECs) lining the vessel wall. The classical leukocyte adhesion cascade involves

three main steps: leukocyte capture and rolling, activation and arrest, and transmigration. ECM-

Extracellular Matrix.

1.1.3 Chemoattractants

A variety of chemoattractants recruit leukocytes to sites of injury and inflammation.

Chemoattractants are divided in two groups. The “classical” chemoattractants include bacterial

derived N-formyl peptides, complement factors such as C3a and C5a, leukotrienes such as

leukotriene B4 and platelet activating factor. These chemoattractants non-specifically recruit

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leukocytes to inflammatory foci. On the other hand, chemokines are a family of small, secreted

peptides of 70 to 80 amino acids that specifically recruit leukocyte subsets to the inflammatory

site. There are approximately 50 human chemokines which are divided into 4 families (CC,

CXC, CX3C and C) according to positioning of first 2, highly conserved cysteines of the amino

acid sequence (Zlotnik and Yoshie 2000).

Most chemokines belong to the CXC (α) or the CC (β) family. The CXC chemokines have

one amino acid residue between the two conserved N-terminal cysteine residues and act

primarily to recruit neutrophils. CXCL8 (IL-8) is the prototypic chemokine of this group. In CC

chemokines, the first two of four N-terminal cysteine residues are adjacent to each other and

attract mononuclear cells to sites of inflammation. These chemokines include CCL2 (monocyte

chemoattractant protein-1, MCP-1), CCL3 (macrophage inflammatory protein-1α, MIP-1α),

CCL4 (macrophage inflammatory protein 1-β, MIP-1β) and CCL5 (regulated and normal T cell

expressed and secreted, RANTES). The CX3C chemokine family has only one member CX3CL1

(fractalkine) (Bazan, Bacon et al. 1997). The fourth family of C (γ) chemokines is composed of

only 2 members: XCL1 (chemokine (C motif) ligand or lymphotactin) and XCL2 (Chemokine

(C motif) ligand 2), which are lymphocyte specific chemokines (Kelner, Kennedy et al. 1994).

Most chemokines are secreted, while two, CX3CL1 and CXCL16, also exist as

transmembrane molecules which act as both chemoattractants and adhesion molecules (Charo

and Ransohoff 2006). The specific effects of chemokines on different leukocyte subsets are

mediated by binding to serpentine G-protein coupled receptors (GPCR’s). Likewise, other

soluble chemoattractants such as complement factors and leukotrienes also bind to and activate

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serpentine GPCR on the surface of neutrophils. Coupled with the differences in receptor

expression on leukocyte subsets, this diversity allows the immune system to activate specific

leukocyte subsets during specific inflammatory conditions/processes.

1.2 The Neutrophil

Polymorphonuclear leukocytes or neutrophils are the key players in innate immune responses

and are the first line of defense against pathogens (Segal 2005). Indeed neutropenias can lead to

severe infection and sepsis (Bodey, Buckley et al. 1966). Neutrophils are rapidly recruited to the

site of inflammation by chemokines or bacterial products (Nathan 2006). Once recruited to the

site of injury, neutrophils generate chemotactic signals to attract monocytes and dendritic cells.

They also generate proteolytic enzymes and reactive oxygen species (Nathan 2006). These

responses help to control infection and to promote tissue repair, but when unchecked can also

lead to excessive tissue injury. Neutrophils have been implicated in several inflammatory

diseases such as rheumatoid arthritis, chronic obstructive airway disease and ischemia

reperfusion injury (Linas, Whittenburg et al. 1995; Pillinger and Abramson 1995; Stockley

2002). In order to understand the balance between the beneficial and deleterious effects of

impact of neutrophil mediated destruction of pathogens and host tissue it is important to

understand the steps involved in neutrophil recruitment.

1.2.1 Capture and Rolling

Leukocyte capture and rolling is mediated by cell surface glycoproteins called selectins (Tedder,

Steeber et al. 1995). The selectin family of leukocyte adhesion molecules consists of three

known members: L-selectin, P-selectin, and E-selectin (Bevilacqua and Nelson 1993). L-selectin

15

is expressed on a multiple leukocyte subsets, including neutrophils. P-selectin is constitutively

stored in the Weibel-Palade bodies of endothelial cells and in the alpha granules of platelets (Ley

2001). E-selectin is expressed on endothelial cells activated by the pro-inflammatory cytokines

interleukin-1 (IL-1) and TNF-α (Ley 2003). All three selectins have a extracellular region

composed of an N-terminal lectin domain, an epidermal growth factor (EGF) domain, two to

nine short consensus repeat (SCR) units homologous to domains found in complement binding

proteins, a transmembrane domain and a cytoplasmic domain (Patel, Cuvelier et al. 2002).

Several ligands of L-selectin have been identified. These include: P-selectin glycoprotein

ligand-1 (PSGL-1), GlyCAM-1, MAdCAM-1 and CD34 (Patel, Cuvelier et al. 2002). P-selectin

ligands include PSGL-1 and CD24. Ligands proposed for E-selectin include PSGL-1, E-selectin

ligand-1 (ESL-1) and CD44 (Sperandio 2006) PSGL-1 is the most important P-selectin and L-

selectin ligand. E-selectin also binds to PSGL-1, however its major ligand remains to be

discovered (Chamoun, Burne et al. 2000). PSGL-1 knockout mice show delayed neutrophil

recruitment and moderate neutrophilia (Yang, Hirata et al. 1999; Xia, Sperandio et al. 2002).

The binding of leukocyte L-selectin to PSGL-1 facilitates secondary leukocyte capture by

adherent leukocytes (Sperandio, Smith et al. 2003).

Generation of mice deficient in individual selectin molecules has provided important

information about their individual roles. L-selectin knockout mice have the most severe

phenotype and display impaired leukocyte recruitment in inflammation (Arbones, Ord et al.

1994; Tedder, Steeber et al. 1995). Lymphocytes from these mice fail to bind to high endothelial

venules of the lymph nodes and there is reduced cellularity in peripheral lymph nodes (Arbones,

16

Ord et al. 1994; Tedder, Steeber et al. 1995). P-selectin deficient mice have mild neutrophilia

and display reduced injury in models of atherosclerosis, transplantation and IRI (Connolly,

Winfree et al. 1997; Johnson, Chapman et al. 1997; Naka, Toda et al. 1997). E-selectin deficient

mice display no impairment in leukocyte recruitment in inflammation and contact

hypersensitivity models but have higher rates of mortality secondary to bacteremia than wild

type mice when exposed to Streptococcus pneumoniae (Labow, Norton et al. 1994; Munoz,

Hawkins et al. 1997).

