Recruitment Integrin Regulation During Leukocyte

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Integrin Regulation during Leukocyte Recruitment Jan Herter and Alexander Zarbock This information is current as J Immunol 2013; 190:4451-4457; ; of September 24, 2021. doi: 10.4049/jimmunol.1203179 http://www.jimmunol.org/content/190/9/4451 Downloaded from References This article cites 76 articles, 40 of which you can access for free at: http://www.jimmunol.org/content/190/9/4451.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Integrin Regulation during Leukocyte Recruitment Jan Herter*,†,‡ and Alexander Zarbock*,† Integrins are recognized as vital players in leukocyte re- integrin activation: modulating the expression of signaling cruitment. Integrin malfunction causes severe disease receptors and ligands, curbing signaling pathways, and antag- patterns characterized by the inability to fight patho- onizing ligand binding of integrins. gens. Although inflammatory reactions are beneficial Although several details of the molecular mechanisms have and necessary for host defense, these reactions have to been unraveled, the current knowledge of the involved pathways be controlled to prevent tissue destruction and harmful is still fragmentary. Some parts, such as selectin-mediated slow sequelae. In this review, we discuss the different signaling leukocyte rolling and transmigration, are better understood than, pathways leading to the change of integrin adhesiveness for instance, G protein–coupled receptor (GPCR) signaling leading to integrin activation. Several in vitro findings, such as in neutrophils, monocytes, and lymphocytes. We thereby Downloaded from focus on the importance of integrin activation for the dif- rolling of monocytes at the site of inflammation, have yet to be ferent steps of the leukocyte recruitment cascade, in- confirmed under physiological conditions. Furthermore, the interaction among different integrins and their mutual activation cluding rolling, adhesion, postadhesion strengthening, during crawling and transmigration are still poorly defined. intravascular crawling, and transmigration, as each step Genetic mutation seen in patients with leukocyte adhesion necessitates the proper functioning of a distinct set of deficiency (LAD) syndromes provides a demonstration of the integrin molecules that has to be activated specifically. importance of these pathways: LAD-I is caused by mutations http://www.jimmunol.org/ Additionally, we discuss endogenous mechanisms that in the b2-integrin family that is predominantly involved in balance and counteract integrin activation and limit leukocyte/endothelial interactions (5). These patients present leukocyte recruitment at the site of inflammation. Fur- with recurrent infections, as leukocytes are unable to adhere ther insight into these complex mechanisms may provide and recruit to the site of inflammation. Patients with LAD-III new approaches for developing new anti-inflammatory additionally present with bleeding disorders. This mystery was therapies. The Journal of Immunology, 2013, 190: solved when the responsible mutations were found to affect 4451–4457. kindlin-3, a protein essential not only for b2-integrin activation on leukocytes, but also for b3-integrin activation on by guest on September 24, 2021 recise targeting of leukocytes to their marked battle- platelets (5). grounds is a key strategy for victory by the immune Multiple animal studies have demonstrated promising clini- P defense (1). Although this complex process has been cal potential of therapeutic use of targeting integrin activation. a subject of intensive research for decades (2), many of the However, clinical trials have not shown the anticipated success. involved signaling pathways are still poorly understood (3). One reason for this observation could be the importance of Different sequential recruitment steps have been identified integrins in other key physiological systems (6). A better un- that each necessitates distinct functioning of particular derstanding of the involved molecular signaling pathways and members of the integrin family of adhesion molecules (1). physiological regulation mechanisms may unravel opportuni- These plasma membrane proteins feature a dual role of ties to tackle leukocyte recruitment and