Regulation of leukocyte recruitment by the long pentraxin PTX3.

Pentraxins are a superfamily of conserved proteins involved in the acute-phase response and innate immunity. Pentraxin 3 (PTX3), a prototypical member of the long pentraxin subfamily, is a key component of the humoral arm of innate immunity that is essential for resistance to certain pathogens. A regulatory role for pentraxins in inflammation has long been recognized, but the underlying mechanisms remain unclear. Here we report that PTX3 bound P-selectin and attenuated neutrophil recruitment at sites of inflammation. PTX3 released from activated leukocytes functioned locally to dampen neutrophil recruitment and regulate inflammation. Antibodies have glycosylation-dependent regulatory effect on inflammation. Therefore, PTX3, which is an essential component of humoral innate immunity, and immunoglobulins share functional outputs, including complement activation, opsonization and, as shown here, glycosylation-dependent regulation of inflammation.

GEF-H1 Is Required for Colchicine Inhibition of Neutrophil Rolling and Recruitment in Mouse Models of Gout.

Gout is a painful arthritic inflammatory disease caused by buildup of monosodium urate (MSU) crystals in the joints. Colchicine, a microtubule-depolymerizing agent that is used in prophylaxis and treatment of acute gout flare, alleviates the painful inflammatory response to MSU crystals. Using i.p. and intra-articular mouse models of gout-like inflammation, we found that GEF-H1/GEF-H1/AHRGEF2, a microtubule-associated Rho-GEF, was necessary for the inhibitory effect of colchicine on neutrophil recruitment. GEF-H1 was required for neutrophil polarization in response to colchicine, characterized by uropod formation, accumulation of F-actin and myosin L chain at the leading edge, and accumulation of phosphorylated myosin L chain, flotillin-2, and P-selectin glycoprotein ligand-1 (PSGL-1) in the uropod. Wild-type neutrophils that were pre-exposed to colchicine failed to roll or accumulate on activated endothelial monolayers, whereas GEF-H1 knockout (GEF-H1-/-) neutrophils were unaffected by treatment with colchicine. In vivo, colchicine blocked MSU-induced recruitment of neutrophils to the peritoneum and the synovium in wild-type mice, but not in GEF-H1-/- mice. Inhibition of macrophage IL-1ß production by colchicine was independent of GEF-H1, supporting a neutrophil-intrinsic mode of action. Our results suggest that the anti-inflammatory effects of colchicine in acute gout-like inflammation can be accounted for by inhibition of neutrophil-rolling interactions with the inflamed vasculature and occurs through GEF-H1-dependent neutrophil stimulation by colchicine. These results contribute to our understanding of the therapeutic action of colchicine, and could inform the application of this drug in other conditions.

Tissue instruction for migration and retention of TRM cells.

During infection, a subset of effector T cells seeds the lymphoid and non-lymphoid tissues and gives rise to tissue-resident memory T cells (TRM). Recent findings have provided insight into the molecular and cellular mechanisms underlying tissue instruction of TRM cell homing, as well as the programs involved in their retention and maintenance. We review these findings here, highlighting both common features and distinctions between CD4 TRM and CD8 TRM cells. In this context we examine the role of memory lymphocyte clusters (MLCs), and propose that the MLCs serve as an immediate response center consisting of TRM cells on standby, capable of detecting incoming pathogens and mounting robust local immune responses to contain and limit the spread of infectious agents.

CD43 Functions as an E-Selectin Ligand for Th17 Cells In Vitro and Is Required for Rolling on the Vascular Endothelium and Th17 Cell Recruitment during Inflammation In Vivo.