Hydrodynamic shear flow is a critical determinant of neutrophil endothelial interactions within a

given vascular bed. (Finger, Puri et al. 1996). Leukocytes must experience a minimum

threshold wall shear stress to tether and roll on selectins. As the wall shear stress is increased,

the number of rolling cells increases initially and then decreases in a biphasic manner. L-selectin

requires threshold shear stress to support leukocyte rolling on P-selectin glycoprotein ligand-1

(PSGL-1). The bond lifetimes initially increases with force, indicating the presence of catch

bonds. After reaching a maximum, the lifetime decreases with force, indicating slip bonds.

(Marshall, Long et al. 2003). Besides selectins, integrins also take part in rolling. The co-

expression of ICAM-1 with L-selectin ligand fucosyltransferase VII in human vascular

endothelial cell line led to increased leukocyte rolling and slower rolling velocities (Kadono,

Venturi et al. 2002). Monocytes also roll on immobilized VCAM-1 via very late antigen 4

(VLA-4; α4β1-integrin) engagement (Berlin, Bargatze et al. 1995). Additionally, slow rolling in

vivo was shown not only to require selectins but also β2 integrins; LFA-1 and macrophage

receptor 1 (Mac-1; αMβ2-integrin). LFA-1 and Mac-1 knockout mice display elevated leukocyte

rolling velocity and subsequent reduced leukocyte adhesion (Dunne, Ballantyne et al. 2002).

17

1.2.2 Adhesion

Firm adhesion of neutrophils on activated endothelial cells is a pre-requisite for eventual

neutrophil transmigration towards inflammatory foci. This firm adhesion is integrin-mediated.

Integrins are large transmembrane glycoproteins that connect the extracellular environment with

the cytoskeleton of leukocytes. Integrins exist as heterodimers of α and β subunits. The β2

family of integrins is expressed only on leukocytes and play an important role in neutophil

adhesion. The β2 integrins are composed of a variable α subunit (CD11a, -b, and -c) and a

common β subunit (CD18). The two most important integrins on neutrophils are CD11a/CD18

(LFA-1, lymphocyte function associated antigen-1) and CD11b/CD18 (Mac-1 or complement

receptor 3). Both LFA-1 and Mac-1 bind to ICAM-1. Mac-1 also binds to fibrinogen, heparin,

factor X and ic3b fragment of complement factor 3 (Diamond, Staunton et al. 1991).

Following binding of soluble chemoattractants to their GPCR on the surface of

leukocytes, leukocytes become activated, initiating rapid inside-out signaling which ultimately

leads to integrin activation. The intracellular signaling pathways from GPCR activation to

integrin activation are ill understood and are shown in Figure 1.3 A. Phosphotidyl-inositol-3-

kinase (PI3K) and phospholipase C (PLC) are activated downstream of GPCR binding. PI3K

activation leads to activation of cytohesin-1(Kinashi 2005). Cytohesin-1 in turn leads to integrin

activation (Weber, Weber et al. 2001). Activation of PLC leads to production of inositol

triphosphate (IP3) and diacylglycerol (DAG), and an increase in intracellular calcium. This

calcium flux and the production of DAG activates guanine nucleotide exchange factors (GEFs)

such as CALDAG and Vav-1, which in turn activate RAP-1 and Rho-family GTPases (Anthis

and Campbell ; Gakidis, Cullere et al. 2004). RhoA also activates protein kinase-ζ which leads

18

to integrin activation (Giagulli, Scarpini et al. 2004). The final common pathway involving

integrin activation requires association actin binding protein talin, and lead to integrin affinity

up-regulation (Sampath, Gallagher et al. 1998; Vielkind, Gallagher-Gambarelli et al. 2005;

Wegener, Partridge et al. 2007).

Talin is a 250 kDa cytoskeletal protein that is a key player in integrin activation and links

intergrin to actin cytoskeleton. Talin has an N- terminal head region and an elongated helical

rod. The head contains a FERM (protein 4.1, ezrin, radixin and moesin) domain that directly

associates with NPxY motif (where x denotes any amino acid) of the β-tail domain of the

integrins (Moser, Legate et al. 2009). When the head of talin containing FERM domain is

overexpressed, it increases adhesion of LFA-1 expressing cells to ICAM-1(Kim, Carman et al.

2003). The C-terminal end of talin has a F-actin binding site and thus provides a direct link

between β-integrin tail and actin cytoskeleton (Moser, Legate et al. 2009).

In addition to GPCR-dependent inside-out signaling, binding of ligands to integrins also

induces outside-in signaling cascades, Figure 1.3 B (Hynes 1992). Paxillin is a signalling

adaptor molecule which binds to integrins (Schaller 2001). Ligand induced integrin clustering

activates several tyrosine kinases including FAK and Src-family kinases. These phosphorylate

paxillin allowing the recruitment of downstream effectors, including ADP-ribosylating factor

GTPase activating protein (ArfGAP) which restricts Rac activation to the laeading edge of the

polarising cell (Nishiya, Kiosses et al. 2005). Paxillin also recruits PAK interacting exchange

factor (PIX) which activates Cdc42 (DeMali, Wennerberg et al. 2003). Src-family kinases also

activate Vav1 which leads to activation of Rho GTPases (DeMali, Wennerberg et al. 2003).

19

Activation of Rho GTPases leads to mobilization of the actin cytoskeleton and formation of

adhesive contacts. Both GPCR- induced inside-out signaling and ligand-induced outside-in

signaling link β2 integrins on the neutrophil surface to adhesion molecules on the endothelium,

and/or extracellular matrix.

20

Figure 1.3: Schematic of “inside out” and “outside in” signaling A) Phosphotidyl-inositol-3-kinase (PI3K) and phospholipase C (PLC) are activated downstream

of GPCR binding. PI3K activation leads to activation of cytohesin-1 which in leads to integrin

activation. Activation of PLC leads to activaton of guanine nucleotide exchange factors (GEFs),

such as CALDEG and Vav, and results in the recruitment and activation of Rap-1 and Rho-

family GTPases including Rho A. Rho A then activates PKC-ζ. The final common pathway

involving integrin activation requires association actin binding protein talin, and lead to integrin

affinity up-regulation B) Ligand-binding induces integrin clustering and intracellular signaling.

