Role of Chemokines in Leukocyte Trafficking

Chemokines

Chemokines (a clipping blend of chemo tactic cyto kines ) are critical molecular messengers in the complex cellular communication network used by the immune system. Almost 50 human chemokine genes have been identified to date ( Table 12.1 ). The two major subclasses of chemokines are designated CC- or CXC-, reflecting the relative position of the two proximal to the N-terminus canonical cysteines, being either adjacent or separated by a single amino acid, respectively. XCL1 and XCL2, and CX3CL1 represent additional structural chemokine forms with one cysteine and three amino acids between the two canonical cysteines, respectively. CX3CL1 and another chemokine, CXCL16, are associated with a cell membrane via a long spacer sequence and anchored by a transmembrane domain; however, both these chemokines can be cleaved off their stalks and give rise to functional soluble molecules. All other chemokines are secreted proteins of 67 to 127 amino acids. Historically, chemokines have been grouped into functional subfamilies termed inflammatory and homeostatic chemokines. Former are induced by inflammatory signals and control the recruitment of effector leukocytes in infection, inflammation, tissue injury, and malignancies, whereas those belonging to the latter group navigate leukocytes during hematopoiesis in the BM and in the thymus, during initiation of adaptive immune responses in SLOs and in immune surveillance of healthy peripheral tissues. However, it is now clear that such functional distinction is largely blurred, as many “inflammatory” chemokines are produced under physiologic conditions and the expression of “homeostatic” chemokines is upregulated in inflammation.

Chemokine signals are transmitted through specific cell-surface G protein-coupled receptors (GPCRs) with seven transmembrane domains. The human chemokine receptor repertoire identified at present consists of 20 different GPCRs ( Table 12.2 ). The tremendous specificity and plasticity of leukocyte homing and tissue localization is largely determined by the interactions of chemokines with their cognate receptors. Individual leukocyte subsets express highly characteristic fingerprints of chemokine receptors that determine their complex responses to chemokines and define their migratory paths in the body with different receptors playing either nonredundant, combinatorial or overlapping roles. The interactions of individual chemokines and receptors are nonrandom and have been comprehensively characterized. According to new findings, still pending independent confirmation, the last remaining orphan chemokine, CXCL14, known to bind and allosterically modify chemokine receptors, including CXCR4, but not directly signal per se, triggers its own GPCR and GPR85. Each chemokine receptor binds either one chemokine or a defined set of them and any individual chemokine can ligate either one unique or several different receptors resulting in some overlap in specificities. However, there are multiple reasons why the apparent “promiscuity” of chemokine-receptor interactions is not a sign of chemokine redundancy and does not reflect a tautology of their signals. First, while the receptor use by different chemokines might overlap, the full spectra of receptors triggered by any particular chemokine are mostly distinct and characteristic for a chemokine, especially within the CC subfamily. Thus, individual chemokines can unmistakably be recognized, albeit in many cases not by one receptor, but by their entire system. Second, messages encoded by individual chemokines that ligate the same receptor are largely unique, because different chemokines have highly disparate binding affinities, interact with distinct receptor moieties and consequently lead to biased signaling, i.e., they induce a dissimilar “texture” of intracellular secondary effectors, activating some and inhibiting others, ultimately resulting in unique spectra of downstream molecular and cellular responses. For example, homologous chemokines CCL17 and CCL22 signaling through their cognate receptor CCR4 leads to differential molecular and functional outcomes. CCL19 and CCL21 provide another example of differential, biased signaling through their common receptor CCR7, whereby individually or in combination they can induce variations of directed migratory cell responses, including chemotaxis, haptotaxis or chemorepulsion. Third, chemokines with an apparent overlap of their receptor specificity can behave fundamentally dissimilar due to their differential physicochemical properties, e.g., occurring primarily as soluble versus substrate-bound, as again exemplified by CCL19 and CCL21. Fourth, individual chemokines that ligate the same receptor are produced in vivo in different tissues and/or derive from different cells and/or localize in alternate tissue microenvironments, thus do not act simultaneously but induce distinct, often sequential, steps of leukocyte migratory paths. This was recently shown for two CXCR2 ligands, CXCL1 and CXCL2, which are produced in the vessel wall and by migrating neutrophils, respectively, localize in distinct microanatomical foci thus induce sequential steps of neutrophil emigration. In summary, there are no two chemokines alike and their endless possible combinations allow these structurally homologous signaling molecules to convey most diverse cellular messages acting as building blocks of a universal cell language.

