Introduction

The ability of leukocytes to phagocytose, produce superoxide, and selectively lyse cells is the backbone of the mammalian innate immune system. Knockout studies have demonstrated that the movement of neutrophils, monocytes and natural killer cells to sites of insult is central to the maintenance of intact innate immunity, and can mean the difference between routine clearance of an offending organism or a fatal infection. Beyond inflammation, strategic leukocyte homing to immunologically distal sites such as the gut and central nervous system is necessary to main

*SEB and KAH contributed equally to this work.

tain adequate innate immunity in these sites. Homing mechanisms also regulate homeostasis of innate cells; how these cells maintain adequate numbers in the periphery and are cleared during senescence. An important function of cellular innate immunity is to provide stimulating signals for the activation of adaptive immunity, and migratory mechanisms regulate the movement of innate cells to the appropriate location in secondary lymphoid cells.

During haematopoiesis in the bone marrow and T cell differentiation in the thymus, leukocyte homing is critical for the manoeuvring of leukocytes into specific compartments for maturation. Furthermore, effective immune surveillance by T and B lymphocytes requires a constant cycle of migration between the primary and secondary lymphoid tissues, which allows for effective antigen presentation and initiation of adaptive immune responses.

Primarily, two families of molecules, chemokines plus their cognate receptors, and adhesion molecules regulate leukocyte migration. Chemokine gradients dictate the direction in which a cell will migrate and signal actin rearrangements within the cell, while adhesion molecules establish the cell-to-cell and cell-to-matrix interactions necessary for this movement.

The importance of these molecules is underscored by the observation that homozygous deletion of the chemokine CXCL12/SDF-1 in mice results in a lethal phenotype with defects in haematopoietic and cerebellar development [1,2]. This review will detail the current understanding of the role of chemokines and adhesion molecules in leukocyte homing and trafficking.

1.1. Chemokines and chemokine receptors

Chemokines are a subfamily of cytokines with chemotactic properties. While capable of acting on a variety of cell types including fibroblasts, endothelial cells, and smooth muscle cells, chemokines are best known for their ability to induce leukocyte chemotaxis. Chemokines are classified based on the positioning of conserved cysteine residues [3-5], The CC, CXC and CX3C families contain a set of four conserved cysteines in their amino-terminal regions and are distinguished by the absence (CC) or the presence of one (CXC) or three (CX3C) intervening amino acids between the first two cysteines. The C family of chemokines (XC) contains only two conserved cysteines that correspond to the second and fourth cysteines of the other families [3], Chemokines induce migration upon binding to their specific receptors. Chemokine receptors belong to the rhodopsin-like superfamily of seven transmembrane-spanning G protein-coupled receptors. The receptors themselves are classified based upon ligand specificity rather than amino acid similarity. Certain receptors are promiscuous, binding multiple chemokines and likewise certain chemokines can bind multiple receptors.

Chemokines and their receptors can be roughly divided based on their functionality. The receptors CXCR1, CXCR2, CXCR3, CCR1, CCR2, CCR3, CCR5, and CCR6 are typically associated with the inflammatory response, whereas the receptors CXCR4, CXCR5, CCR4, CCR7, CCR9, and CCR10 are regarded as homeostatic receptors [6], In general, chemokines involved in leukocyte homing and trafficking bind only a single chemokine receptor.

1.2 Adhesion molecules

Adhesion molecules are proteins that regulate cell-to-cell and cell-to-matrix interactions. The binding of adhesion molecules allows cells to move along a substratum or to maintain contact with a target cell. Adhesion molecules can be divided into four families: selectins, integrins, vascular addressins, and immunoglobulins. The selectin and integrin subfamilies play the largest role in homeostatic immunity, particularly in T cell homing. Selectins are expressed both on leukocytes (CD62L/L-selectin) and on the endothelium (P- and E-selectin). Selectins share a core protein structure, but possess different extracellular lectin domains that determine specificity. L-selectin, for example, binds the sulphated sialyl Lewis" sugar of the mucin-like vascular addressins common to the endothelium. Selectins provide the initial contact between leukocytes and vascular endothelium needed for leukocyte homing into tissues. This weak interaction participates in leukocyte rolling and acts in concert with integrins, which provide stronger adhesions capable of facilitating cell arrest and extravasation.