Srcfamily kinases activate Rho GEFs either directly (Vav1) or through Paxillin (Pix). Paxillin

also recruits ArfGAP, which suppresses Rac activity. Modulation of Rho GTPase activity allows

for actin remodeling and development of adhesive contacts.

21

1.2.3 Transmigration

Transmigration is the final step in neutrophil emigration into the inflamed tissue.

Emigrating neutrophils cross three distinct barriers which include endothelial cells, the

underlying basement membrane and the pericytes (Voisin, Woodfin et al. 2009).

1.2.3.1 Crawling:

During transendothelial transmigration, adherent leukocytes first crawl laterally to seek

preferred sites of transendothelial migration, namely intercellular junctions. This intravascular

crawling is Mac-1 and ICAM-1- dependent and its blockade seems to reduce overall

transmigration (Phillipson, Heit et al. 2006). Mac-1 deficient mice show reduced transmigration

which occurs preferentially through the transcellular as opposed to the paracellular route

(Phillipson, Heit et al. 2006). The mechanisms that mediate the transition from firm adhesion to

intraluminal crawling have not been fully elucidated. However, a role for the Rho-GEF Vav-1

has been recently suggested. Analysis of leukocyte responses in Vav-1 deficient mice by

intravital microcopy demonstrated defective neutrophil arrest, reduced intra-luminal crawling

and reduced transmigration (Phillipson, Heit et al. 2009).

Endothelial cells are actively involved in facilitating leukocyte transmigration.

Engagement of endothelial ICAM-1 by neutrophil LFA-1 induces formation of microvilli-like

endothelial projections rich in ICAM-1, VCAM-1, cytoskeletal “linker” ERM (ezrin, radixin and

moesin) proteins and cytoskeletal components (such as α-actinin, vinculin and talin-1). These

“docking structures” or “transmigratory cups” then embrace the leukocytes and guide the

leukocyte transmigration via paracellular or transcellular route (Barreiro, Yanez-Mo et al. 2002;

22

Carman and Springer 2004). Rho GTPases activate the ERM proteins and are involved in

generating and maintaining these docking structures (Barreiro, Yanez-Mo et al. 2002).

1.2.3.2 Paracellular Migration:

During paracellular migration leukocytes must pass through endothelial cell-cell

junctions (Figure 1.4). Multiple endothelial-leukocyte adhesion molecules are engaged and

cluster by leukocyte binding, tranducing intracellular signals which ultimately conclude in

cytoskeletal remodeling and junctional disruption. These include E-selectin, ICAM-1 and

VCAM-1(Hordijk 2006). Besides being involved in leukocyte rolling, E-selectin also transduces

signals into the endothelial cell. E-selectin- dependent leukocyte adhesion leads to activation of

the mitogen-activated protein kinase (MAPK) signaling cascade in human umbilical venous

endothelial cells thus suggesting its role in transendothelial migration (Hu, Kiely et al. 2000; Hu,

Szente et al. 2001).

ICAM-1 is the key integrin ligand involved in leukocyte transendothelial migration,

particularly of neutrophils and lymphocytes. Ligation of leukocyte integrins with endothelial

ICAM-1 is associated with increased intracellular calcium and activation of Rho GTPases

(Huang, Manning et al. 1993; Adamson, Etienne et al. 1999; Etienne-Manneville, Manneville et

al. 2000; Thompson, Randi et al. 2002; Greenwood, Amos et al. 2003). Figure 1.5 describes the

signaling events downstream of ICAM-1 engagement. Rho A activates Rho-kinases (ROCK),

which subsequently inactivate myosin-specific phosphatase-1 (MYPT-1) through

phosphorylation of its regulatory unit (Riento and Ridley 2003). This leads to increased

phosphorylation of myosin light chain (MLC) and triggers the formation of stress fibres and

actomyosin contractility (Chrzanowska-Wodnicka and Burridge 1996; Riento and Ridley 2003).

23

This in turn leads to increased endothelial cell contractility and opening of inter-endothelial

contacts thus facilitating leukocyte transendothelial migration (Garcia, Verin et al. 1998; Saito,

Minamiya et al. 1998). Inhibition of Rho A activity is associated with reduced leukocyte

migration (Adamson, Etienne et al. 1999; Wojciak-Stothard, Williams et al. 1999). In addition

to activating RhoA, ICAM-1 cross-linking also leads to phosphorylation of many endothelial cell

proteins including paxillin, FAK (focal adhesion kinase), Src, p38 MAPK, ezrin, cortactin and

VE-cadherin (Etienne, Adamson et al. 1998; Wang and Doerschuk 2001; van Buul and Hordijk

2004; Yang, Kowalski et al. 2006; Allingham, van Buul et al. 2007; Wittchen 2009). Cortactin

(cortical actin binding protein) is a scaffold protein that mediates actin remodeling via Arp 2/3

complex (Weaver, Young et al. 2003). Cortactin further links ICAM-1 engagement during

leukocyte adhesion with downstream clustering of E-selectin and ICAM-1 on the endothelial cell

surface (Yang, Kowalski et al. 2006). Tyrosine phosphorylation of the adherens junction protein

VE-cadherin causes endothelial cell junctional disassembly (Lampugnani, Corada et al. 1997;

Garcia, Schaphorst et al. 2000). FAK phosphorylation promotes focal adhesion turnover and

perhaps reduced focal adhesions leading to increased transmigration (Mullaly, Moyse et al. 2002;

Millan and Ridley 2005).

VCAM-1 mediates adhesion of leukocytes expressing VLA-4 to the endothelium.

VCAM-1 mediated Rac activation leads to ROS generation via NADPH oxidase and transient

adherens junction disruption (van Buul and Hordijk 2004). Blockade of VCAM-1 alone is not

sufficient to significantly reduce monocyte transmigration, but blocking both ICAM-1 and

VCAM-1 has an additive effect (Ronald, Ionescu et al. 2001).