inflammation more sensing and interacting with the surrounding environment specifically and, ultimately, more successfully. (4). Even though various stimuli are known to activate integrins in this context, the molecular details of the involved Integrin physiognomy signaling pathways are the subject of ongoing research. A Integrins are adhesion proteins affecting a wide variety of comparably new aspect is the idea of integrin regulation by cellular functions (7). Beyond their integral role in immune surrounding tissues at the site of inflammation. Recent pub- surveillance and leukocyte trafficking, integrins are critical lications identified physiological interceptions on all levels of players in development, hemostasis, and cancer and influence

*Department of Anesthesiology, Intensive Care, and Pain Medicine, University of ing A1, University of Mu¨nster, 48149 Mu¨nster, Germany. E-mail address: zarbock@ Mu¨nster, 48149 Mu¨nster, Germany; †Max Planck Institute for Molecular Biomedi- uni-muenster.de cine, 48149 Mu¨nster, Germany; and ‡Center for Excellence in Vascular Biology, De- Abbreviations used in this article: ADAP, adhesion and degranulation–promoting adap- partment of Pathology, Brigham and Women’s Hospital, and Harvard Medical School, tor protein; CalDAG-GEFI, Calcium diacylglycerol guanine nucleotide exchange factor Boston, MA 02115 I;Del-1,developmentalendotheliallocus-1;DHA,docosahexaenoicacid;GDF-15, Received for publication November 19, 2012. Accepted for publication February 17, growth differentiation factor-15; GEF, guanine nucleotide exchange factor; GPCR, G 2013. protein–coupled receptor; LAD, leukocyte adhesion deficiency; PLC, phospholipase C; PTX-3, pentraxin-3; SLP76, Src homology 2 domain–containing leukocyte phospho- This work was supported by German Research Foundation Grants AZ 428/3-1, AZ 428/ protein of 76 kDa; Syk, spleen tyrosine kinase. 6-1, SFB 1009/A5 (to A.Z.), and HE-6810/1-1 (to J.H.) and by a grant from the Interdisciplinary Center of Clinical Research (to A.Z.). Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 Address correspondence and reprint requests to Dr. Alexander Zarbock, Department of Anesthesiology, Intensive Care, and Pain Medicine, Albert Schweitzer Campus 1, Build- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1203179 4452 BRIEF REVIEWS: INTEGRIN REGULATION DURING LEUKOCYTE RECRUITMENT key cell cycle events (7). During inflammation, integrins are Furthermore, regulation of avidity is an important feature of linked to a diverse set of crucial processes that include an- integrin adhesiveness that has received increasing attention choring leukocytes to the extracellular matrix (inside–out sig- (13). However, the contribution of avidity to adhesiveness, its naling) and mediating signals in response to the surroundings investigation in vivo, and the cell’s active contribution to its either by binding of extracellular matrix proteins or ligands ex- regulation still pose a challenge for researchers, as changes of pressed on the surface of other cells (outside–in signaling) (4). avidity and affinity physiologically often go hand in hand To fulfill these duties, integrin assembly constitutes obligate (13). Formation of artificial clusters by crosslinking of in- noncovalently-bound heterodimers (8). At least 18 a- and 8 tegrins is commonly used to study outside–in signaling as it b-subunits have been identified in mammals, which generate evokes a strong stimulus into the cell (14). For LFA-1, high- 24 distinct integrins. However, several different splice variants resolution mapping of its surface distribution has revealed at are known, suggesting that the biological diversity of the least three different avidity patterns: randomly distributed integrin family is greater than currently recognized (7). The molecules, ligand-independent nanoclusters, and ligand- various leukocyte subsets in the circulation display different triggered macroclusters (15). Only preformed nanoclusters combinations of integrins. Neutrophils express predominantly can be dynamically recruited to the cell/cell interface to form b2-integrins, but also low amounts of b1- and b3-integrins. macroclusters (15). Monocytes express b1- and b2-integins, whereas lymphocytes Leukocyte recruitment possess a pattern of b1-, b2-, and b7-integrins varying