Integrin Regulation during Leukocyte Recruitment

Precise targeting of leukocytes to their marked battlegrounds is a key strategy for victory by the immune defense (1). Although this complex process has been a subject of intensive research for decades (2), many of the involved signaling pathways are still poorly understood (3). Different sequential recruitment steps have been identified that each necessitates distinct functioning of particular members of the integrin family of adhesion molecules (1). These plasma membrane proteins feature a dual role of sensing and interacting with the surrounding environment (4). Even though various stimuli are known to activate integrins in this context, the molecular details of the involved signaling pathways are the subject of ongoing research. A comparably new aspect is the idea of integrin regulation by surrounding tissues at the site of inflammation. Recent publications identified physiological interceptions on all levels of integrin activation: modulating the expression of signaling receptors and ligands, curbing signaling pathways, and antagonizing ligand binding of integrins.

Although several details of the molecular mechanisms have been unraveled, the current knowledge of the involved pathways is still fragmentary. Some parts, such as selectin-mediated slow leukocyte rolling and transmigration, are better understood than, for instance, G protein–coupled receptor (GPCR) signaling leading to integrin activation. Several in vitro findings, such as rolling of monocytes at the site of inflammation, have yet to be confirmed under physiological conditions. Furthermore, the interaction among different integrins and their mutual activation during crawling and transmigration are still poorly defined.

Genetic mutation seen in patients with leukocyte adhesion deficiency (LAD) syndromes provides a demonstration of the importance of these pathways: LAD-I is caused by mutations in the β₂-integrin family that is predominantly involved in leukocyte/endothelial interactions (5). These patients present with recurrent infections, as leukocytes are unable to adhere and recruit to the site of inflammation. Patients with LAD-III additionally present with bleeding disorders. This mystery was solved when the responsible mutations were found to affect kindlin-3, a protein essential not only for β₂-integrin activation on leukocytes, but also for β₃-integrin activation on platelets (5).

Multiple animal studies have demonstrated promising clinical potential of therapeutic use of targeting integrin activation. However, clinical trials have not shown the anticipated success. One reason for this observation could be the importance of integrins in other key physiological systems (6). A better understanding of the involved molecular signaling pathways and physiological regulation mechanisms may unravel opportunities to tackle leukocyte recruitment and inflammation more specifically and, ultimately, more successfully.

Integrins are adhesion proteins affecting a wide variety of cellular functions (7). Beyond their integral role in immune surveillance and leukocyte trafficking, integrins are critical players in development, hemostasis, and cancer and influence key cell cycle events (7). During inflammation, integrins are linked to a diverse set of crucial processes that include anchoring leukocytes to the extracellular matrix (inside–out signaling) and mediating signals in response to the surroundings either by binding of extracellular matrix proteins or ligands expressed on the surface of other cells (outside–in signaling) (4).

To fulfill these duties, integrin assembly constitutes obligate noncovalently-bound heterodimers (8). At least 18 α- and 8 β-subunits have been identified in mammals, which generate 24 distinct integrins. However, several different splice variants are known, suggesting that the biological diversity of the integrin family is greater than currently recognized (7). The various leukocyte subsets in the circulation display different combinations of integrins. Neutrophils express predominantly β₂-integrins, but also low amounts of β₁- and β₃-integrins. Monocytes express β₁- and β₂-integins, whereas lymphocytes possess a pattern of β₁-, β₂-, and β₇-integrins varying with subtype and state of activation (9). We focus in this review on β₂-integrins and α₄β₁, as these are the predominant molecules mediating interactions with the endothelium.

Integrins are composed of two noncovalently-linked type I transmembrane glycoproteins, termed α- and β-subunits. Both subunits possess a large extracellular domain, a transmembrane domain, and a short cytoplasmic tail (10). The intracellular tail of the β-subunit links the molecule to the cytoskeleton. Close association of the α- and β-chains was found to aid in keeping the molecule in a quiescent state. The extracellular chains lie in close proximity, forming a ligand-binding site at the N-terminal ends featuring an I domain (or A domain) that may lie on either the α-chain or on the β-chain. This metal ion-dependent adhesion binding site is crucial for ligand binding and requires the presence of calcium or magnesium as coenzyme.