24

Besides endothelial-leukocyte adhesion molecules, many junctional molecules are

involved in paracellular migration. The evidence in support of the role of these molecules in

leukocyte transendothelial migration comes from use of neutralizing antibodies and gene-

targeted mice. There is redistribution of these junctional molecules in the inflamed endothelial

cells that favours transendothelial migration. The molecules involved include junctional

molecules belonging to immunoglobulin superfamily members, platelet endothelial cell adhesion

molecule-1 (PECAM-1), junctional adhesion molecules (JAMs) and endothelial cell-selective

adhesion molecule (ESAM) as well as non-immunoglobulin molecule CD99 (Wegmann, Petri et

al. 2006; Bixel, Petri et al. 2007; Woodfin, Reichel et al. 2007; Woodfin, Voisin et al. 2009).

PECAM-1 is expressed on endothelial cells, platelets, neutrophils, monocytes and some T

cells (Newman, Berndt et al. 1990). Blocking antibodies to PECAM-1 inhibit neutrophil

transendothelial migration in vivo (Vaporciyan, DeLisser et al. 1993). PECAM-1-deficient mice,

however showed only limited problems in model of inflammation (Duncan, Andrew et al. 1999).

Later studies have shown that this may be dependent on the mice strain (C57/BL6) used.

C57/BL6 is uniquely able to compensate for the loss of PECAM function unlike other mice

strains (Schenkel, Chew et al. 2004).

Junctional adhesion molecules (JAM-A, JAM-B, JAM-C) are expressed in leukocytes,

platelets, endothelial and epithelial surfaces. JAMs interact with tight junction associated

proteins including zona occludens-1 (ZO-1), afadin (AF-6) and Multi-PDZ Domain Protein-

1(MUPP-1) via their intra-cellular domain (Ebnet, Suzuki et al. 2004). JAMs also engage with

leukocyte integrins via their extracellular domain; JAM-A binds to LFA-1, JAM-B associates

25

with VLA-4 and JAM-C interacts with Mac-1 (Choi, Santoso et al. 2009). Antibody blocking

JAM-A inhibits neutrophil recruitment in mice meningitis model (Del Maschio, De Luigi et al.

1999). JAM-A deficient neutrophils show reduced transendothelial migration in murine models

of peritonitis and cardiac IRI (Corada, Chimenti et al. 2005). JAM-C deficient mice display

delayed neutrophil recruitment, increased susceptibility to infection, growth retardation and poor

survival (Imhof, Zimmerli et al. 2007).

ESAM-1 is expressed on endothelial tight junctions and platelets. ESAM-1 deficient

mice display reduced neutrophil transendothelial migration in murine models of peritonitis

(Wegmann, Petri et al. 2006). CD99 is a heavily glycosylated protein expressed by most

leukocytes and endothelial cells (Muller 2009). CD99 blocking antibodies inhibit

transendothelial migration in vitro (Schenkel, Mamdouh et al. 2002). Different junctional

molecules mediate leukocyte transmigration in either a stimulus-specific or leukocyte-specific

manner. For instance, PECAM-1 and JAM-A mediate leukocyte transmigration in response to

interleukin-1β (IL-1β) but not TNF (Nourshargh, Krombach et al. 2006). ESAM-1 does not

show a stimulus specific role but appears to mediate neutrophil rather than T cell transmigration

(Wegmann, Petri et al. 2006).

Finally, adherens junction proteins play important role in transendothelial migration.

Adherens junctions are present along the paracellular cleft and act as a major barrier to leukocyte

transendothelial migration. VE-cadherin is the predominant transmembrane protein forming

inter-cellular contacts. VE-cadherin blocking antibodies cause increased leukocyte

transendothelial migration both in vitro and in vivo (Gotsch, Borges et al. 1997; Corada,

26

Marriotti et al. 1999; Hordijk, Anthony et al. 1999). The intracytoplamic domain of VE-cadherin

complexes with α-, β-, and p120-catenin and subsequently associates with actin cytoskeleton

(Hordijk, Anthony et al. 1999). VE-cadherin is physically displaced from adherans junctions

during transendothelial migration and re-localises within minutes once the leukocyte

transendothelial migration is completed (Shaw, Bamba et al. 2001). Tyrosine phosphorylation of

VE-cadherins occurs downstream of ICAM-1 cross-linking and has been implicated in the

junctional disassembly (Allingham, van Buul et al. 2007).

2.3.3 Transcellular Migration

In transcellular migration, leukocytes migrate directly through a single endothelial cell

(Figure 1.6) (Feng, Nagy et al. 1998). This route represents a small percentage (5-20%) of all

leukocyte migration (Carman and Springer 2004). During transcellular migration, ICAM-1

enriched caveolae link together to form vesiculo-vacuolar organelles (VVOs) in the endothelial

cells, creating an intracellular passage through which leukocytes can migrate (Dvorak and Feng

2001). Transcellular pathway is more readily observed in vivo, while it is a less preferred route

in vitro. Signaling pathways which preferentially drive paracellular vs. transcellular migration

are not fully elucidated. Microvascular endothelial cells support transcellular migration more

readily than macrovascular endothelial cells (Carman, Sage et al. 2007). Interestingly,

incubation of endothelial cells with TNF-α for longer periods or overexpression of ICAM-1 has

been shown to increase the relative contribution of the transcellular vs paracellular neutrophil

transmigration (Yang, Froio et al. 2005). The transcellular pathway may also become more

prominent when intravascular crawling is disabled in vivo (Phillipson, Heit et al. 2006).

27

1.2.3.4 Migration through the endothelial basement membrane and pericyte sheath:

After penetrating the endothelial cell barrier, leukocytes then cross the endothelial

basement membrane and the pericyte sheath. The endothelial basement membrane is composed

of two protein networks composed of vascular laminins and collagen type IV, which are

connected by interactions with proteins such as nidogen-2 and the heparan sulfate proteoglycan

perlecan (Hallmann, Horn et al. 2005). Wang et al reported the existence of regions of low

expression of matrix proteins (such as laminin 10 and collagen IV) within the endothelial

basement membrane in unstimulated mouse cremastric venules (Wang, Voisin et al. 2006).

These low- expression regions co-localized with gaps between pericytes, allowing neutrophil

migration to occur via the path of least resistance, namely these gaps (Wang, Voisin et al. 2006).

28

Figure 1.4: Schematic diagram of leukocyte paracellular transendothelial migration

Ligation of ICAM-1 is associated with increased intracellular calcium and activation of Rho-

family GTPases and p38 mitogen MAPK pathway which collectively lead to activation of

myosin light chain kinase (MLCK). This may lead to endothelial cell contraction and opening of

interendothelial junctions. Besides ICAM-1, several other junctional proteins participate in this

process.