with subtype and state of activation (9). We focus in this review on Leukocyte recruitment into inflamed tissue follows a well- Downloaded from b2-integrins and a4b1, as these are the predominant mole- defined cascade of events, beginning with capturing of free- cules mediating interactions with the endothelium. flowing leukocytes to the vessel wall, followed by rolling, Integrins are composed of two noncovalently-linked type I adhesion to endothelial cells, postadhesion strengthening, transmembrane glycoproteins, termed a-andb-subunits. crawling, and finally transmigration (Fig. 1A). During these Both subunits possess a large extracellular domain, a trans- steps, different integrins have to be activated via inside–out membrane domain, and a short cytoplasmic tail (10). The signaling. Each step necessitates a different set of integrins that http://www.jimmunol.org/ intracellular tail of the b-subunit links the molecule to the differs among leukocyte subsets. Similarly, different activating cytoskeleton. Close association of the a- and b-chains was stimuli trigger distinct signaling pathways during every step. found to aid in keeping the molecule in a quiescent state. The Capturing and rolling. Capturing of leukocytes from the blood- extracellular chains lie in close proximity, forming a ligand- stream is initially mediated by selectins displayed on the endo- binding site at the N-terminal ends featuring an I domain (or thelial luminal surface and their corresponding counterreceptors A domain) that may lie on either the a-chain or on the on leukocytes (16). This initial contact triggers a signaling cas- b-chain. This metal ion-dependent adhesion binding site is cade leading to the activation of integrins on the rolling leuko- crucial for ligand binding and requires the presence of calcium cyte to an intermediate-affinity conformation effectively slowing by guest on September 24, 2021 or magnesium as coenzyme. down the rolling velocity of the cells (hence the term “slow Ligand affinity and avidity are actively altered by activation rolling”) (16, 17). of characteristic signaling pathways in an inside–out fashion Integrin activation in leukocyte rolling is best understood (3). “Affinity” is a term for the strength of monovalent in neutrophils rolling on E-selectin. E-selectin binding to protein/ligand interaction, that is, the inverse likelihood of PSGL-1 induces the phosphorylation of the ITAM containing protein/ligand dissociation. “Avidity” describes the ability to adaptor proteins DAP12 and FcRg (18). This step is defective form multiple interactions and describes the combined, syn- in mice lacking either the Src kinase Fgr or both Hck and ergistic formation of bonds. Binding of ligands to the integrin Lyn (19): neutrophils from these mice fail to consecutively in turn evokes signaling in an outside–in direction, causing phosphorylate the spleen tyrosine kinase (Syk) (19). Syk profound alterations to cell physiology (4). activates a protein complex of the Src homology 2 domain– On resting cells, integrins are displayed in their closed or containing leukocyte phosphoprotein of 76 kDa (SLP76) “bent” conformation, offeringalowbindingaffinityfor and the adhesion and degranulation–promoting adaptor ligands, representing the inactive form of the molecule (10). protein (ADAP) (20). SLP76, in turn, activates Bruton ty- Upon activation, the conformation of the molecule erects to rosine kinase (20). Both Bruton tyrosine kinase and ADAP an extended shape, thereby adopting a high-affinity con- are needed to signal downstream via two independent formationforligands(10).Inthisconformation,thea-and branches (20): one is phospholipase C (PLC)g2 dependent b-subunits are found farther apart, an observation that may and comprises Calcium diacylglycerol guanine nucleotide reflect active rearrangement by binding of the cytoskeletal exchange factor I (CalDAG-GEFI), p38 MAPK, and Rap1a proteins talin-1 and kindlin-3 to the cytoplasmic b-chain (21). The other pathway is PI3Kg dependent (22). However, (11). However, shear stress was found to passively move the McEver and colleagues (19) found E-selectin–mediated two chains apart and stabilize ligand binding. Therefore, shear rolling to be independent of PI3K by using in vitro assays. stress is likely to constitute a prerequisite for