Endothelial E- and P-selectins mediate lymphocyte trafficking in inflammatory processes by interacting with lymphocyte selectin ligands. These are differentially expressed among different T cell subsets and function alone or in cooperation to mediate T cell adhesion. In this study, we characterize the expression and functionality of E-selectin ligands in Th type 17 lymphocytes (Th17 cells) and report that CD43 functions as a Th17 cell E-selectin ligand in vitro that mediates Th17 cell rolling on the vascular endothelium and recruitment in vivo. We demonstrate Th17 cells express CD44, P-selectin glycoprotein ligand (PSGL)-1, and CD43. Few PSGL-1(-/-)CD43(-/-) Th17 cells accumulated on E-selectin under shear flow conditions compared with wild-type cells. CD43(-/-) Th17 cell accumulation on E-selectin was impaired as compared with wild-type and PSGL-1(-/-), and similar to that observed for PSGL-1(-/-)CD43(-/-) Th17 cells, indicating that CD43 alone is a dominant ligand for E-selectin. Notably, this finding is Th17 cell subset specific because CD43 requires cooperation with PSGL-1 in Th1 cells for binding to E-selectin. In vivo, Th17 cell recruitment into the air pouch was reduced in CD43(-/-) mice in response to CCL20 or TNF-α, and intravital microscopy studies demonstrated that CD43(-/-) Th17 cells had impaired rolling on TNF-α-treated microvessels. Furthermore, CD43(-/-) mice were protected from experimental autoimmune encephalomyelitis and had impaired recruitment of Th17 cells in the spinal cord. Our findings demonstrate that CD43 is a major E-selectin ligand in Th17 cells that functions independent of PSGL-1, and they suggest that CD43 may hold promise as a therapeutic target to modulate Th17 cell recruitment.

The role of junctional adhesion molecules in vascular inflammation.

Junctional adhesion molecules (JAMs) of the immunoglobulin superfamily are important in the control of vascular permeability and leukocyte transmigration across endothelial-cell surfaces, by engaging in homophilic, heterophilic and lateral interactions. Through their localization on the endothelial-cell surface and expression by platelets, JAMs contribute to adhesive interactions with circulating leukocytes and platelets. Antibody-blocking studies and studies using genetically modified mice have implicated these functions of JAMs in the regulation of leukocyte recruitment to sites of inflammation and ischaemia-reperfusion injury, in growth-factor-mediated angiogenesis, atherogenesis and neointima formation. The comparison of different JAM-family members and animal models, however, shows that the picture remains rather complex. This Review summarizes recent progress and future directions in understanding the role of JAMs as 'gate keepers' in inflammation and vascular pathology.

Replacing the Promoter of the Murine Gene Encoding P-selectin with the Human Promoter Confers Human-like Basal and Inducible Expression in Mice.

In humans and mice, megakaryocytes/platelets and endothelial cells constitutively synthesize P-selectin and mobilize it to the plasma membrane to mediate leukocyte rolling during inflammation. TNF-α, interleukin 1ß, and LPS markedly increase P-selectin mRNA in mice but decrease P-selectin mRNA in humans. Transgenic mice bearing the entire human SELP gene recapitulate basal and inducible expression of human P-selectin and reveal human-specific differences in P-selectin function. Differences in the human SELP and murine Selp promoters account for divergent expression in vitro, but their significance in vivo is not known. Here we generated knockin mice that replace the 1.4-kb proximal Selp promoter with the corresponding SELP sequence (Selp(KI)). Selp(KI) (/) (KI) mice constitutively expressed more P-selectin on platelets and more P-selectin mRNA in tissues but only slightly increased P-selectin mRNA after injection of TNF-α or LPS. Consistent with higher basal expression, leukocytes rolled more slowly on P-selectin in trauma-stimulated venules of Selp(KI) (/) (KI) mice. However, TNF-α did not further reduce P-selectin-dependent rolling velocities. Blunted up-regulation of P-selectin mRNA during contact hypersensitivity reduced P-selectin-dependent inflammation in Selp(KI) (/-) mice. Higher basal P-selectin in Selp(KI) (/) (KI) mice compensated for this defect. Therefore, divergent sequences in a short promoter mediate most of the functionally significant differences in expression of human and murine P-selectin in vivo.

Postarrest stalling rather than crawling favors CD8(+) over CD4(+) T-cell migration across the blood-brain barrier under flow in vitro.