Ligand affinity and avidity are actively altered by activation of characteristic signaling pathways in an inside–out fashion (3). “Affinity” is a term for the strength of monovalent protein/ligand interaction, that is, the inverse likelihood of protein/ligand dissociation. “Avidity” describes the ability to form multiple interactions and describes the combined, synergistic formation of bonds. Binding of ligands to the integrin in turn evokes signaling in an outside–in direction, causing profound alterations to cell physiology (4).

On resting cells, integrins are displayed in their closed or “bent” conformation, offering a low binding affinity for ligands, representing the inactive form of the molecule (10). Upon activation, the conformation of the molecule erects to an extended shape, thereby adopting a high-affinity conformation for ligands (10). In this conformation, the α- and β-subunits are found farther apart, an observation that may reflect active rearrangement by binding of the cytoskeletal proteins talin-1 and kindlin-3 to the cytoplasmic β-chain (11). However, shear stress was found to passively move the two chains apart and stabilize ligand binding. Therefore, shear stress is likely to constitute a prerequisite for physiological β₂- and α₄β₁-integrin functioning at the vessel wall (12). Crystallographic analysis has revealed a third, intermediate conformation for LFA-1, giving rise to the current model of at least three distinct activation states (10). It has been postulated that each of these conformations only describes a favored conformation for a defined activation level whereas the other conformations may also exist at the same time (10).

Furthermore, regulation of avidity is an important feature of integrin adhesiveness that has received increasing attention (13). However, the contribution of avidity to adhesiveness, its investigation in vivo, and the cell’s active contribution to its regulation still pose a challenge for researchers, as changes of avidity and affinity physiologically often go hand in hand (13). Formation of artificial clusters by crosslinking of integrins is commonly used to study outside–in signaling as it evokes a strong stimulus into the cell (14). For LFA-1, high-resolution mapping of its surface distribution has revealed at least three different avidity patterns: randomly distributed molecules, ligand-independent nanoclusters, and ligand-triggered macroclusters (15). Only preformed nanoclusters can be dynamically recruited to the cell/cell interface to form macroclusters (15).

Leukocyte recruitment into inflamed tissue follows a well-defined cascade of events, beginning with capturing of free-flowing leukocytes to the vessel wall, followed by rolling, adhesion to endothelial cells, postadhesion strengthening, crawling, and finally transmigration (Fig. 1A). During these steps, different integrins have to be activated via inside–out signaling. Each step necessitates a different set of integrins that differs among leukocyte subsets. Similarly, different activating stimuli trigger distinct signaling pathways during every step.

Capturing of leukocytes from the bloodstream is initially mediated by selectins displayed on the endothelial luminal surface and their corresponding counterreceptors on leukocytes (16). This initial contact triggers a signaling cascade leading to the activation of integrins on the rolling leukocyte to an intermediate-affinity conformation effectively slowing down the rolling velocity of the cells (hence the term “slow rolling”) (16, 17).

Integrin activation in leukocyte rolling is best understood in neutrophils rolling on E-selectin. E-selectin binding to PSGL-1 induces the phosphorylation of the ITAM containing adaptor proteins DAP12 and FcRγ (18). This step is defective in mice lacking either the Src kinase Fgr or both Hck and Lyn (19): neutrophils from these mice fail to consecutively phosphorylate the spleen tyrosine kinase (Syk) (19). Syk activates a protein complex of the Src homology 2 domain–containing leukocyte phosphoprotein of 76 kDa (SLP76) and the adhesion and degranulation–promoting adaptor protein (ADAP) (20). SLP76, in turn, activates Bruton tyrosine kinase (20). Both Bruton tyrosine kinase and ADAP are needed to signal downstream via two independent branches (20): one is phospholipase C (PLC)γ2 dependent and comprises Ca²⁺ and 1,2-diacylglycerol–regulated guanine nucleotide exchange factor (GEF)I, p38 MAPK, and Rap1a (21). The other pathway may be PI3Kγ dependent (22). However, McEver and colleagues (19) found E-selectin–mediated rolling to be independent of PI3K by using in vitro assays. These differences can be explained by the fact that isolated neutrophils were used. The isolation process activates neutrophils, which may have hampered the detection of a partial phenotype. In another study, no differences in rolling velocities were obtained in vivo (23). However, in this assay no blocking anti–P-selectin Ab and no pertussis toxin were used to demask a defect in E-selectin–mediated slow leukocyte rolling.