Integrins are heterodimeric proteins composed of a and (3 chains. The heterodimers are not specific, so that a particular (3 chain may associate with several different a chains and vice versa. Classification of the composite integrin is based on the (3 chain. Nearly all leukocytes express (32 integrins on their surface, however, T cells also express (31 integrins. These (31 integrins are termed very late activation antigens (VLAs) because they are only expressed late in the maturation process. Integrin binding partners also vary substantially. The integrin lymphocyte function-associated antigen (LFA)-l is expressed on T-cells and binds to three members of the immunoglobulin superfamily: intercellular adhesion molecules (ICAM)-l,-2 and -3. The integrin vascular cell adhesion molecule (VCAM)-l, on the other hand, is expressed on activated endothelium and binds the T-cell VLA-4 integrin. The interaction between VCAM-1 and VLA-4 on lymphocytes provides a much stronger adhesion than selectins and allows for firm adhesion and arrest on the endothelium. Integrins and chemokines act in concert to regulate leukocyte homing: surface expression of integrins can be induced by signal transduction events initiated by chemokine receptor activation; conversely, cross-linking of integrins can stimulate chemokine production capable of acting in an autocrine fashion. For a comprehensive review of adhesion molecules in lymphocyte homing see Fabbri et al. [7].

2. MECHANISMS OF NEUTROPHIL HOMING 2.1. Introduction

Human polymorphonuclear neutrophils (PMNs) arc the most numerous of the circulating phagocytes, composing 40-65% of the white blood cells [8]. Its phylogeny vastly predates lymphocytes, and as such PMNs represent an important arm of innate immunity. PMNs are the earliest leukocytes to arrive at a site of infection and function primarily to provide a rapid defence against invading pathogens and clearance of inflammatory debris [8]. In response to a diverse array of pro-inflammatory stimuli including cytokines, chemoattractants, opsonins and highly conserved bacterial structures, PMNs activate two central antimicrobial effector mechanisms: oxidative respiratory burst and mobilization of cytotoxic granules [9]. The respiratory burst mechanism consists of the formation of a multi-subunit NADPH oxidase complex in phago-somal membranes and the plasmalemma of the neutrophil, which exerts its antibiotic activity by the production of highly reactive superoxides [10], Granules are intracellular vesicles containing primarily bactericidal and degradative enzymes [11,12]. Upon neutrophil activation, granules are mobilized and migrate from deep within the cytoplasm to fuse with phagosomes and the plasma membrane, releasing their components to the extracellular space and phagosomal lumen [11-13],

2.2. Regulation of neutrophil chemotaxis during inflammation 2.2.1. Adhesion molecules in neutrophil transendothelial migration

PMNs are segregated into two compartments: a mature subset circulating throughout the peripheral blood and an immature pool within the bone marrow. Because of their central role in acute inflammation, the mechanisms regulating neutrophil migration and effector function have been intensely studied. Migration of PMNs is regulated by three groups of surface molecules: adhesion molecules, receptors for pro-inflammatory cytokines/stimuli, and chemoattractant receptors [14].

Current models of the process by which leukocytes cross the endothelial cell wall and exit the circulation into inflamed tissue are separated into discrete steps, each governed by distinct molecular mechanisms. Monocytes, lymphocytes and granulocytes all utilize a similar process to emigrate from the peripheral blood, frequently utilizing related molecules at each stage. Upon encounter with a chemotactic stimulus, the initial stage in leukocyte migration is margination, whereby flowing cells "capture" the endothelial wall and commence "rolling" along the vessel wall [4], PMNs utilize selectins for both capture and rolling. L-selectin is constitutively expressed on the surface of PMNs [15], L-selectin on the surface of lymphocytes binds to endothelial CD34 [16]; the endothelial binding partner for L-selectin on PMNs is poorly characterized, but known to be a member of a family of sialomucin oligosaccharides [17], Capture and rolling of PMNs has been demonstrated under non-inflammatory conditions, suggesting constitutive expression of an L-selectin endothelial ligand [18], However, endothelial ligands for PMN L-selectin can also be induced by treatment with LPS and pro-inflammatory cytokines [19], P-selectin and E-selectin regulate PMN capture by endothelial cells (ECs). P-selectin protein is stored within Weibel-Palade bodies of endothelial cells: stimulation by cytokines, complement components or superoxide products can induce their surface expression [20], P-selectin glycoprotein ligand-1 (PSGL-1) is constitutively expressed on PMNs and likely contributes to the rolling process to re-enforce the more transient L-selectin mediated interactions [14,21], E-selectin appears much later (4-6 h) on the endothelial surface, as it is transcribed de novo after stimulation with proinflammatory cytokines [22,23], No definite PMN ligand for P-selectin has been described, but it is believed to belong to the same family of proteins as the PMN ligand for L-selectin [24], Because of its late onset of surface presentation, E-selectin is believed to maintain rolling after P-selectin has been downregulated.