physiological b2- These differences can be explained by the fact that isolated and a4b1-integrin functioning at the vessel wall (12). Crys- neutrophils were used. The isolation process activates neu- tallographic analysis has revealed a third, intermediate con- trophils, which may have hampered the detection of a partial formation for LFA-1, giving rise to the current model of at phenotype. In another study, no differences in rolling ve- least three distinct activation states (10). It has been postu- locities were obtained in vivo (23). However, in this assay no lated that each of these conformations only describes a favored blocking anti–P-selectin Ab and no pertussis toxin were used conformation for a defined activation level whereas the other to demask a defect in E-selectin–mediated slow leukocyte conformations may also exist at the same time (10). rolling. The Journal of Immunology 4453

FIGURE 1. (A) Integrins in leukocyte recruitment. In each step of the leukocyte recruitment cascade, integrins play a crucial role. During rolling, integrins mediate interaction with the endothelium. After this contact, integrin activation mediates cell arrest, and accumulation of integrins at the endothelial/leukocyte border marks postadhesion strengthening. Leukocytes then crawl over the endothelium before they transmigrate and ﬁnally detach. Integrins found to be involved in each step are listed below. (B) Ways

of integrin regulation. 1) Interaction of endothe- Downloaded from lial selectins with their respective counterreceptors triggers inside–out signaling, leading to integrin activation. 2) Decrease in expression of receptors and ligands: galectin-1, lipoxin A4, and DHA. 3) Blunted upregulation of integrins: lipoxin A4, resolvin D2, resolvin D1. 4) Antagonizing receptor engagement: PTX-3. 5) Interception of inside–out http://www.jimmunol.org/ signaling: GDF-15. 6) Antagonizing integrin engagement: Del-1. by guest on September 24, 2021

The importance of integrins in monocyte and T cell rolling the vessel wall: without further stimuli the cell detaches. During is less well studied. In vitro studies suggest a predominant rolling, however, leukocytes pick up inflammatory signals pre- role of VLA-4 for monocyte rolling, as blockade of b2- sented on the endothelial cells that may trigger arrest (31). On integrins by mAbs had no effect on rolling flux and cell ad- a cellular level, leukocyte arrest is mediated by the activation of hesion on IL-4–activated HUVECs (24). However, IL-4 GPCRs that mediate activation of integrins to their extended, induces VCAM-1 expression in HUVECs without affecting high-affinity conformation, resulting in binding of ligands such E-selectin or ICAM-1 expression (24). Similar results were as ICAM-1 or VCAM-1 (32). found on atherosclerotic endothelium (25), on which VCAM- Lateral movement of integrins on the cell surface following 1 is also highly expressed. In an in vitro model of monocyte arrest results in an accumulation of integrins at the site of en- adhesion to the injured vessel wall, Kuijper et al. (26) found dothelial ligand presentation, forming an integrin “cluster” at that Mac-1 is the predominant integrin mediating monocyte the leukocyte/endothelial border. This increase in avidity may rolling on ECM alone and that the interaction of PSGL-1 represent a trigger event for outside–in signaling, thus marking with P-selectin as well as Mac-1 accounts for nearly all the a crucial event for postadhesion strengthening (33). Lateral rolling interactions on activated platelets bound to ECM sub- movement of LFA-1 in T cells requires active release from cy- strates at low shear stress. toskeletal constraints after GPCR engagement (34). Further- CD4+ T cells were also found to roll on P- and E-selectin more, Ca2+ flux was shown to be essential for integrin clustering (27). CD8+ T cells roll on TNF-a– and IFN-g–treated vessels (35). Striking differences of GPCR-mediated alterations of in a VLA-4–dependent manner (28). VLA-4 seems to be the integrin avidity for VLA-4/VCAM-1 and LFA-1/ICAM-1 have predominant integrin mediating lymphocyte rolling (29); how- been observed on lymphocytes: whereas chemokine-induced ever, physiological relevance of monocyte and T cell rolling rearrangement of GPCRs within cholesterol rafts was a prereq- for recruitment