The importance of integrins in monocyte and T cell rolling is less well studied. In vitro studies suggest a predominant role of VLA-4 for monocyte rolling, as blockade of β₂-integrins by mAbs had no effect on rolling flux and cell adhesion on IL-4–activated HUVECs (24). However, IL-4 induces VCAM-1 expression in HUVECs without affecting E-selectin or ICAM-1 expression (24). Similar results were found on atherosclerotic endothelium (25), on which VCAM-1 is also highly expressed. In an in vitro model of monocyte adhesion to the injured vessel wall, Kuijper et al. (26) found that Mac-1 is the predominant integrin mediating monocyte rolling on ECM alone and that the interaction of PSGL-1 with P-selectin as well as Mac-1 accounts for nearly all the rolling interactions on activated platelets bound to ECM substrates at low shear stress.

CD4⁺ T cells were also found to roll on P- and E-selectin (27). CD8⁺ T cells roll on TNF-α– and IFN-γ–treated vessels in a VLA-4–dependent manner (28). VLA-4 seems to be the predominant integrin mediating lymphocyte rolling (29); however, physiological relevance of monocyte and T cell rolling for recruitment to peripheral tissues and involved signaling pathways affecting integrin affinity and avidity are still unclear. Details of lymphocyte rolling on HEV and details of lymphocyte trafficking in lymph nodes are described elsewhere (30).

In most tissues rolling or tethering is insufficient to permanently target the leukocyte to the vessel wall: without further stimuli the cell detaches. During rolling, however, leukocytes pick up inflammatory signals presented on the endothelial cells that may trigger arrest (31). On a cellular level, leukocyte arrest is mediated by the activation of GPCRs that mediate activation of integrins to their extended, high-affinity conformation, resulting in binding of ligands such as ICAM-1 or VCAM-1 (32).

Lateral movement of integrins on the cell surface following arrest results in an accumulation of integrins at the site of endothelial ligand presentation, forming an integrin “cluster” at the leukocyte/endothelial border. This increase in avidity may represent a trigger event for outside–in signaling, thus marking a crucial event for postadhesion strengthening (33). Lateral movement of LFA-1 in T cells requires active release from cytoskeletal constraints after GPCR engagement (34). Furthermore, Ca²⁺ flux was shown to be essential for integrin clustering (35). Striking differences of GPCR-mediated alterations of integrin avidity for VLA-4/VCAM-1 and LFA-1/ICAM-1 have been observed on lymphocytes: whereas chemokine-induced rearrangement of GPCRs within cholesterol rafts was a prerequisite for subsequent enhancement of VLA-4 avidity, disruption of these rafts did not affect LFA-1 avidity changes (36).

Although the GPCR signaling triggered by various chemokines is a key step in leukocyte recruitment, molecular details of this integrin-activating pathway remain elusive (3). The elevation of intracellular calcium, the calcium-binding messenger protein calmodulin, and inositol-1,4,5-triphosphate receptors as downstream events of PLC activation were found to be involved in GPCR-mediated VLA-4 activation following stimulation with fMLP or CXCL12 in monocytes (37). Neither p38 MAPKs, PI3K, or, most interestingly, PKC was necessary to achieve VLA-4 activation and subsequent binding to VCAM-1 (37). In contrast, neutrophil response to chemokines uses distinct PI3K isoforms over time: early responses were found to be PI3Kγ dependent, whereas prolonged recruitment of neutrophils following MIP-2 or TNF-α superfusion were PI3Kδ dependent (23).