The total number of unique chemokines is further increased by single nucleotide polymorphisms of genes encoding them and their several known spliced variants. Also, practically all chemokines undergo post-translational modifications of their main secreted forms with specific enzymes and protein-modifying agents inducing truncation, degradation, nitration or citrullination of chemokines. Many of such modified chemokine isoforms have altered affinity and spectrum of agonistic activities on different receptors and, in some cases, are rendered completely inactive by their processing or gain receptor antagonistic profile. Proteases prominently implicated in processing chemokines include dipeptidyl peptidase IV, also known as CD26, which cleaves two NH2-terminal amino acids of chemokines with alanine or proline at the N-terminal penultimate position, sometimes sequentially, elastase, the ADAM family, as well as matrix metalloproteases (MMPs), a family of more than 20 enzymes with important functions in matrix degradation and multifaceted roles in inflammatory leukocyte recruitment. Conversely, some chemokines including CCL14, CCL15, CCL16, and CCL23 are secreted as precursors and their receptor interactive forms are generated upon proteolytic processing by serine proteases and MMPs abundant in the inflammatory tissue environments. Also, chemokine processing by glutaminyl cyclase, the modifications to pyroglutamate of their N-terminal amino acids in CCL2 and CX3CL1, is required for protecting these chemokines from proteolysis in vivo and is required for their biological activity. Further intricacy of chemokine signaling is achieved by the propensity of chemokines to dimerize and oligomerize with chemokine monomers and dimers differentially ligating receptors and causing biased signaling, as has been shown recently for CXCL12. Moreover many, but not all, chemokines form heterodimers within and across their structural sub-family boundaries following a recently mapped pattern of nonrandom specificity. The resultant chemokine heterodimers shift the use of their receptors as compared to the individual monomeric chemokines.

Signaling by Chemokine Receptors

Chemokine receptors, just like other GPCRs, function as allosteric molecular relays where chemokine binding to the extracellular portion modifies the receptor’s tertiary structure. This allows the intracellular domain of the engaged receptor to bind to and activate heterotrimeric G proteins. In response, the activated G-proteins exchange GDP for GTP and in the process, dissociate into Gα and Gβγ subunits. Curiously, different chemokine receptors are able to associate with different subsets of Gα subtypes, though any particular functional relevance of such biased signaling is not yet clear. The dissociated Gβγ subunits mediate large parts of chemokine-induced signals by activating different phosphatidylinositol 3-kinase (PI3K) isoforms, leading to the formation of phosphatidyl-3,4,5-triphosphate (PIP ₃ ). PI3K and its product PIP ₃ then translocate to the pseudopod at the leading edge of migrating leukocytes, where they colocalize with the small GTPase Rac. PIP ₃ activates Rac through specific guanine nucleotide exchange factors. Rac in turn acts through the downstream effectors p21-activated kinase and the Wiskott-Aldrich protein homologue WAVE, which stimulate actin-related protein 2/3. Together, this process induces focal polymerization, required for the development and forward extension of the pseudopod, a critical step in leukocyte chemotaxis. The importance of PI3K-dependent signaling for leukocyte chemotaxis is evidenced by the lack of migration of myeloid leukocytes to chemokines in mice lacking PI3Kγ Notably, though, distinct signaling pathways or at least other PI3K isoforms appear to be involved in the trafficking of immune cells. For example, neutrophil and B-cell migration requires PI3Kδ, whereas T-cell chemotaxis is not impaired in PI3K-deficient mice, but depends on the Rac guanine exchange factor DOCK2.