to peripheral tissues and involved signaling uisite for subsequent enhancement of VLA-4 avidity, disruption pathways affecting integrin affinity and avidity are still un- of these rafts did not affect LFA-1 avidity changes (36). clear. Details of lymphocyte rolling on HEV and details Although the GPCR signaling triggered by various chemo- of lymphocyte trafficking in lymph nodes are described else- kines is a key step in leukocyte recruitment, molecular details of where (30). this integrin-activating pathway remain elusive (3). The ele- Adhesion and postadhesion strengthening. In most tissues rolling or vation of intracellular calcium, the calcium-binding messenger tethering is insufficient to permanently target the leukocyte to protein calmodulin, and inositol-1,4,5-triphosphate receptors 4454 BRIEF REVIEWS: INTEGRIN REGULATION DURING LEUKOCYTE RECRUITMENT as downstream events of PLC activation were found to be in- HUVECs were found to actively move to cellular junctions to volved in GPCR-mediated VLA-4 activation following stimu- start transmigration within seconds (47). However, blocking lation with fMLP or CXCL12 in monocytes (37). Neither p38 of CD18, ICAM-1, or ICAM-2 rendered the monocytes unable MAPKs, PI3K, or, most interestingly, PKC was necessary to to locate endothelial cell junctions and caused the cells to wander achieve VLA-4 activation and subsequent binding to VCAM-1 during 90 min without effectively initiating transmigra- (37). In contrast, neutrophil response to chemokines uses dis- tion (47). Blockade of ICAM-1 and ICAM-2 still allowed tinct PI3K isoforms over time: early responses were found to be movement of monocytes, whereas blockade of CD11a and PI3Kg dependent, whereas prolonged recruitment of neu- CD11b resulted in immobile monocytes “pirouetting” on trophils following MIP-2 or TNF-a superfusion were PI3Kd their uropod (47). Monocytes crawling along the vasculature dependent (23). in vivo have also been termed “patrolling” (48). In contrast Farther downstream, the small GTPase Rap1 was found to to the in vitro findings on TNF-a– or IL-1b–stimulated be involved in Mac-1 activation in a similar assay (38). In CD3+ HUVECs, injection of a blocking LFA-1, but not Mac-1, Ab T cells Rap1 is required for LFA-1 and VLA-4 activation (32). completely and rapidly detached crawling monocytes in vivo, GTPases in turn are activated by guanine nucleotide exchange indicating a predominant role of LFA-1 in vivo (48). Also, factors (GEFs). In neutrophils stimulated with leukotriene B4, mice lacking CX3CR1 revealed a decrease of adherent mono- Rap1 is activated by CalDAG-GEFI, which is activated by Ca2+ cytes by two-thirds, 50% reduction in average crawling and DAG, two products of PLC (39). In contrast, another study distance while maintaining crawling velocities of wild-type using fMLP stimulation revealed that the GEFs Vav1 and P- animals (48). These data suggest a crucial role for GPCR- Downloaded from Rex1 redundantly mediate the activity of the GTPase Rac (40), mediated integrin activation in monocyte crawling. A recent suggesting that the different stimuli activate distinctly different study suggests that monocyte crawling may reflect expression pathways. A similar pathway was also demonstrated in human of surface proteins: on unstimulated venules, monocytes were CD3+ T cells following the stimulation with CXCL12 leading found to crawl in an LFA-1–dependent manner, but after stim- to LFA-1, but not VLA-4, activation (32). However, VLA-4 ulation with TNF-a and upregulation of adhesion molecules on activation was dependent on Rac1 activation by Vav1 alone endothelial cells, crawling became Mac-1 dependent (49). This http://www.jimmunol.org/ following stimulation with CXCL12 (41). A recently published could potentially explain the differences seen in vitro and in vivo. study demonstrated for the first time that the GTPase Cdc42 Similarly, neutrophils crawl on the endothelium following negatively regulates LFA-1 activation upon chemokine stimu- firm arrest in vivo (50). Interestingly, crawling was unaffected lation (42). This observation was very surprising because of the by blocking ICAM-2, but injection of a blocking ICAM-1 high homology of the different GTPases. The band 4.1/ezrin/ Ab almost completely abolished neutrophil crawling (50). radixin/moesin domain-containing proteins kindlin-3 and In contrast to the in vivo findings for monocyte crawling, talin-1 were found to be necessary for CXCL1-triggered LFA-1 blockade of LFA-1 did not affect neutrophil crawling. How-