29

Figure 1.5: Schematic diagram of signaling events initiated downstream of ICAM-1 ligation Leukocyte binding to ICAM-1 triggers multiple intracellular signaling pathways within the

endothelial cells. Rho family-GTPase activation, calcium signaling, production of ROS and

phosphorylation of target proteins are the key pathways involved. These pathways then

contribute to actin remodeling and / or junctional disruption that allows transendothelial

migration

30

Figure 1.6: Schematic diagram of leukocyte transcellular migration

Transcellular migration occurs through a single endothelial cell. During the transcellular

migration, ICAM-1 translocates to regions rich in actin and caveolae. ICAM- containing

caveolae link together to form vesiculo-vacuolar organelles (VVOs) that form an intracellular

channel through which leukocytes can migrate. Ezrin, radixin and moesin (ERM) proteins may

act as linkers between ICAM-1 and cytoskeletal proteins such as actin and vimentin, causing

them to localize around the channel.

31

1.3 Slit2: a guidance cue for migrating cells

During the development of the central nervous system (CNS), neurons navigate over long

distances to make contact with their target cells. Neuronal migration is precisely regulated by

repulsive or attractive cues which coordinate and direct the axonal path-finding (Tessier-Lavigne

et al., 1996). The guidance cues can either promote or repel migration of neurons and axonal

projections. Slit family of secreted proteins and their trans-membrane receptor Roundabout

(Robo) were identified through large scale mutant screens for CNS midline crossing defects in

Drosophila melanogaster (Rothberg, Hartley et al. 1988; Seeger, Tear et al. 1993). During

development, axons normally cross the midline once before projecting towards their synaptic

targets. However, the Drosophila Robo mutants exhibit repeated and random crossing of axons

in the midline (Kidd, Brose et al. 1998). The Slit mutants demonstrate a complete collapse of

commissural and longitudinal axon scaffold onto ventral midline (Rothberg, Jacobs et al. 1990).

There are three known isoforms of Slit (Slit1, 2 and 3) and 4 known isoforms of Robo (Robo-1

to 4) and will be described in more detail later (Wu, Feng et al. 2001; Wong, Park et al. 2002).

1.3.1 Slit and Robo Structure

Slits are secreted glycoproteins (~190-200 kDa) and exhibit a high degree of evolutionary

conservation between species (Brose, Bland et al. 1999). All Slit proteins possess an N-terminal

signal peptide, a long stretch of epidermal growth factor (EGF) repeats, four leucine-rich repeats

(LRRs), a further one or three EGF-like domains in invertebrates and vertebrates, respectively,

and a C-terminal cysteine knot (Fig 1.3) (Hohenester 2008). The EGF repeats and LRR allow

the Slit proteins to interact with extracellular matrix components, such as glypican-1 (Ronca,

32

Andersen et al. 2001). Slit2 is proteolytically cleaved after the fifth EGF repeat generating a 140

kDa N-terminal (Slit-N) and a 55-60 kDa C-terminal (Slit-C) (Brose, Bland et al. 1999). The

identity of protease/s cleaving Slit2 remains elusive. The Slit-N includes the four LRR and the

first five EGF repeats. The four LRRs are necessary and sufficient for interaction with Robo and

the downstream signaling (Battye, Stevens et al. 2001; Chen, Wen et al. 2001). Thus both full

length Slit and the Slit-N retain their repellent activity (Nguyen Ba-Charvet, Brose et al. 2001).

The cleaved fragments have different cell-association properties. Full length Slit2 and Slit-N are

tightly membrane bound whereas Slit-C is diffusible. Furthermore, Slit-C binds with higher

affinity than Slit-N to the heparin sulfate proteoglycan, glypican-1 (Liang, Annan et al. 1999).

The Slit receptor, Robo, belongs to the immunoglobulin (Ig) superfamily of transmembrane

signaling receptors. The extracellular region of Robo contains five immunoglobulin (Ig) repeats

and three fibronectin type III domains. The large cytosolic domain of Robo contains four

conserved sequence motifs designated CC0, CC1, CC2 and CC3 (Kidd, Brose et al. 1998; Legg,

Herbert et al. 2008; Dickinson and Duncan 2010). The molecular weight of Robo-1 is 190-

200kDa, and recent studies reveal that it is sequentially cleaved by metalloproteinases and then

γ-secretase (Seki, Watanabe et al. 2010). Seki et al also demonstrated nuclear accumulation of

Robo-1, which is abolished by either a metalloproteinase inhibitor TAPI-1 or a γ-secretase

inhibitor suggesting that the released intracellular fragment of Robo-1 may translocate to the

nucleus (Seki, Watanabe et al. 2010). Structural and biochemical analysis has revealed that the

second LRR domain of Slit binds to the first and the second Ig domains of Robo (Howitt, Clout

et al. 2004; Morlot, Thielens et al. 2007). The intracellular CC motifs of Robo mediate the

repulsive response to Slit binding. Deletion of each of the CC motif compromises but does not

33

eliminate the repulsive response, suggesting their important but redundant role (Bashaw and

Goodman 1999). Robo-4 (also known as magic roundabout) shows significant differences from

other Robo members with the extracellular domain containing only two immunoglobulin and two

fibronectin domains (Legg, Herbert et al. 2008).

Figure 1.7: Structure of Mammalian Slit2 and Robo-1 proteins

Mammalian Slit2 contains four leucine rich repeats (LRRs), nine epidermal growth factor (EGF)

repeats, a laminin G (G) domain, and a cysteine rich C terminus. The Robo-1 receptor contains

five immunoglobulin (Ig) repeats, three fibronectin (FN) type III, a transmembrane Domain

(TM) and four conserved cytoplasmic (CC) signaling motifs

34

1.3.2 Slit and Robo expression

The expression of the Slit genes has been demonstrated in many organisms, including

Drosophila (Battye, Stevens et al. 1999), Caenorhabditis elegans (Hao, Yu et al. 2001), Xenopus

(Chen, Wu et al. 2000), chickens (Holmes and Niswander 2001), mice (Holmes, Negus et al.

1998), rats (Marillat, Cases et al. 2002) and humans (Itoh, Miyabayashi et al. 1998).