Although CD8(+) T cells have been implied in the pathogenesis of multiple sclerosis (MS), the molecular mechanisms mediating CD8(+) T-cell migration across the blood-brain barrier (BBB) into the central nervous system (CNS) are ill defined. Using in vitro live cell imaging, we directly compared the multistep extravasation of activated CD4(+) and CD8(+) T cells across primary mouse brain microvascular endothelial cells (pMBMECs) as a model for the BBB under physiological flow. Significantly higher numbers of CD8(+) than CD4(+) T cells arrested on pMBMECs under noninflammatory and inflammatory conditions. While CD4(+) T cells polarized and crawled prior to their diapedesis, the majority of CD8(+) T cells stalled and readily crossed the pMBMEC monolayer preferentially via a transcellular route. T-cell arrest and crawling were independent of G-protein-coupled receptor signaling. Rather, absence of endothelial ICAM-1 and ICAM-2 abolished increased arrest of CD8(+) over CD4(+) T cells and abrogated T-cell crawling, leading to the efficient reduction of CD4(+) , but to a lesser degree of CD8(+) , T-cell diapedesis across ICAM-1(null) /ICAM-2(-/-) pMBMECs. Thus, cellular and molecular mechanisms mediating the multistep extravasation of activated CD8(+) T cells across the BBB are distinguishable from those involved for CD4(+) T cells.

Multi-Inhibitory Effects of A2A Adenosine Receptor Signaling on Neutrophil Adhesion Under Flow.

A2A adenosine receptor (A2AAR) signaling negatively regulates inflammatory responses in many disease models, but the detailed mechanisms remain unclear. We used the selective A2AAR agonist, ATL313, to examine how A2AAR signaling affects human and murine neutrophil adhesion under flow. Treating neutrophils with ATL313 inhibited selectin-induced, ß2 integrin-dependent slow rolling and chemokine-induced, ß2 integrin-dependent arrest on ICAM-1. ATL313 inhibited selectin-induced ß2 integrin extension, which supports slow rolling, and chemokine-induced hybrid domain "swing-out," which supports arrest. Furthermore, ATL313 inhibited integrin outside-in signaling as revealed by reduced neutrophil superoxide production and spreading on immobilized anti-ß2 integrin Ab. ATL313 suppressed selectin-triggered activation of Src family kinases (SFKs) and p38 MAPK, chemokine-triggered activation of Ras-related protein 1, and ß2 integrin-triggered activation of SFKs and Vav cytoskeletal regulatory proteins. ATL313 activated protein kinase A and its substrate C-terminal Src kinase, an inhibitor of SFKs. Treating neutrophils with a protein kinase A inhibitor blocked the actions of ATL313. In vivo, ATL313-treated neutrophils rolled faster and arrested much less frequently in postcapillary venules of the murine cremaster muscle after TNF-α challenge. Furthermore, ATL313 markedly suppressed neutrophil migration into the peritoneum challenged with thioglycollate. ATL313 did not affect A2AAR-deficient neutrophils, confirming its specificity. Our findings provide new insights into the anti-inflammatory mechanisms of A2AAR signaling and the potential utility of A2AAR agonists in inflammatory diseases.

Mutation in the CD45 inhibitory wedge modulates integrin activation and leukocyte recruitment during inflammation.

Neutrophil recruitment to the site of inflammation plays a pivotal role in host defense. Src family kinases (SFKs) activation is required for integrin and chemokine signaling as well as immune cell function. The receptor-like protein tyrosine phosphatase CD45 positively regulates chemoattractant signaling acting on SFK activity. To further investigate the role of CD45 in neutrophil recruitment and function, we analyzed transgenic mice carrying a single point mutation (CD45E613R), which constitutively activates CD45. By using intravital microscopy experiments, we demonstrated that different steps of the leukocyte recruitment cascade were affected in CD45E613R mutant mice. The rolling velocity of CD45E613R mutant neutrophils was decreased compared with wild-type neutrophils that subsequently resulted in an increased number of adherent cells. The analysis of ß2 integrins LFA-1 and macrophage-1 Ag (Mac-1) showed that in CD45E613R mutant neutrophils LFA-1 adhesiveness was impaired, and avidity was enhanced, whereas Mac-1 adhesiveness was increased. Because of the increased Mac-1 adhesiveness, neutrophil crawling was impaired in CD45E613R mutant compared with wild-type neutrophils. In an Escherichia coli lung infection model, CD45E613R mice displayed a decreased neutrophil recruitment into the alveolar compartment, which resulted in an increased number of CFUs in the lung. Our data demonstrate that the CD45E613R mutation modulates integrin activation and leukocyte recruitment during inflammation.