activation in neutrophils (43). Surprisingly, only talin-1 is re- ever, injection of a blocking Mac-1 Ab significantly decreased by guest on September 24, 2021 quired for inducing LFA-1 extension, which corresponds to the percentage of crawling cells and dramatically decreased intermediate affinity and induces neutrophil slow rolling (43). distance and velocity (50). Based on these data, Mac-1, and not Kindlin-3 was also found to be involved in clustering of LFA-1 LFA-1, is the central integrin mediating neutrophils crawling on lymphocytes (44), a finding that also explains the defect in in vivo (50). Neutrophils lacking the GEF Vav1 were found to integrin clustering seen in patients with leukocyte adhesion have an impaired ability to crawl perpendicular to flow (51). deficiency III syndrome (45). Because Vav1 has been shown to be involved in LFA-1 out- Another surpising finding was the paradoxical role of side–in signaling mediating cytoskeletal rearrangements in cytohesin-1 upon activation of the b2-integrins LFA-1 and Mac- neutrophils (52), these findings led to the hypothesis that ac- 1 in neutrophils. Following GPCR stimulation with fMLP, tivation of Mac-1 via LFA-1–mediated outside–in signaling cytohesion-1 interacts with the cytoplasmatic integrin tail, pro- is a prerequisite for crawling (51). However, the molecular moting activation of LFA-1 and hampering Mac-1 activation pathways leading to initiation of crawling and activation of (46), suggesting closely related yet specific regulation of each Mac-1 in this context are currently unknown. integrin following GPCR stimulation. Lymphocyte crawling was also observed in vitro. Interest- In summary, VLA-4 and LFA-1 seem to be the predominant ingly, crawling of T cells on HUVECs is LFA-1 dependent and integrins mediating T cell adhesion, whereas monocytes involves very distinct mechanics: multiple scattered traction adhere in a VLA-4– or b2-integrin–dependent manner and sites form by engagement of high-affinity LFA-1 with endo- neutrophil adhesion is LFA-1 dependent. Furthermore, the thelial ICAM-1, and these form in a rapid turnover fashion published data suggest that PLC activation seems to be a key event resulting in “millipede-like” translocation over the endothelium in GPCR-mediated integrin activation (32, 37). The available that is triggered by chemokine CXCL12 (53). Also, it was data indicate that the involved GEFs and GTPases not only vary found that Cdc42 and Rap1 are involved in T cell crawling on among cell types but also among signaling pathways targeting HUVECs but are independent of Src kinases (53). different integrins. However, the signaling mechanics leading to These findings suggest different modes of movement for dif- integrin activation after chemokine stimulation remain poorly ferent leukocyte subtypes, ranging from the scattered forming of understood. Future studies, especially on the signaling molecules high-affinity LFA-1/ICAM-1 microclusters seen in T cells to directly interacting with integrins, such as talin, kindlin, and crawling seen in neutrophils that use the integrin Mac-1. How- cytohesin-1, will provide us with a clearer picture of what allows ever, the molecular events involved in the transition from differential activation of specific integrins after different stimuli. adhesion to crawling are still largely elusive. Intravascular crawling. Crawling was first observed in vitro and Transmigration and detachment. At the preferred site of transmi- initially termed “locomotion”: monocytes that adhered to gration, integrin binding to ICAM-1 and VCAM-1 constitutes The Journal of Immunology 4455 central players in the orchestra of protein interactions in- Integrin regulation at the site of inflammation volved in both para- and transcellular transmigration (54). An increasing number of studies have investigated the regu- Activated endothelium forms ring-like membrane structures lation of integrins at the site of inflammation. In the following enriched with ICAM-1 and VCAM-1 that are maintained for text we discuss mechanisms that physiologically regulate