Several pathways have been identified that can terminate chemokine signaling through their GPCRs. The Gα subunit possesses an intrinsic GTPase activity to hydrolyze GTP. In a negative-feedback loop, this GTPase activity allows the Gα subunits to reassociate with the Gβγ subunits, thereby restoring the heterotrimeric G protein to its inactive state. In addition, another class of molecules, known as regulators of G protein signaling (RGS), also modulates signaling through chemokine GPCRs. RGS are a large and diverse protein family initially identified as GTPase-activating proteins of heterotrimeric G- protein Gα-subunits. At least some RGS can also influence Gα activity through either effector antagonism by competing with effector molecules for GTP-bound Gα-subunits or by acting as guanine nucleotide dissociation inhibitors. To date, over three dozen genes have been identified within the human genome that encode proteins containing an RGS or RGS-like domain. Additionally, chemokine-induced receptor signaling is terminated by specific cytosolic enzymes known as GPCR kinases or GRKs. GRKs are able to rapidly phosphorylate the intracellular domains of the receptors thus, on one hand, abolishing G-protein-mediated signaling and, on the other hand, permitting the association of β-arrestins with the receptors. Such engagement of β-arrestins promotes the internalization of chemokine-triggered GPCRs from the cell membrane into intracellular vesicular compartments with the internalized chemokines targeted for lysosomal degradation and GPCRs either being also degraded or, alternatively, recycled back onto the cell membrane. Thus, pathways that involve GRKs and β-arrestins importantly mediate receptor “desensitization.” The activation of Src-family kinases downstream of the chemokine receptors via not yet fully mapped pathways involving G-proteins- and β-arrestins also contribute to the negative regulation of chemokine-induced migratory responses of myeloid cells. In addition to its well-documented role in shutting down chemokine receptor signaling and contributing to receptor desensitization, β-arrestins also play important roles in mediating some of the cellular effects downstream of chemokine receptors by triggering the activation of multiple intracellular molecular signals, thus “redirecting” signaling from G-proteins to the G-protein-independent pathways.

Remarkably, the molecular mechanisms involving RGS proteins and GRKs contributing to the “negative regulation” of chemokine signaling through their GPCRs might actually be required for optimal chemokine-induced in vitro and in vivo migratory leukocyte responses and their functions in host defense. A mutation (G184S) in Gαi2 protein abolishes its interactions with RGS proteins, thus allowing it to continuously activate the downstream effectors. Neutrophils of mice with such (G184S) mutation showed enhanced rate baseline locomotion but had reduced chemotactic responses in vitro and defective migratory responses in vivo. Analogously, neutrophils deficient of GRK2, the enzyme dominantly involved in desensitization of their chemotactic migration in response to swarming-inducing autocrine chemoattractants, migrated faster, hence covered larger field areas but were functionally less effective, e.g., in locating and killing bacteria.