Cross-Talk between Shp1 and PIPKIγ Controls Leukocyte Recruitment.

Neutrophil recruitment to the site of inflammation plays a pivotal role in host defense. However, overwhelming activation and accumulation of neutrophils in the tissue may cause tissue damage and autoimmunity due to the release of cytokines, oxidants, and proteases. Neutrophil adhesion in acute inflammation is initiated by activation of αLß2 (LFA-1), which can be induced by rolling on E-selectin (slowly) or by exposure to the chemokine CXCL1 (rapidly). Despite the clinical importance, cell-intrinsic molecular mechanisms of negative regulation of integrin adhesiveness and neutrophil recruitment are poorly understood. Mice deficient in the tyrosine phosphatase Src homology 2 domain-containing protein tyrosine phosphatase 1 (Shp1) show increased leukocyte adhesion, but the interpretation of these data is limited by the severe global phenotype of these mice. In this study, we used mice with global and myeloid-restricted deletion of Shp1 to study neutrophil arrest, adhesion, crawling, and transendothelial migration in vitro and in vivo. Shp1 deficiency results in increased neutrophil adhesion in vivo; however, neutrophil crawling, transmigration, and chemotaxis were reduced in these mice. Mechanistically, Shp1 binds and controls PIPKIγ activity and, thereby, modulates phosphatidylinositol (4,5)-bisphosphate levels and adhesion. Thus, Shp1 is involved in the deactivation of integrins and regulation of neutrophil recruitment into inflamed tissue.

High-resolution imaging of intravascular atherogenic inflammation in live mice.

RATIONALE: The inflammatory processes that initiate and propagate atherosclerosis remain poorly understood, largely because defining the intravascular behavior of immune cells has been technically challenging. Respiratory and pulsatile movements have hampered in vivo visualization of leukocyte accumulation in athero-prone arteries at resolutions achieved in other tissues. OBJECTIVE: To establish and to validate a method that allows high-resolution imaging of inflammatory leukocytes and platelets within the carotid artery of atherosusceptible mice in vivo. METHODS AND RESULTS: We have devised a procedure to stabilize the mouse carotid artery mechanically without altering blood dynamics, which dramatically enhances temporal and spatial resolutions using high-speed intravital microscopy in multiple channels of fluorescence. By applying this methodology at different stages of disease progression in atherosusceptible mice, we first validated our approach by assessing the recruitment kinetics of various leukocyte subsets and platelets in athero-prone segments of the carotid artery. The high temporal and spatial resolution allowed the dissection of both the dynamic polarization of and the formation of subcellular domains within adhered leukocytes. We further demonstrate that the secondary capture of activated platelets on the plaque is predominantly mediated by neutrophils. Finally, we couple this procedure with triggered 2-photon microscopy to visualize the 3-dimensional movement of leukocytes in intimate contact with the arterial lumen. CONCLUSIONS: The improved imaging of diseased arteries at subcellular resolution presented here should help resolve many outstanding questions in atherosclerosis and other arterial disorders.

Adiponectin inhibits tumor necrosis factor-α-induced vascular inflammatory response via caveolin-mediated ceramidase recruitment and activation.