leu- the duration of transmigration (55). Recent studies have found kocyte integrin activation at the site of inflammation. Impair- evidence that neutrophil integrin engagement of ICAM-1 may ment of these local responses may cause constitutive integrin trigger the disassembly of the VE-cadherin and consecutive activation leading to autoimmune diseases, unnecessary tissue encapsulation of the transmigrating leukocyte in “domes,” as damage, and deterioration of organ function (20). Mecha- mice lacking cortactin, a cytoskeletal molecule serving as an nisms that limit leukocyte recruitment by regulating the ac- adaptor protein to ICAM-1, failed to initiate transmigration tivation of integrins were identified at several cellular levels (56). For a detailed review of the endothelial molecules in- (Fig, 1B). Some of these molecules hamper integrin activation volved in this step, see Williams et al. (54). Recent studies have by altering the transcription, decreasing the expression of revealed that extravasated neutrophils crawl in the space between endothelial ligands, activating leukocyte receptors (galectin-1, the endothelial abluminal surface and pericytes in a Mac-1– lipoxin A , docosahexaenoic acid [DHA]), or inhibiting the and LFA-1–dependent manner (57). Following subendothelial 4 upregulation of integrins (lipoxin A , resolvin D , resolvin crawling, leukocytes maintain connection to the endothelial 4 1 D ), whereas some interfere with the interaction of integrins abluminal surface, leading to uropod elongation and a signif- 2 with their ligands (developmental endothelial locus-1 [Del-1]) icantly delayed detachment (up to .20 min) of neutrophils, Downloaded from and others interfere with inside–out signaling (growth dif- monocytes, and lymphocytes (58). Leukocytes in mice deficient ferentiation factor-15 [GDF-15]). Also, different mechanisms in VLA-3 were found to have impaired detachment, as they failed act at different time points of inflammation; for example, to elongate by active movement of the leading edge of the cell galectins act early on, whereas many resolvins predominantly (58). In contrast, LFA-1–deficient leukocytes did not elongate act late in inflammation. and detached quickly (58). These data suggest that once extravasated, LFA-1/ICAM-1 maintain binding of the uropod to Galectins. One important regulation mechanism is the alter- http://www.jimmunol.org/ the abluminal surface of endothelium while VLA-3 mediates ation of integrin expression or the expression of their receptors. movement of the front of the cell and thus facilitates leukocyte Galectin-1 is an endothelial-derived protein of the b-galactoside– emigration through the basement membrane (58). binding lectin family. Preincubation of neutrophils with galectin- 1 decreased the rolling flux of neutrophils on HUVECs (66). Outside–in signaling in leukocyte recruitment. Following engage- This effect is partially mediated by a pronounced decrease in ment of ligands, integrins signal into the cell and elicit an the expression of PSGL-1, CD11b (Mac-1), and L-selectin on array of responses, some of which represent distinct cellular the cell surface of neutrophils (66). These findings were con- functions (4). Outside–in signaling triggers reactive oxygen firmed in a mouse model of IL-1b–induced inflammation of species production and degranulation, stabilizes the immune by guest on September 24, 2021 the m. cremaster that revealed increased numbers of recruited synapse, and mediates cytokine excretion in T cells and helps neutrophils in galectin-1–deficient mice (66). Although galectin- to differentiate and migrate macrophages (4). Physiologically, 3 has been found to improve the clinical picture of experimental however, inside–out and outside–in signaling rarely occur models of LPS-induced inflammation (67), the underlying separately (4). Many signaling molecules found in outside– mechanisms remain unclear, especially because galectin-3 has in signaling are also involved in inside–out signaling (4). The been shown to possess proinflammatory qualities (68). ITAM-containing adaptor proteins DAP12 and FcRg,Src kinases, as well as Syk have found to be involved in outside–in Resolvins and lipoxins. Lipid mediators including resolvins and signaling in neutrophils, macrophages, and platelets (59). lipoxins