Atypical Chemokine Receptors

In addition to GPCRs, most chemokines also bind to one or multiple among the “atypical” chemokine receptors (ACKRs). Currently four ACKRs have been included in the nomenclature ( Table 12.3 ), which were shown to bind a broad range of chemokine ligands. It is now clear that at least one additional ACKR exists, pending its inclusion in the systemic nomenclature. Analogously to chemokine GPCRs, ACKRs are serpentine membrane receptors with seven transmembrane domains. However, unlike GPCRs, ACKRs lack or have an altered DRYLAIV consensus motive in the second intracellular loop, which is required for G-protein coupling and signaling through G-proteins. Therefore, upon chemokine binding ACKRs do not trigger signaling events characteristic of GPCRs and cannot mediate cell migration. However, ACKRs internalize chemokines and may target them either into lysosomes or alternatively into transcytotic cellular pathways. ACKRs may also transmit intracellular signals, independently of G-proteins, for example, through triggering biochemical cascades downstream of β-arrestins or affect signaling through classical chemokine GPCRs expressed by the same cell, possibly through receptor heterodimerization or the consumption of secondary signaling molecules. Curiously, ACKRs can heterodimerize with the GPCRs which they share with chemokine ligands, e.g., CXCR4 and ACKR3 or do not share any, e.g., CXCR2 and CCRL2, although, despite several reports, the latter might not be a bona fide ACKR as only its chemerin- but not chemokine-binding activity was independently confirmed. Generally, the main role of ACKRs is to determine in which tissue microenvironments chemokines may or may not exert their activities, achieved either through chemokine scavenging or chemokine transport and presentation in salient tissue microenvironments. Based on the chemokine-scavenging activity of some ACKRs, it has been postulated that the key role of this group of ACKRs is to counteract chemokine effects by reducing the chemokine availability in the tissues and in the organism as a whole. However, all ACKRs are expressed in discrete and often sparse cellular microenvironments, and they most effectively modify chemokine availability within the microenvironments of their cellular expression; although, of note, ACKRs expressed in tissues can affect the levels of chemokines in plasma, with broadly variable impact on leukocyte responses. Mechanistically, scavenging of chemokines by ACKRs also might buffer chemokine “noise” and keep chemokine concentrations within the range of optimal dissociation constants (K _d ) for their cognate GPCRs. Alternatively, by acting as chemokine sinks, ACKRs that are expressed in juxtaposition to the cellular sources of chemokines can facilitate the formation of functional chemokine gradients. Both of such mechanistic outcomes have been suggested for ACKR4, and despite involving chemokine scavenging support the functions of cognate ligands in mediating cell homing into the lymph nodes (LNs) and spleen.

Chemokine Interaction With Glycosaminoglycans

In addition to their classical and atypical receptors, chemokines importantly bind to sulfated glycosaminoglycans (GAG) polysaccharides, heparan sulfate, chondroitin sulfate, and dermatan sulfate. GAGs decorate cell membrane proteoglycans on multiple cells, most notably, endothelial cells where they importantly contribute to the function of the apical surface glycocalyx. Extracellular matrices are also rich in GAGs and their immobilization of chemokines allows for the formation of haptotactic, i.e., substrate-immobilized chemokine gradients that facilitate extravascular leukocyte locomotion. Such haptotactic gradients also spontaneously develop in vitro as chemokines avidly bind to plastics, and therefore Boyden-type and other in vitro chemotaxis assays measure haptotaxis and not chemotaxis, without recognizing this fact. Chemokine binding to GAGs is characterized by a considerable selectivity on parts of both different GAGs molecules and different chemokines, thus facilitating defined patterns of anatomical and microanatomical chemokine localization and availability. Critical involvement of GAGs supporting chemokine functions in vivo is illustrated by abrogated leukocyte homing to LNs and disrupted chemokine-dependent growth and function of the thymus that can be observed in the absence of local heparan sulfate expression. Mechanistically, GAG binding can augment chemokine activities in vitro and in vivo, by protecting them from proteolytic cleavage and promoting their oligomerization, or alternatively, in a diametrically opposite scenario, depending on a cellular context, interfere with chemokine binding to GPCRs. It has been suggested that trimolecular complexes of GAG-chemokine-GPCRs are not likely to exist, at least not for some chemokines, e.g., CXCL8. If indeed, some chemokines are unable to bind to charged surfaces and GPCRs simultaneously, chemokine “presentation” would entail merely their retention on cells and in matrices and therefore should require chemokine release into solution to enable the subsequent binding to GPCRs. However, this cannot be universally true as CCL21 induces in vitro haptotaxis when firmly substrate-attached, even printed on the surfaces as a stable photo-immobilized gradient. In addition to GAGs, other charged molecules can also specifically bind chemokines. These include anionic phospholipid phosphatidylserine that decorates apoptotic cells, thus allowing for their rapid detection by phagocytes and collagen IV, thus allowing chemokine immobilization around LN high endothelial venules (HEVs).