The second stage of leukocyte emigration across the endothelial barrier, "firm adhesion", is mediated by establishing associations with the endothelial wall that are longer lived than those mediated by selectins. This second step is mediated by integrins expressed on leukocytes and their cognate binding to endothelial-expressed ICAMs. PMN-endothelium are binding is predominately regulated by the [37 integrin Mac-1 (ocMP7, CD1 lb/CD 18), but is also mediated to a lesser degree by LFA-1 (aLP„ CD1 la/CD 18) and pi50, 95 (ocx|37, CD1 lc/CD18) [20,25], Mac-1 is stored as a presynthesized protein within the membranes of secondary/specific and tertiary/gelatinase granules and secretory vesicles, and can be rapidly transported to the surface when cells are exposed to various secretagogues [12], Inflammatory stimuli such as lipopoly-saccharide (LPS) and pro-inflammatory cytokines are not classical stimuli of degranulation in that they are incapable of mobilizing the large, deeply embedded azurophil or specific granules; however they have been demonstrated to induce surface presentation of Mac-1 by inducing the release of the smaller secretory vesicles [12], Upregulation of surface Mac-1, however, is not sufficient for activation of adhesion. Intracellular Mac-1 is unphosphorylated, but upon surface presentation, the CD 18 subunit is phosphorylated [26], Mac-1 activation can be stimulated by IL-8 treatment, selectin engagement and pro-inflammatory cytokine treatment [27,28], The intracellular molecules regulating integrin activation are equally diverse: the small G protein Rho, cyohesin-1 and the actin-bundling protein L-plastin have all been implicated in integrin activation [27,29], The ligand for Mac-1 is ICAM-1, expressed constitutively on endothelium, but with upregulation inducible by proinflammatory cytokines [7],

After establishing a strong association with the endothelial wall, leukocytes must pass between endothelial cells in a process termed "extravasation". This stage involves a receptor switch, as selectins are downregulated and a protein of the immunoglobulin family, platelet-endothelial cell adhesion molecule (PECAM-1) has been demonstrated to be vital to cell passage through endothelium [14], PECAM-1 can form intercellular homodimers; thus, can act as its own ligand [30], The role of PECAM-1 was most definitively demonstrated in studies involving neutralising anti-PECAM-1 antibodies; PMN extravasation within in vitro [31] and animal models [32] was inhibited, while cell adhesion was unaffected.

Extravascular passage of leukocytes, or transmigration, within the underlying tissues involves adhesion to both extracellular matrix and cell surfaces. Gelatinase granules within PMNs are released readily in response to secretagogues; the release of gelatinase B, a potent collagenase, has been postulated to facilitate neutrophil movement through the extracellular matrix [12], The ability of protease inhibitors to reduce PMN chemotaxis in vitro has strengthened this hypothesis [33], Counter to this model are observations that mice genetically deficient in gelatinase B do not show any abnormalities in PMN migration to lungs, skin or peritoneum [34],

The mechanisms by which PMNs adhere to resident cells as they navigate the subendothelial space has some redundancy with those used in the vascular lumen: ICAM-1 surface expression can be detected on parenchymal tissues [35], as well, antibodies against PECAM-1 are able to inhibit PMN movement across the basement membrane [36], Activated PMNs express the integrins very late antigen 4 (VLA-4, a4(3,), VLA-5 (a5P,), VLA-6 (a6p,) and VLA-9 (agP,), all of which have been demonstrated to be required for PMN migration through lung and synovial barriers [37,38], VLA-4 binds vascular VCAM-1 on endothelial cells in vitro', however, VLA-5 and 6 bind fibronectin and laminin, respectively [14], VLA-9 is the most abundant p( integrin on PMNs, and is not expressed on monocytes or lymphocytes [38], VLA-9 is capable of binding fibronectin, VCAM-1 and tenascin, making it an attractive candidate for regulation of PMN transmigration.

As described above, transendothelial migration of PMNs utilizes many different surface molecules. However, the timing of expression and activation of these receptors at the appropriate stages is equally important. The intracellular signalling pathways regulating neutrophil adhesion to endothelium have been intensely studied (for a full review see Wang and Doerschuk [39]). At each stage, the activation and expression of adhesion molecules can be modulated by chemoat-tractants and cytokines, however, this activation/upregulation is also regulated, at least partially, by adhesion molecules themselves in a domino like fashion, whereby receptors active at one stage serve to activate those regulating the next. Activation of L-selectin stimulates p2 integrin-dependent adhesion, and this adhesion can be blocked by tyrosine kinase, and protein kinase C inhibitors, suggesting a putative pathway between L-selectin and Mac-1. In turn, activation of P2 integrins can stimulate the phosphorylation of PECAM-1. It is unlikely that the complete process of transendothelial migration could be regulated autonomously by endogenous adhesion molecules; however it is clear that this internal cross-talk is necessary.