Invertebrates have a single Slit protein, however vertebrates have 3 isoforms of Slit, namely Slit

1, 2 and 3. Slit1 is predominantly expressed in the developing CNS while Slit2 and Slit3 are also

expressed outside the CNS, in the lung, kidney and heart, and in immune cells (Yuan, Zhou et al.

1999; Wu, Feng et al. 2001).

Robo expression has been demonstrated in Drosophila (Kidd, Brose et al. 1998), mice

(Yuan, Zhou et al. 1999) and humans (Kidd, Brose et al. 1998). Caenorhabditis elegans has

single Robo isoform, Drosophila has 3 Robo isoforms and vertebrates have four isoforms of

Robo (Robo-1, Robo-2, Robo-3 and Robo-4) (Dickinson and Duncan 2010). Robo-1 is most

highly expressed outside the CNS, including on immune cells (Wu, Feng et al. 2001). Robo-2

can be detected in the spleen, liver, thymus, kidney, ovaries and brain of adult organisms and in

embryonic tissue (Dallol, Dickinson et al. 2005). Robo-3 is almost exclusively expressed in the

brain (Liu, Hou et al. 2006). Robo4 is specifically expressed by endothelium and is implicated

in angiogenesis (Park, Morrison et al. 2003; Suchting, Heal et al. 2005). The tissue expression of

Slit and Robo are complementary, suggesting a functional ligand-receptor relationship (Yuan,

Zhou et al. 1999).

35

1.3.3 Slit and Robo Function

In addition to their role as axonal guidance cues, Slit and Robo also play a crucial role in

development of other organ systems. For example, studies in Drosophila have demonstrated that

Slit and Robo mutants have disruption of cardiac polarity, cell polarity and cell migration (Qian,

Liu et al. 2005; MacMullin and Jacobs 2006). Slit1 homozygous mutant mice appear

phenotypically normal while Slit2 homozygous deficiency is lethal (Plump, Erskine et al. 2002).

Slit3 mice are viable but their morbidity and mortality increases beyond 30 post-natal days.

These mice have higher incidences of congenital diaphragmatic hernia, renal agenesis and

cardiac defects (Liu, Zhang et al. 2003; Yuan, Rao et al. 2003). Robo-1 homozygous mutant

mice die at birth due to incomplete lung development (Xian, Clark et al. 2001). Robo-2 and

Robo-3 homozygous mutant mice also die soon after birth (Grieshammer, Le et al. 2004;

Sabatier, Plump et al. 2004).

Slit and Robo play important roles in nephrogenesis. Renal abnormalities are observed in

Slit2, Slit3 and Robo-2 deficient mice (Liu, Zhang et al. 2003; Grieshammer, Le et al. 2004).

Kidney development is normal in Robo1 mutant homozygotes. During nephrogenesis, the

ureteric bud arises from the nephric duct in response to glial cell line-derived neurotrophic factor

(GDNF) secreted by the adjacent nephrogenic mesenchyme. Posterior restriction of GDNF is

critical for correct ureteric bud positioning. Slit2 and Robo-2 deficient mice display abnormal

patterns of GDNF secretion and supernumerary ureteric buds that remain inappropriately

connected to nephric duct thus leading to hydroureter (Grieshammer, Le et al. 2004).

Furthermore, variations in the human Robo-2 gene have been associated with familial

36

vesicoureteral reflux (Bertoli-Avella, Conte et al. 2008), a condition with improper insertion of

ureters into the bladder resulting in retrograde flow of urine from the bladder to the kidney.

Slit2/Robo1 expression persists in the adult organism, suggesting a role for Slit proteins

beyond embryogenesis. Slit2/Robo has been shown to inhibit aortic smooth muscle cell

migration toward a gradient of platelet-derived growth factor (PDGF) (Liu, Hou et al. 2006).

Slit2 has also been shown to inhibit cancer cell migration, thus preventing cancer cell metastasis.

Human breast cancer cells express both Robo and the chemokine receptor, CXCR4. Slit2

inhibited breast cancer cell chemotaxis towards the CXCR4 ligand, CXCL12 and cancer cell

adhesion and chemo-invasion in one study (Prasad, Fernandis et al. 2004). Slit2 promoter gene

is frequently inactivated due to hypermethylation thus suggesting a tumour suppressor role

(Dallol, Da Silva et al. 2002; Dallol, Morton et al. 2003). Furthermore, Slit2 has been shown to

inhibit colony formation in colorectal, breast and lung cancer cell lines (Dallol, Da Silva et al.

2002; Dallol, Morton et al. 2003). Collectively these studies suggest a critical role of Slit and

Robo outside of the developing central nervous system.

1.3.4 Slit2/Robo1 intracellular signal transduction

Studies in neuronal tissue have demonstrated that Robo-1 signals via cytoplasmic CC motifs

through two major pathways: Enabled (Ena) protein and Rho GTPases. Ena and its mammalian

homologue (Mena) modulate the actin cytoskeleton rearrangement by binding to prolifin, an

actin binding protein which regulates actin polymerisation (Pantaloni and Carlier 1993). Ena has

been demonstrated to be a substrate for the Abelson (Abl) kinase, and is implicated in normal

axonal guidance (Gertler, Bennett et al. 1989). Genetic and biochemical evidence has revealed a

37

role for Abl and Ena in Slit/Robo signaling during axonal guidance (Bashaw, Kidd et al. 2000).

Ena binds to Robo via the CC1 and CC2 motif whereas Abl binds the CC3 motif. Ena mediates

part of Robo repulsive function whereas Abelson antagonises it (Bashaw, Kidd et al. 2000).

Interruption in Ena binding to Robo leads to impaired Robo function, while a mutation in

conserved cytoplasmic tryrosine, which can be phosphorylated by Abl, leads to Robo

hyperactivity suggesting opposite roles of Ena and Abl on Slit-Robo signaling (Bashaw, Kidd et

al. 2000).