are involved in the resolution of inflammation (69). However, striking differences enable distinct activation of These mediators modulate the expression of integrins and outside–in signaling events: in neutrophils, the Src kinases receptors involved in integrin activation. Lipoxin A4 down- Hck and Fgr are necessary for outside–in-mediated adhesion regulates the expression of endothelial P-selectin and Mac-1 on strengthening but not for GPCR-mediated integrin activation neutrophils (69). In a similar fashion, resolvin D2 is able to (33). In contrast to this signaling pathway, only Fgr or Hck blunt the upregulation of b2-integrins on neutrophils evoked and Lyn are required for LFA-1 activation following E-selectin by platelet-activating factor superfusion (70). Resolvin D1 can engagement (18, 19), showing that the different Src family counteract leukotriene B4–stimulated expression of b2-integrins kinases expressed in neutrophils have distinct functions. In via the GPCRs GPR32 and ALX (formyl peptide receptor 2) Jurkat T cells, crosslinking of LFA-1 results in formation of (71). The omega-3 polyunsaturated fatty acid DHA was an actin cloud that is dependent on the presence of the Src described to reduce the expression of endothelial ICAM-1 and kinase Lyn (60). Downstream of Syk, phosphorylation of FAK VCAM-1 after proinflammatory stimuli (71). However, many and Pyk was found to be impaired upon integrin engagement in actions of the aforementioned molecules require further in- Syk-deficient neutrophils and macrophages (61). Also, SLP76 vestigation. In particular, resolvins have complex impacts on (62), ADAP (63), and its homolog PRAM-1 (64) were inflammation, as they are capable of preventing organ damage identified to be essential for intact outside–in signaling events while promoting clearance of intruding organisms (69). in neutrophils, suggesting a dual role for SLP76 and ADAP for Pentraxin-3. Pentraxin-3 (PTX-3), a member of the family of outside–in and inside–out signaling. Farther downstream, long pentraxins, directly inhibits leukocyte/endothelial inter- activation of PI3K and ERK was found to mediate b1- but action and thus indirectly prevents integrin activation. Being not b2-integrin–mediated NF-kB activation in monocytes a recognized player in the innate immune system for several following crosslinking, suggesting specific signaling pathways years, PTX-3 was found to protect mice in a model of acute for different integrins despite intriguing similarities (65). myocardial infarction (72), whereas deficiency in PTX-3 4456 BRIEF REVIEWS: INTEGRIN REGULATION DURING LEUKOCYTE RECRUITMENT elicited overshooting neutrophil infiltration in pleural inflammation of which only a fraction of players are known today. mation and acid-induced acute lung injury (73). However, the Future research on the mechanics of integrin activation responsible mechanism was just identified recently. Leukocyte- and regulation are needed to complete our picture of its derived PTX-3 binds to P-selectin on endothelial cells and physiological detail and hopefully reveal opportunities to thus blocks the interaction between P-selectin and PSGL-1 on clinically target integrins in leukocyte recruitment more suc- leukocytes, leading to an inhibition of leukocyte capturing and cessfully. rolling and integrin activation via inside–out signaling (73). Del-1. Upregulation of integrin affinity and avidity and subse- Acknowledgments quent binding of the activated integrin to ligands is a hallmark 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 lim- of inside–out signaling. The 52-kDa glycoprotein Del-1 has been itations. 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 Disclosures The authors have no financial conflicts of interest. functions as a competitive antagonist, effectively hampering leukocyte adhesion (74). Accordingly, mice lacking endothelial Del-1 displayed increased neutrophil infiltration in a model of References 1. Petri, B., M. Phillipson, and P. Kubes. 2008. The physiology of leukocyte re- LPS-induced pulmonary infiltration (74). cruitment: an in vivo perspective. J. Immunol. 180: 6439–6446. GDF-15. A recent publication identified the TGF-b superfamily 2. Springer, T. A., D. B. Teplow, and W. J. Dreyer. 1985. 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