2.2.2. Chemokine regulation of neutrophil inflammation

Several soluble cytokines are capable of influencing PMN migration and can be divided into two categories [14], The first are pro-inflammatory cytokines, which can stimulate expression/activation of adhesion receptors but are incapable of inducing chemotaxis alone. TNF-a, IL-1 and IFN-y are representative members of this group. The second group, chemoattractants, induce trafficking independent of other stimuli. PMNs migrate in response to several stimuli: platelet activating factor (PAF), complement protein C5a, leukotriene B4 (LTB4), formylated bacterial peptides (fMLP) and chemokines. Of these, the regulatory mechanisms for chemokine and chemokine regulation have the most developed models.

Human PMNs express the chemokine receptors CXCR1, CXCR2, CCR1, CCR2 and CXCR4 [4,40], CXCL8/IL-8 was the first chemokine to be discovered, based on its ability to induce PMN migration after LPS treatment of neutrophils; as such it has been the most intensely studied chemokine regulating PMN function [41], CXCL8/IL-8 binds to both CXCR1 and CXCR2. Despite their prevalence in regulating human PMN biology, no murine counterpart to either molecule has been characterized [4], IL-8 expression has been detected by endothelium, epithelium, monocytes, platelets, and a variety of parenchymal cells. In addition to IL-8, human PMNs also respond to the CXCL7/chemokines neutrophil-activating peptide-2 (NAP-2), 3 forms of melanoma growth stimulating activity (GRO-a,(3,y)/CXCLl, CXCL2, CXCL3, and CXCL5/ epithelial cell-derived neutrophil-activating peptide-78 (ENA-78) [4],

PMNs in mice and rats respond to the chemokines CXCL1/KC (a homologue of human GRO-p and y), CXCL2/macrophage inflammatory protein-2 (MIP-2), CXCL15/LIX (an ENA-78 homologue) [4] and two novel chemokines in rat termed cytokine induced neutrophil chemoattractants, CINC-2a and CINC-2p, have been described [42J. CXCL1/KC and CXCL2/MIP-2 have been demonstrated to regulate PMN migration in rat models of lung and intestinal inflammation [43,44],

Granulocytes differ from lymphocytes in that the molecular signals for migration are closely linked to activation of their effector function; in PMNs, the same ligand can initiate migration, but can also stimulate degranulation and superoxide production. At low concentrations, (1 nM) IL-8 is capable of inducing PMN migration through classical hctcrotrimer signalling pathways [45]. At higher concentrations of IL-8, CXCR1 is desensitized to ligand, and rendered incapable of signalling along this pathway. Receptors are subsequently phosphorylated on serine residues within their cytoplasmic tails by G-protein coupled receptor kinases (GRKs), providing a signal for the association of the adapter molecule p-arrestin, which serves to internalize the CXCR1 from the membrane [45], This model of receptor desensitization has been demonstrated for CCR5, CXCR1, CXCR4 and CCR2B [45^18], After internalization, p-arrestin associated with CXCR1 has been demonstrated to bind to the activated form of the Src tyrosine-kinase Hck [49] and this complex could be detected on azurophil granules. Further, RBL-2H3 cells, a cell line model of PMNs, cotransfected with CXCR1 and dominant negative molecules of p-arrestin, demonstrated inhibited granule release in response to IL-8 stimulation. Thus, P-arrestin mediated signalling may represent a mechanism by which PMNs can migrate in response to lower ligand concentrations that might be present at sites distal from inflammation, and upon encounter with higher ligand concentrations that would be present at proximal locations, stop migration and switch to effector function.

3.3. Chemokine regulation of neutrophil homeostasis

Relative to lymphocytes, which have complex migratory pathways through the periphery, primary and secondary lymphoid organs, PMNs follow a simple route: development in the bone marrow, release to the periphery where they circulate until they are recruited into inflammatory sites. Bone marrow contains a large reserve of mature PMNs. Pro-inflammatory cytokines and chemoattractants in the blood can initiate rapid mobilization of this reserve compartment and increase the number of circulating PMNs [50], Approximately 1011 PMNs are released into the blood per day [8], Blood PMNs are short-lived (t = 6 hr), thus a highly active homeostatic mechanism is required to maintain adequate PMN levels and clearance. At inflammatory sites, neutrophils have been demonstrated to undergo apoptosis and be phagocytosed by macrophages [51]. Circulating PMNs entering senescence have been postulated to undergo clearance by the liver, spleen and bone marrow. The expression of CXCR4 on PMNs has been debated, however conclusive evidence has demonstrated that PMNs maintain baseline levels of the receptor upon entry into the periphery, but that marked upregulation occurs as they age [40], Using an in vivo bone marrow perfusion system, Martin et al demonstrated that the CXCR4 inhibitor, AMD-3100, could stimulate the migration of bone marrow resident PMNs into the circulation [40], Further, ex vivo cultured PMNs drastically upregulated CXCR4. When re-introduced to mice either by IV or bone marrow perfusion, the cultured, CXCR41,igh PMNs were sequestered within the marrow at significantly higher numbers than freshly isolated CXCR4l0w PMNs.

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