Farther downstream, the small GTPase Rap1 was found to be involved in Mac-1 activation in a similar assay (38). In CD3⁺ T cells Rap1 is required for LFA-1 and VLA-4 activation (32). GTPases in turn are activated by GEFs. In neutrophils stimulated with leukotriene B₄, Rap1 is activated by Ca²⁺ and 1,2-diacylglycerol–regulated GEFI, which is activated by Ca²⁺ and DAG, two products of PLC (39). In contrast, another study using fMLP stimulation revealed that the GEFs Vav1 and P-Rex1 redundantly mediate the activity of the GTPase Rac (40), suggesting that the different stimuli activate distinctly different pathways. A similar pathway was also demonstrated in human CD3⁺ T cells following the stimulation with CXCL12 leading to LFA-1, but not VLA-4, activation (32). However, VLA-4 activation was dependent on Rac1 activation by Vav1 alone following stimulation with CXCL12 (41). A recently published study demonstrated for the first time that the GTPase Cdc42 negatively regulates LFA-1 activation upon chemokine stimulation (42). This observation was very surprising because of the high homology of the different GTPases. The band 4.1/ezrin/radixin/moesin domain-containing proteins kindlin-3 and talin-1 were found to be necessary for CXCL1-triggered LFA-1 activation in neutrophils (43). Surprisingly, only talin-1 is required for inducing LFA-1 extension, which corresponds to intermediate affinity and induces neutrophil slow rolling (43). Kindlin-3 was also found to be involved in clustering of LFA-1 on lymphocytes (44), a finding that also explains the defect in integrin clustering seen in patients with leukocyte adhesion deficiency III syndrome (45).

Another surpising finding was the paradoxical role of cytohesin-1 upon activation of the β₂-integrins LFA-1 and Mac-1 in neutrophils. Following GPCR stimulation with fMLP, cytohesion-1 interacts with the cytoplasmatic integrin tail, promoting activation of LFA-1 and hampering Mac-1 activation (46), suggesting closely related yet specific regulation of each integrin following GPCR stimulation.

In summary, VLA-4 and LFA-1 seem to be the predominant integrins mediating T cell adhesion, whereas monocytes adhere in a VLA-4– or β₂-integrin–dependent manner and neutrophil adhesion is LFA-1 dependent. Furthermore, the published data suggest that PLC activation seems to be a key event in GPCR-mediated integrin activation (32, 37). The available data indicate that the involved GEFs and GTPases not only vary among cell types but also among signaling pathways targeting different integrins. However, the signaling mechanics leading to integrin activation after chemokine stimulation remain poorly understood. Future studies, especially on the signaling molecules directly interacting with integrins, such as talin, kindlin, and cytohesin-1, will provide us with a clearer picture of what allows differential activation of specific integrins after different stimuli.

Crawling was first observed in vitro and initially termed “locomotion”: monocytes that adhered to HUVECs were found to actively move to cellular junctions to start transmigration within seconds (47). However, blocking of CD18, ICAM-1, or ICAM-2 rendered the monocytes unable to locate endothelial cell junctions and caused the cells to wander during 90 min without effectively initiating transmigration (47). Blockade of ICAM-1 and ICAM-2 still allowed movement of monocytes, whereas blockade of CD11a and CD11b resulted in immobile monocytes “pirouetting” on their uropod (47). Monocytes crawling along the vasculature in vivo have also been termed “patrolling” (48). In contrast to the in vitro findings on TNF-α– or IL-1β–stimulated HUVECs, injection of a blocking LFA-1, but not Mac-1, Ab completely and rapidly detached crawling monocytes in vivo, indicating a predominant role of LFA-1 in vivo (48). Also, mice lacking CX₃CR1 revealed a decrease of adherent monocytes by two-thirds and 50% reduction in average crawling distance while maintaining crawling velocities of wild-type animals (48). These data suggest a crucial role for GPCR-mediated integrin activation in monocyte crawling. A recent study suggests that monocyte crawling may reflect expression of surface proteins: on unstimulated venules, monocytes were found to crawl in an LFA-1–dependent manner, but after stimulation with TNF-α and upregulation of adhesion molecules on endothelial cells, crawling became Mac-1 dependent (49). This could potentially explain the differences seen in vitro and in vivo.