Apart from inducing leukocyte migration, chemokines play multiple additional roles in health and disease. Several chemokines, including CXCL1, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CCL1, CCL19, CCL20, CCL21, CCL25, and CCL28, or their naturally occurring cleaved fragments have been ascribed direct microbicidal activities, albeit to a variable extent and at concentrations orders of magnitude higher than those required for inducing optimal leukocyte migration. Some of these chemokines are generated in infections, and thus might contribute directly to the first line of anti-microbial host defenses. Others, however, are secreted constitutively, e.g., by epithelial cells of exocrine glands and secreted in sweat, saliva, tears, and colostrum and milk, thus could play a prophylactic role in infections and in regulating skin and mucosal microbiota balance. Furthermore, chemokines are fundamentally involved in homeostatic brain functions, pathophysiology of reproduction and birth, embryogenesis, including the development of heart, brain, and lymphatic vasculature. Furthermore, chemokines mediate angiogenic repair, control hematopoiesis, induce cell survival and apoptosis in leukocytes, and other cells, including tumor cells, etc. Curiously “the mother of all chemokines,” CXCL12, is involved in many of such effects outside leukocyte trafficking. As remarkable is the breadth of different chemokine activities, their main and best studied functions involve the regulation of leukocyte trafficking.

In general, leukocyte trafficking can be classified into three distinct patterns of migration: (1) entry into tissues from the circulation; (2) migration within tissues ; and (3) exit from tissues . The following sections will discuss each of these steps in leukocyte trafficking.

Leukocyte Entry Into Tissues

In order to leave the circulation and enter target tissues, leukocytes engage in several sequential steps of adhesion to the endothelial cells, which most often take place in the postcapillary and small venules. Discrete adhesion steps are mediated by binding interactions of pairs of adhesion receptors and their counter-ligands expressed in trans-geometry by leukocytes and endothelial cells. The initial tethering of leukocytes to the endothelial cell is induced by adhesion molecules, which can rapidly bind their ligands with high tensile strength. Prime initiators of leukocyte tethering are selectins, expressed on leukocytes (L-selectin), endothelial cells (E- and P-selectin) and platelets (P-selectin). Selectins have constitutive optimal affinity for their counter-ligands, sialomucins that are decorated with oligosaccharides related to sialyl-Lewis ^x , including P-selectin glycoprotein ligand (PSGL)-1 and the peripheral node addressin (PNAd). Selectin-mediated adhesion bonds that are formed in the bloodstream are transient and do not allow prolonged, firm leukocyte arrest. As tethered leukocytes are pushed along the vessel wall by the blood flow, selectin bonds continuously dissociate at the cells’ upstream end and new ones form downstream, resulting in slow rolling motion characteristic for the leukocyte tethering. To undergo firm adhesion, the rolling leukocyte must engage additional adhesion receptors that belong to the integrin family, particularly CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1) and the α4 integrins, α4β1 (VLA-4), and α4β7. Without exception, individual integrins are expressed by the subsets of leukocytes and their counter-ligands by the endothelial cells.