A second pathway through which Slit/Robo mediates cell repulsion is through

modulation of Rho-family GTPase activity. Wong et al discovered a family of GTPase

activating proteins, Slit Robo GTPase activating proteins (srGAPs), which bind Robo (Wong,

Ren et al. 2001). The srGAPs contain a Fer-CIP4 homology (FCH) domain, a Src-homology 3

(SH3) domain and a Rho GTPase activating protein (Gamblin and Smerdon) domain. The SH3

domain is required for binding to the CC3 motif of Robo, the RhoGAP domain regulates the

activity of Rho GTPase Rho, Rac and Cdc42. The function of FCH domain is unknown (Wong,

Ren et al. 2001). In HEK 293 cells not expressing Robo-1, Slit2 did not change levels of the

active form of Cdc42. However in HEK 293 cells expressing Robo-1, srGAP bound to and

inactivated Cdc42 and RhoA, but not Rac1. Furthermore, the expression of a srGAP1 mutant

lacking GAP abolished Slit regulation of RhoA and Cdc42 but not Rac1. In the same study,

transfection of constitutively active Cdc42 was able to rescue the Slit2 mediated migratory defect

of neurons (Wong, Ren et al. 2001). Collectively these data suggest a model where Slit and

Robo binding leads to recruitment of srGAP and inactivation of Rho GTPases, with subsequent

inhibition of actin remodeling and cell motility.

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Figure 1.8: Slit/Robo intracellular signal transduction

Binding of Robo-1 to Slit2 results in recruitment of srGAP, which converts active GTP bound

form of Cdc42 to inactive GDP bound forms, thus inhibiting actin assembly. Enabled protein

also binds to Robo-1 and may contribute to Robo-mediated repulsion whereas Abelson kinase

can phosphorylate Robo and thus antagonise its action

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1.3.5 Slit/Robo in leukocyte trafficking

Both neuronal and leukocyte cell migration share similar features and require the

recognition of guidance cues, generation of cell polarity and mobilization of the actin

cytoskeleton machinery. Thus, not surprisingly, Slit has been also been found to inhibit

leukocyte migration (Guan, Zu et al. 2003; Kanellis, Garcia et al. 2004; Prasad, Qamri et al.

2007).

The effect of Slit2 on leukocyte chemotaxis was first described by Wu et al. This study used

transwell migration assays to demonstrate that Slit2 inhibits the chemotaxis of rat lymphocytes to

stromal derived factor-1α (SDF-1α) and neutrophil-like HL-60 cells to fMLP gradients (Wu,

Feng et al. 2001). Similarly, Kanellis et al demonstrated that Slit2 inhibited RAW 264.7 murine

macrophages and rat glomerulonephritic inflammatory leukocytes towards monocyte

chemoattractant protein-1 (MCP-1) and formyl methionyl-leucyl-phenylalanine (fMLP)

respectively (Kanellis, Garcia et al. 2004). Another study showed that Slit2 inhibited migration

of Langerhans dendritic cells (DCs) thus reducing contact hypersensitivity responses and

decreasing inflammation (Guan, Zu et al. 2003).

Prasad et al demonstrated that Slit2 inhibits chemotaxis of Jurkat T lymphocytes towards

SDF-1α (Prasad, Qamri et al. 2007). Furthermore, our group demonstrated that Slit2 prevents

chemotactic migration of neutrophils to diverse chemoattractants both in vitro and in vivo (Tole,

Mukovozov et al. 2009). Collectively these data demonstrate that Slit2 may have a therapeutic

role as a universal inhibitor of leukocyte migration.

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1.4 Rho GTPases: Rac and Cdc42

The small GTPases of the Rho family are a part of the Ras superfamily of small GTP-

binding proteins. To date, over 20 mammalian Rho-family GTPases have been characterized,

and these can be grouped into several subfamilies: Rac (Rac1, Rac2, Rac3, RhoG), Rho (RhoA,

RhoB, RhoC), Cdc42 (Cdc42, TCL/Rho J, TC10/Rho Q), Rnd (Rnd1, Rnd2, Rnd3/RhoE),

RhoD, Rho BTB (RhoBTB1, RhoBTB2) and TTF/Rho H (Kjoller and Hall 1999; Heasman and

Ridley 2008). Rho GTPases are pivotal regulators of signaling pathways that control diverse

cellular functions including cell polarity, migration, vesicle trafficking and cell cycle progression

(Hall 1998; Hall and Nobes 2000; Heasman and Ridley 2008).

1.4.1 Structure and Regulation

All Rho GTPases contain 2 main structural domains, a catalytic GTP domain and the C-

terminal ‘CAAX’ motif (cysteine (C) followed by two aliphatic amino acids (AA) and a terminal

amino acid (X). The GTPases undergo post-translational modifications at the C-terminal

‘CAAX’ motif, involving covalent addition of isoprenoid moieties to the cysteine residue,

carboxy-terminal proteolysis of the AAX residues followed by carboxy-methylation. The

modified C-terminal domain then allows the protein to associate with membrane lipids (Casey,

Solski et al. 1989; Gutierrez, Magee et al. 1989; Fujiyama and Tamanoi 1990). The catalytic

domain of Rho GTPases consists of switch I and switch II, corresponding to different

conformations in GTP-bound and GDP-bound states (Karnoub, Symons et al. 2004). The N-

terminal catalytic domain of GTPases allows for conformational changes, via binding to GDP or

41

GTP. Rho GTPases cycle between the inactive, GDP-bound state and the active GTP-bound

forms (Olofsson 1999; DerMardirossian and Bokoch 2005).

Figure 1.9: Structure of Cdc42/Rac1

Rho GTPases contain 2 main structural domains, a catalytic GTP domain at the N-

terminal and the C-terminal ‘CAAX’ motif (cysteine (C) followed by two aliphatic amino acids

(AA) and a terminal amino acid (X). The N-terminal catalytic domain of GTPases allows for

conformational changes, via binding to GDP or GTP. The catalytic domain of Rho GTPases

consists of switch I and switch II, corresponding to different conformations in GTP-bound and

GDP-bound states. The C-terminal domain allows the protein to associate with lipid.

Attachment of the lipids at the C-terminal then facilitates membrane association and subcellular

localisation of Rho GTPases.

42

The active GTP-bound form of the protein can transduce signals via interactions with

downstream targets or effector molecules to produce a cellular response (Kjoller and Hall 1999;

Heasman and Ridley 2008). Activity of Rho GTPases is tightly controlled by three classes of

regulatory molecules: guanine nucleotide exchange factors (GEFs), guanine nucleotide

dissociation inhibitors (GDIs) and GTPase-activating proteins (GAPs). GEFs catalyze the

exchange of GDP for GTP, leading to activation of Rho GTPases. Rho GEFs are characterised

by the presence of a DH (Dbl) homology domain followed by a PH (pleckstrin homology)

domain. The DH domain of Rho GTPases is critical for the recognition of GEF’s by their

specific targets. The PH domain targets GEFs to specific subcellular membranes through

interaction with lipids (Schmidt and Hall 2002; Rossman, Der et al. 2005).