Similarly, neutrophils crawl on the endothelium following firm arrest in vivo (50). Interestingly, crawling was unaffected by blocking ICAM-2, but injection of a blocking ICAM-1 Ab almost completely abolished neutrophil crawling (50). In contrast to the in vivo findings for monocyte crawling, blockade of LFA-1 did not affect neutrophil crawling. However, injection of a blocking Mac-1 Ab significantly decreased the percentage of crawling cells and dramatically decreased distance and velocity (50). Based on these data, Mac-1, and not LFA-1, is the central integrin mediating neutrophils crawling in vivo (50). Neutrophils lacking the GEF Vav1 were found to have an impaired ability to crawl perpendicular to flow (51). Because Vav1 has been shown to be involved in LFA-1 outside–in signaling mediating cytoskeletal rearrangements in neutrophils (52), these findings led to the hypothesis that activation of Mac-1 via LFA-1–mediated outside–in signaling is a prerequisite for crawling (51). However, the molecular pathways leading to initiation of crawling and activation of Mac-1 in this context are currently unknown.

Lymphocyte crawling was also observed in vitro. Interestingly, crawling of T cells on HUVECs is LFA-1 dependent and involves very distinct mechanics: multiple scattered traction sites form by engagement of high-affinity LFA-1 with endothelial ICAM-1, and these form in a rapid turnover fashion resulting in “millipede-like” translocation over the endothelium that is triggered by chemokine CXCL12 (53). Also, it was found that Cdc42 and Rap1 are involved in T cell crawling on HUVECs but are independent of Src kinases (53).

These findings suggest different modes of movement for different leukocyte subtypes, ranging from the scattered forming of high-affinity LFA-1/ICAM-1 microclusters seen in T cells to crawling seen in neutrophils that use the integrin Mac-1. However, the molecular events involved in the transition from adhesion to crawling are still largely elusive.

At the preferred site of transmigration, integrin binding to ICAM-1 and VCAM-1 constitutes central players in the orchestra of protein interactions involved in both para- and transcellular transmigration (54). Activated endothelium forms ring-like membrane structures enriched with ICAM-1 and VCAM-1 that are maintained for the duration of transmigration (55). Recent studies have found evidence that neutrophil integrin engagement of ICAM-1 may trigger the disassembly of the VE-cadherin and consecutive encapsulation of the transmigrating leukocyte in “domes,” as mice lacking cortactin, a cytoskeletal molecule serving as an adaptor protein to ICAM-1, failed to initiate transmigration (56). For a detailed review of the endothelial molecules involved in this step, see Williams et al. (54). Recent studies have revealed that extravasated neutrophils crawl in the space between the endothelial abluminal surface and pericytes in a Mac-1– and LFA-1–dependent manner (57). Following subendothelial crawling, leukocytes maintain connection to the endothelial abluminal surface, leading to uropod elongation and a significantly delayed detachment (up to >20 min) of neutrophils, monocytes, and lymphocytes (58). Leukocytes in mice deficient in VLA-3 were found to have impaired detachment, as they failed to elongate by active movement of the leading edge of the cell (58). In contrast, LFA-1–deficient leukocytes did not elongate and detached quickly (58). These data suggest that once extravasated, LFA-1/ICAM-1 maintain binding of the uropod to the abluminal surface of endothelium while VLA-3 mediates movement of the front of the cell and thus facilitates leukocyte emigration through the basement membrane (58).