To efficiently bind their endothelial cell counter-ligands, leukocyte integrins first need to be activated. Such activation is induced by chemoattractant signals that trigger a rapid reversible change in integrin conformation (leading to enhanced ligand binding affinity) and in integrin clustering (enhancing avidity), or both. A subset of chemokines can associate with the luminal surface of microvascular endothelial cells, allowing them to trigger integrin activation and efficiently induce leukocyte arrest, hence designated arrest chemokines . The retention of chemokines on the vessel endothelium is mediated by GAGs in the luminal multifunctional glycocalyx, heparan sulfate in particular. Such apical GAG-immobilized chemokine gradients also drive intraluminal crawling of leukocytes, allowing them to find transmigration hotspots, loci optimal for breaching the endothelial barrier, and migration into the extravascular space. Many chemokines are produced by endothelial cells themselves and either secreted immediately or stored in specialized organelles, the Weibel-Palade bodies, and other storage vesicles and upon secondary stimulation released in the process of regulated exocytosis. Chemokines that are produced extravascularly can be transported across the endothelial barrier to the luminal surface. This is triggered by chemokine binding to the atypical chemokine receptor ACKR1 normally expressed in venular, but not capillary or arterial endothelial cells but upregulated in endothelia of different vascular segments in inflammation. Such chemokine transcytosis by ACKR1 is especially important in microvascular beds characterized by tightest adhesions between EC, e.g., in the brain vasculature. Similar transport mechanisms exist for other tissue-derived chemoattractant as C5a transcytosis across endothelium is accomplished by its atypical receptor C5aR2. However, soluble tissue-derived chemokines, being small proteins, also can freely diffuse from the extravascular space through the endothelial cell junctions into the blood. As a consequence, the chemokine-encoded signals lose their attribution to the sites of their origin and become systemic stimuli in blood. This process is counteracted by ACKR1, which salvages escaping into circulation chemokines by capturing them and presenting them in luminal and junctional membrane microdomains. Importantly, chemokine binding to ACKR1, in contrast to other ACKRs, does not lead to chemokine targeting into lysosomes and their degradation. ACKR1 has been initially described on the red blood cells as the Duffy blood group antigen/receptor for chemokines or DARC where it acts as a chemokine sink and reservoir as well as entry receptor of malarial parasite Plasmodium vivax. Erythrocytes are devoid of transcription and translation, hence ACKR1 is expressed already by their precursors, nucleated erythroid cells of the BM, in which it mediates their cell contacts with HSCs and HPCs and thus contributes to the regulation of hematopoiesis. ACKR1 is the most promiscuous of all chemokine receptors but binds its ligands within a very broad spectrum of affinities. Due to differential affinities of individual chemokines and their monomeric versus dimeric forms, chemokines can compete for ACKR1 binding, shifting the equilibria of free and bound chemokines in the tissues and in plasma.

Chemokines signaling via the Gα _i subfamily of large heterotrimeric G proteins can be inhibited by pertussis toxin (PTX). Consequently, intravital microscopy studies have shown that PTX-treated lymphocytes undergo normal tethering and rolling interactions in HEV in LNs and Peyer patches (PPs) but are unable to undergo integrin-dependent firm arrest. Chemokine receptor activation precipitates a cascade of intracellular signaling and adapter proteins, including Kindlin-3 and RAP-RAPL, that are involved in the so-called inside-out signaling that results in integrin activation. Modifications at the cytoplasmic tails of the integrin α and β chains are critical to regulating leukocyte adhesion to integrin ligands, such as the binding and spreading of neutrophils on intercellular cell adhesion molecule-1 (ICAM-1) and the complement C3 activation product, iC3b. Surprisingly, some chemokines, e.g., CX3CL1 and CXCL12 can directly bind to the alternative bindings sites of leukocyte integrins αvβ3 and α4β1 and allosterically activate them without signaling through their respective GPCRs. Once firmly adherent, leukocytes rapidly polarize and slowly migrate within the vessel in random directions. The intralumenal crawling is thought to be essential to enable leukocytes to find exit points within the vessel through which they can leave the vasculature. A subset of monocytes crawl within normal microvessels under steady-state conditions. These patrolling monocytes are poised to provide immune surveillance of the endothelial cell surfaces and clear the intravascular debris but may also enter the extravascular space in response to damage and infection. Once emigrated, some mononuclear cells may differentiate into macrophages or dendritic cells. In some tissues and organs, intestine, monocyte emigration contributes throughout life to the replenishing of the resident macrophage pool. In other tissues, macrophage and dendritic cell precursors home only during the embryonic period from either the liver or yolk sack and the resident mature cells proliferate in situ to give rise to their progeny.

The interactions between β2-integrins, LFA-1, and Mac-1, with endothelial ICAMs are required for intravascular adhesion and crawling. However, the specificity of these interactions differs between different leukocytes, e.g., neutrophils and monocytes. Neutrophil luminal crawling is mainly mediated by Mac-1, whereas monocytes and T cells use LFA-1, Recent studies using blocking antibodies against Mac-1 and LFA-1 showed that crawling patterns of monocytes and neutrophils differ at steady-state versus inflammatory conditions; both LFA-1 and Mac-1 contribute to monocyte crawling; however, the LFA-1–dependent crawling in unstimulated venules becomes Mac-1 dependent upon inflammation. By contrast, Mac-1 alone is responsible for neutrophil crawling in both unstimulated and cytokine-stimulated venules. This indicates that differences in monocyte and neutrophil crawling behavior result from involvement of different β2 integrins and consequently affect the next step of the leukocyte migration cascade, the transendothelial migration.