RATIONALE: Anti-inflammatory and vascular protective actions of adiponectin are well recognized. However, many fundamental questions remain unanswered. OBJECTIVE: The current study attempted to identify the adiponectin receptor subtype responsible for adiponectin's vascular protective action and investigate the role of ceramidase activation in adiponectin anti-inflammatory signaling. METHODS AND RESULTS: Adiponectin significantly reduced tumor necrosis factor (TNF)α-induced intercellular adhesion molecule-1 expression and attenuated TNFα-induced oxidative/nitrative stress in human umbilical vein endothelial cells. These anti-inflammatory actions were virtually abolished by adiponectin receptor 1 (AdipoR1-), but not AdipoR2-, knockdown (KD). Treatment with adiponectin significantly increased neutral ceramidase (nCDase) activity (3.7-fold; P<0.01). AdipoR1-KD markedly reduced globular adiponectin-induced nCDase activation, whereas AdipoR2-KD only slightly reduced. More importantly, small interfering RNA-mediated nCDase-KD markedly blocked the effect of adiponectin on TNFα-induced intercellular adhesion molecule-1 expression. AMP-activated protein kinase-KD failed to block adiponectin-induced nCDase activation and modestly inhibited adiponectin anti-inflammatory effect. In contrast, in caveolin-1 KD (Cav1-KD) cells, >87% of adiponectin-induced nCDase activation was lost. Whereas adiponectin treatment failed to inhibit TNFα-induced intercellular adhesion molecule-1 expression, treatment with sphingosine-1-phosphate or SEW (sphingosine-1-phosphate receptor agonist) remained effective in Cav1-KD cells. AdipoR1 and Cav1 colocalized and coprecipitated in human umbilical vein endothelial cells. Adiponectin treatment did not affect this interaction. There is weak basal Cav1/nCDase interaction, which significantly increased after adiponectin treatment. Knockout of AdipoR1 or Cav1 abolished the inhibitory effect of adiponectin on leukocyte rolling and adhesion in vivo. CONCLUSIONS: These results demonstrate for the first time that adiponectin inhibits TNFα-induced inflammatory response via Cav1-mediated ceramidase recruitment and activation in an AdipoR1-dependent fashion.

Heterotropic modulation of selectin affinity by allosteric antibodies affects leukocyte rolling.

Selectins are a family of adhesion receptors designed for efficient leukocyte tethering to the endothelium under shear. As a key property to resist premature bond disruption, selectin adhesiveness is enhanced by tensile forces that promote the conversion of a bent into an extended conformation of the N-terminal lectin and epidermal growth factor-like domains. Conformation-specific Abs have been invaluable in deciphering the activation mechanism of integrins, but similar reagents are not available for selectins. In this study, we show that the anti-human L-selectin mAbs DREG-55 and LAM1-5 but not DREG-56, DREG-200, or LAM1-1 heterotropically modulate adhesion presumably by stabilizing the extended receptor conformation. Force-free affinity assays, flow chamber, and microkinetic studies reveal a ligand-specific modulation of L-selectin affinity by DREG-55 mAb, resulting in a dramatic decrease of rolling velocity under flow. Furthermore, secondary tethering of polymorphonuclear cells was blocked by DREG-200 but significantly boosted by DREG-55 mAb. The results emphasize the need for a new classification for selectin Abs and introduce the new concept of heterotropic modulation of receptor function.

Identification of activators of ERK5 transcriptional activity by high-throughput screening and the role of endothelial ERK5 in vasoprotective effects induced by statins and antimalarial agents.

Because ERK5 inhibits endothelial inflammation and dysfunction, activating ERK5 might be a novel approach to protecting vascular endothelial cells (ECs) against various pathological conditions of the blood vessel. We have identified small molecules that protect ECs via ERK5 activation and determined their contribution to preventing cardiac allograft rejection. Using high-throughput screening, we identified certain statins and antimalarial agents including chloroquine, hydroxychloroquine, and quinacrine as strong ERK5 activators. Pitavastatin enhanced ERK5 transcriptional activity and Kruppel-like factor-2 expression in cultured human and bovine ECs, but these effects were abolished by the depletion of ERK5. Chloroquine and hydroxychloroquine upregulated ERK5 kinase activity and inhibited VCAM-1 expression in an ERK5-dependent but MAPK/ERK kinase 5- and Kruppel-like factor 2/4-independent manner. Leukocyte rolling and vascular reactivity were used to evaluate endothelial function in vivo, and we found that EC-specific ERK5 knockout (ERK5-EKO) mice exhibited increased leukocyte rolling and impaired vascular reactivity, which could not be corrected by pitavastatin. The role of endothelial ERK5 in acute cardiac allograft rejection was also examined by heterotopic grafting of the heart obtained from either wild-type or ERK5-EKO mice into allomismatched recipient mice. A robust increase in both inflammatory gene expression and CD45-positive cell infiltration into the graft was observed. These tissue rejection responses were inhibited by pitavastatin in wild-type but not ERK5-EKO hearts. Our study has identified statins and antimalarial drugs as strong ERK5 activators and shown that ERK5 activation is preventive of endothelial inflammation and dysfunction and acute allograft rejection.