The second class of regulatory proteins, GDIs, sequester the Rho-family GTPases in the

cytosol and inhibit their activity in several ways. First, GDIs maintain the GTPases in an

inactive form by preventing dissociation of GDP from the GTPases. Second GDIs can bind to

isoprenyl moieties in the C-terminus of GTPases in order to sequester them in the cytosol thus

preventing their membrane localization (Olofsson 1999). Third, although Rho GDIs usually bind

to GDP-bound GTPases with high affinity, they can also interact with the GTP-bound forms and

prevent their activation by GEFs. All of these actions of GDIs prevent the activation of

RhoGTPases.

The third class of regulatory proteins, GAPs suppress activity of Rho-family GTPases by

enhancing the intrinsic rate of GTP hydrolysis to GDP. To date, more than 70 eukaryotic Rho

GAPs have been identified (Tcherkezian and Lamarche-Vane 2007). Although GTPases have

43

intrinsic GTPase activity (Vetter and Wittinghofer 2001), the rate of GTP hydrolysis is relatively

very low, but can be accelerated by order of magnitude upon interaction with Rho GAPs (Vetter

and Wittinghofer 2001). There exists a large diversity in primary sequence of the various GAPs,

but their tertiary structure as well as the basic GTPase-activating mechanism is similar. Rho

GAPs bind to the nucleotide-contacting core of Rho GTPases and lead to a conformational

structural change. Consequently, an essential arginine residue of GAPs together with a

glutamine residue of the GTPases is responsible for positioning a water molecule in the vicinity

of GTP, thereby triggering hydrolysis to inactivate the GTPases (Moon and Zheng 2003; Bos,

Rehmann et al. 2007). The specificity of individual GAPs for different Rho-family GTPases is

thought to be determined by residues outside the nucleotide-binding core of the GTPases (Li,

Zhang et al. 1997).

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Figure 1.10: Regulation of Rho GTPases

Rho GTPases cycle between active GTP bound forms and inactive GDP bound forms.

Regulation of this molecular switch mechanism is controlled by opposing activities of guanine

nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). GEFs catalyse the

exchange of GDP for GTP whereas GAPs increase the rate of hydrolysis of GTP to GDP.

Further GDP dissociation factors (GDIs) sequester Rho away from GDP-GTP cycle

45

1.4.2 The role of Rho-family GTPases in regulation of the actin cytoskeleton

The movement of eukaryotic cells relies on coordinated extension of actin-rich lamellipodia in

the leading edge and retraction of the uropod at the rear of the cell. The extension of the lamellae

at the leading edge involves rapid turnover of actin filaments (Symons and Mitchison 1991).

More stable actin-myosin cables can be found in more established protrusions in the middle and

rear of the cell (DeBiasio, Wang et al. 1988). Thus, cell motility requires the co-ordinated

polymerisation of actin in protrusions at the leading edge and contraction of actin-myosin cables

at the middle and rear of the cell. Actin polymerisation is in turn regulated by Rho GTPases

(Machesky and Hall 1997). Most of our understanding on the biological functions of Rho

GTPases has been obtained from studies of RhoA, Rac1 and Cdc42, the three most extensively

characterised family members of Rho GTPases. Activation of Rho A, Rac1 and Cdc42 in

fibroblasts is observed to lead to formation of distinctive cytoskeletal structures. Rho A induces

stress fibres and focal adhesions, while activation of Cdc 42 induces formation of filopodial

microspikes (Nobes and Hall 1995; Hall 1998; Raftopoulou and Hall 2004). Finally, activation

of Rac stimulates the formation of sheet-like lamellipodia (Nobes and Hall 1995; Hall 1998;

Raftopoulou and Hall 2004).

As critical regulators of actin cytoskeleton dynamics and cell morphology, Rho GTPases play an

important role in regulation of neutrophil chemotactic migration and adhesion. For example

generalised blockade of Rho GTPases in human neutrophils with Clostridium difficile toxin A

leads to marked decrease in neutrophil chemotaxis and increased adhesion (Brito, Sullivan et al.

46

2002). Cdc 42 deficient mice neutrophils display markedly reduced chemotaxis (Szczur, Zheng

et al. 2009). Rac2 is the predominant isoform in human neutrophils whereas murine neutrophils

express similar amounts of Rac1 and Rac2 (Li, Yamauchi et al. 2002). Selective deletion in of

Rac 1 in mouse neutrophils results in defective directional and display random migration. They

have normal adhesion but increased cell spreading (Gu, Filippi et al. 2003; Sun, Magalhaes et al.

2007). Mice neutrophils deficient in Rac2 move slowly but are able to migrate towards

chemoattractant gradient. Similar to Rac 1 deficient neutrophils, Rac2 deficient neutrophils

display normal adhesion (Gu, Filippi et al. 2003).

1.4.3 The role of Rho-family GTPases in regulation of innate immune function

Rho GTPases play important role in innate immune responses such as neutrophil chemotaxis,

phagocytosis and production of reactive oxygen species (Bokoch 2005). Neutrophil chemotaxis

and phagocytic destruction of pathogens requires mobilisation of actin cytoskeleton and

consequent cell shape changes mediated by Rho GTPases (Ridley, Schwartz et al. 2003). The

production of reactive oxygen species depends on Rac activation which is modulated by

antagonistic cross-talk with Cdc42 (Roberts, Kim et al. 1999; Kim and Dinauer 2001).

1.5 Rationale, Hypothesis and Objectives

1.5.1 Rationale

Infiltration of leukocytes, especially neutrophils, causes AKI associated with IRI (Okusa

2002).. Neutrophils are recruited to the injured kidney by a number of attractants produced

locally, especially IL-8 (Furuichi, Wada et al. 2002). Macrophages, T lymphocytes and

dendritic cells also play an important, albeit less prominent role. Once recruited to the injured

kidney, neutrophils firmly adhere to the “sticky” endothelium activated by locally produced

47

TNF-α. Neutrophils eventually undergo trans-endothelial migration into the injured kidney and

promote and perpetuate the organ damage by releasing inflammatory mediators (Furuichi, Wada

et al. 2003; Friedewald and Rabb 2004; Fiorina, Ansari