The transendothelial migration or diapedesis is a critical event allowing leukocytes to cross the vascular wall and enter their target tissue. Two main routes of leukocyte diapedesis have been observed: a paracellular route that dominates most extravasation processes and a transcellular route, reported for neutrophils and T cells. Both routes involve the action of apical and junctional endothelial intercellular adhesion molecule-1 (ICAM-1) and, at least in some settings, also vascular cell adhesion molecule-1 (VCAM-1). Under inflammatory conditions, additional junctional endothelial ligands such as PECAM-1, VE-cadherin, ESAM, CD99, CD99L2, and junctional adhesion molecules (JAMs) can contribute to leukocyte diapedesis. Different subsets of leukocytes use variations on these molecular mechanisms to emigrate into the tissues.

After penetration of the endothelial barrier, leukocytes may move further within the interstitium towards their target destinations in the tissue. This locomotion is considered to reflect in vivo chemotaxis, increased rate directed cell locomotion driven by the putative gradients of chemoattractants. Several signaling pathways have been proposed to be involved in this gradient-driven process, the most predominant being the PI3K pathway. Thereby leukocytes use an “internal compass” for sensing the direction of chemotactic gradients, and undergo polarization characterized by the formation of lamellipodia at the leading edge of the cell and an uropod at the trailing edge. Chemokines released by a broad range of tissue cells, epithelial cells, mast cells, smooth muscle cells, fibroblasts, myocytes, and tissue-resident immune cells may form gradients and mediate the leukocyte chemotaxis in the interstitium. In this context, the spatiotemporal formation of chemokine gradients in the interstitial tissue may be supported by GAGs, which immobilize chemokines and, thus, determine the position and temporal persistence of chemokine gradients. It has been shown that immobilized, substrate-bound chemokines are effective in inducing directed leukocyte migration, haptotaxis. It has also been suggested that efficient development of chemokine gradients requires, in addition to the free diffusion of chemokines from their cellular sources, also scavenging by their ACKRs expressed by cells in apposition to chemokine sources. Additionally, responding leukocytes may also scavenge chemokines, thus participating in the formation of their functional gradients. Leukocytes moving within the interstitial tissue receive signals from neighboring cells as well as from the extracellular matrix, activate intracellular processes, release inflammatory mediators, and upregulate adhesion molecules and release enzymes. Neutrophils display enhanced expression of β1-integrins upon transmigration into the tissues, and integrins contribute but are not absolutely required for interstitial leukocyte migration. Importantly, the involvement of integrins is different in 2D versus 3D settings. Integrins are dispensable for migration through the 3D tissue fiber networks that confine and mechanically anchor cells from all sides so that they intercalate alongside and perpendicular to tissue structures as shown for integrin-deficient DCs. However, migration on 2D surfaces was shown to be integrin-dependent.

All consecutively occurring steps of (1) leukocyte tethering and rolling, (2) exposure to a chemotactic stimulus, (3) firm arrest, (4) post-adhesive strengthening and intralumenal crawling, (5) diapedesis, and (6) interstitial migration are essential for leukocytes to migrate to sites of inflammation. Accordingly, genetic defects in any of the molecules involved in either step lead to insufficient host defenses. Patients with leukocyte adhesion deficiency (LAD) syndrome, may have a genetic defect in β2 integrins (type 1) or in fucosylated selectin ligands (type 2) and neutrophils either cannot stop or cannot roll, respectively. LAD syndrome is characterized by marked leukocytosis and frequent and severe soft-tissue infections. Another LAD variant referred to as LAD3 is caused by mutations affecting the FERMT3 gene that encodes focal adhesion protein kindlin-3, resulting either in the lack of this protein or its defective function in activating leukocyte and platelet integrins.