Following engagement of ligands, integrins signal into the cell and elicit an array of responses, some of which represent distinct cellular functions (4). Outside–in signaling triggers reactive oxygen species production and degranulation, stabilizes the immune synapse, and mediates cytokine excretion in T cells and helps to differentiate and migrate macrophages (4). Physiologically, however, inside–out and outside–in signaling rarely occur separately (4). Many signaling molecules found in outside–in signaling are also involved in inside–out signaling (4). The ITAM-containing adaptor proteins DAP12 and FcRγ, Src kinases, as well as Syk have found to be involved in outside–in signaling in neutrophils, macrophages, and platelets (59). However, striking differences enable distinct activation of outside–in signaling events: in neutrophils, the Src kinases Hck and Fgr are necessary for outside–in-mediated adhesion strengthening but not for GPCR-mediated integrin activation (33). In contrast to this signaling pathway, only Fgr or Hck and Lyn are required for LFA-1 activation following E-selectin engagement (18, 19), showing that the different Src family kinases expressed in neutrophils have distinct functions. In Jurkat T cells, crosslinking of LFA-1 results in formation of an actin cloud that is dependent on the presence of the Src kinase Lyn (60). Downstream of Syk, phosphorylation of FAK and Pyk was found to be impaired upon integrin engagement in Syk-deficient neutrophils and macrophages (61). Also, SLP76 (62), ADAP (63), and its homolog PRAM-1 (64) were identified to be essential for intact outside–in signaling events in neutrophils, suggesting a dual role for SLP76 and ADAP for outside–in and inside–out signaling. Farther downstream, activation of PI3K and ERK was found to mediate β₁- but not β₂-integrin–mediated NF-κB activation in monocytes following crosslinking, suggesting specific signaling pathways for different integrins despite intriguing similarities (65).

An increasing number of studies have investigated the regulation of integrins at the site of inflammation. In the following text we discuss mechanisms that physiologically regulate leukocyte integrin activation at the site of inflammation. Impairment of these local responses may cause constitutive integrin activation leading to autoimmune diseases, unnecessary tissue damage, and deterioration of organ function (20). Mechanisms that limit leukocyte recruitment by regulating the activation of integrins were identified at several cellular levels (Fig, 1B). Some of these molecules hamper integrin activation by altering the transcription, decreasing the expression of endothelial ligands, activating leukocyte receptors (galectin-1, lipoxin A₄, docosahexaenoic acid [DHA]), or inhibiting the upregulation of integrins (lipoxin A₄, resolvin D₁, resolvin D₂), whereas some interfere with the interaction of integrins with their ligands (developmental endothelial locus-1 [Del-1]) and others interfere with inside–out signaling (growth differentiation factor-15 [GDF-15]). Also, different mechanisms act at different time points of inflammation; for example, galectins act early on, whereas many resolvins predominantly act late in inflammation.

One important regulation mechanism is the alteration of integrin expression or the expression of their receptors. Galectin-1 is an endothelial-derived protein of the β-galactoside–binding lectin family. Preincubation of neutrophils with galectin-1 decreased the rolling flux of neutrophils on HUVECs (66). This effect is partially mediated by a pronounced decrease in the expression of PSGL-1, CD11b (Mac-1), and L-selectin on the cell surface of neutrophils (66). These findings were confirmed in a mouse model of IL-1β–induced inflammation of the m. cremaster that revealed increased numbers of recruited neutrophils in galectin-1–deficient mice (66). Although galectin-3 has been found to improve the clinical picture of experimental models of LPS-induced inflammation (67), the underlying mechanisms remain unclear, especially because galectin-3 has been shown to possess proinflammatory qualities (68).

Lipid mediators including resolvins and lipoxins are involved in the resolution of inflammation (69). These mediators modulate the expression of integrins and receptors involved in integrin activation. Lipoxin A₄ downregulates the expression of endothelial P-selectin and Mac-1 on neutrophils (69). In a similar fashion, resolvin D₂ is able to blunt the upregulation of β₂-integrins on neutrophils evoked by platelet-activating factor superfusion (70). Resolvin D₁ can counteract leukotriene B₄–stimulated expression of β₂-integrins via the GPCRs GPR32 and ALX (formyl peptide receptor 2) (71). The omega-3 polyunsaturated fatty acid DHA was described to reduce the expression of endothelial ICAM-1 and VCAM-1 after proinflammatory stimuli (71). However, many actions of the aforementioned molecules require further investigation. In particular, resolvins have complex impacts on inflammation, as they are capable of preventing organ damage while promoting clearance of intruding organisms (69).

Pentraxin-3 (PTX-3), a member of the family of long pentraxins, directly inhibits leukocyte/endothelial interaction and thus indirectly prevents integrin activation. Being a recognized player in the innate immune system for several years, PTX-3 was found to protect mice in a model of acute myocardial infarction (72), whereas deficiency in PTX-3 elicited overshooting neutrophil infiltration in pleural inflammation and acid-induced acute lung injury (73). However, the responsible mechanism was just identified recently. Leukocyte-derived PTX-3 binds to P-selectin on endothelial cells and thus blocks the interaction between P-selectin and PSGL-1 on leukocytes, leading to an inhibition of leukocyte capturing and rolling and integrin activation via inside–out signaling (73).

Upregulation of integrin affinity and avidity and subsequent binding of the activated integrin to ligands is a hallmark of inside–out signaling. The 52-kDa glycoprotein Del-1 has been shown to interfere with the binding of the activated integrin to its ligand: as a potential LFA-1 ligand, endothelial-derived Del-1 features a higher affinity compared with ICAM-1 and thus functions as a competitive antagonist, effectively hampering leukocyte adhesion (74). Accordingly, mice lacking endothelial Del-1 displayed increased neutrophil infiltration in a model of LPS-induced pulmonary infiltration (74).

A recent publication identified the TGF-β superfamily member GDF-15 as an endogenous mediator that can directly prevent GPCR-mediated integrin activation and thus limit leukocyte recruitment and inflammation (75). GDF-15 is the first cytokine identified to directly interfere with the chemokine signaling leading to integrin activation (75). GDF-15 was initially found to be upregulated in cardiomyocytes after experimental cardiac infarction. Mice deficient in GDF-15 revealed increased neutrophil infiltration, rates of myocardial rupture, and overall mortality (75). Therefore, GDF-15 is thought to locally limit inflammation in terms of a protective homeostatic mechanism following ischemic injury to the heart (75). Our knowledge at present indicates that GDF-15 engages a currently unknown receptor on leukocytes, effectively activating the small GTPase Cdc42 and thereby counteracting upregulation of integrin affinity and avidity (75). GDF-15 also potently inhibited binding of monocytes to VCAM-1 by interception of VLA-4 activation (75).

Interestingly, GDF-15 can also inhibit integrins on other cells. Pretreating platelets with GDF-15 selectively inhibits agonist-dependent activation of the integrin GPIIb/IIIa without affecting other platelet functions (76).

Our present knowledge of the regulation of integrin adhesiveness in leukocyte recruitment is rudimentary despite significant advancements in recent years. Integrin activation during the different steps of the leukocyte adhesion cascade is the result of a fine-tuned orchestra of activation pathways and local regulatory circuits whose malfunctioning may cause severe disease patterns. Although rolling has been described for neutrophils, monocytes, and lymphocytes, the physiological relevance of nonneutrophil rolling is unclear, and so are pathways affecting integrin adhesiveness in these cells. Although signaling details during the adhesion step are cursory, a common first step after GPCR activation by chemokines appears to be the activation of PLC and the downstream activation of GEFs and small GTPases. During crawling, transmigration, and detachment, corresponding triggers and molecular pathways steering integrin functions remain almost completely elusive.

Several molecules regulating integrin adhesiveness at the site of inflammation have been described, impacting different stages of integrin activation. Taken together, these molecules implicate a complex regulatory network at the site of inflammation of which only a fraction of players are known today. Future research on the mechanics of integrin activation and regulation are needed to complete our picture of its physiological detail and hopefully reveal opportunities to clinically target integrins in leukocyte recruitment more successfully.

We thank Francis W. Luscinskas for critical revision of the manuscript. We apologize to all colleagues whose work we could not cite owing to space limitations.

The authors have no financial conflicts of interest.