Ontogenetic regulation of leukocyte recruitment in mouse yolk sac vessels.

In adult mammals, leukocyte recruitment follows a well-defined cascade of adhesion events enabling leukocytes to leave the circulatory system and transmigrate into tissue. Currently, it is unclear whether leukocyte recruitment proceeds in a similar fashion during fetal development. Considering the fact that the incidence of neonatal sepsis increases dramatically with decreasing gestational age in humans, we hypothesized that leukocyte recruitment may be acquired only late during fetal ontogeny. To test this, we developed a fetal intravital microscopy model in pregnant mice and, using LysEGFP (neutrophil reporter) mice, investigated leukocyte recruitment during fetal development. We show that fetal blood neutrophils acquire the ability to roll and adhere on inflamed yolk sac vessels during late fetal development, whereas at earlier embryonic stages (before day E15), rolling and adhesion were essentially absent. Accordingly, flow chamber experiments showed that fetal EGFP(+) blood cells underwent efficient adhesion only when they were harvested on or after E15. Fluorescence-activated cell sorter analysis on EGFP(+) fetal blood cells revealed that surface expression of CXCR2 and less pronounced P-selectin glycoprotein ligand-1 (PSGL-1) begin to increase only late in fetal life. Taken together, our findings demonstrate that inflammation-induced leukocyte recruitment is ontogenetically regulated and enables efficient neutrophil trafficking only during late fetal life.

Coordinated and unique functions of the E-selectin ligand ESL-1 during inflammatory and hematopoietic recruitment in mice.

Beyond its well-established roles in mediating leukocyte rolling, E-selectin is emerging as a multifunctional receptor capable of inducing integrin activation in neutrophils, and of regulating various biological processes in hematopoietic precursors. Although these effects suggest important homeostatic contributions of this selectin in the immune and hematologic systems, the ligands responsible for transducing these effects in different leukocyte lineages are not well defined. We have characterized mice deficient in E-selectin ligand-1 (ESL-1), or in both P-selectin glycoprotein-1 (PSGL-1) and ESL-1, to explore and compare the contributions of these glycoproteins in immune and hematopoietic cell trafficking. In the steady state, ESL-1 deficiency resulted in a moderate myeloid expansion that became more prominent when both glycoproteins were eliminated. During inflammation, PSGL-1 dominated E-selectin binding, rolling, integrin activation, and extravasation of mature neutrophils, but only the combined deficiency in PSGL-1 and ESL-1 completely abrogated leukocyte recruitment. Surprisingly, we find that the levels of ESL-1 were strongly elevated in hematopoietic progenitor cells. These elevations correlated with a prominent function of ESL-1 for E-selectin binding and for migration of hematopoietic progenitor cells into the bone marrow. Our results uncover dominant roles for ESL-1 in the immature compartment, and a functional shift toward PSGL-1 dependence in mature neutrophils.

Platelet serotonin promotes the recruitment of neutrophils to sites of acute inflammation in mice.

The majority of peripheral serotonin is stored in platelets, which secrete it on activation. Serotonin releases Weibel-Palade bodies (WPBs) and we asked whether absence of platelet serotonin affects neutrophil recruitment in inflammatory responses. Tryptophan hydroxylase (Tph)1­deficient mice, lacking non-neuronal serotonin, showed mild leukocytosis compared with wild-type (WT), primarily driven by an elevated neutrophil count. Despite this, 50% fewer leukocytes rolled on unstimulated mesenteric venous endothelium of Tph1(-/-) mice. The velocity of rolling leukocytes was higher in Tph1(-/-) mice, indicating fewer selectin-mediated interactions with endothelium. Stimulation of endothelium with histamine, a secretagogue of WPBs, or injection of serotonin normalized the rolling in Tph1(-/-) mice. Diminished rolling in Tph1(-/-) mice resulted in reduced firm adhesion of leukocytes after lipopolysaccharide treatment. Blocking platelet serotonin uptake with fluoxetine in WT mice reduced serum serotonin by > 80% and similarly reduced leukocyte rolling and adhesion. Four hours after inflammatory stimulation, neutrophil extravasation into lung, peritoneum, and skin wounds was reduced in Tph1(-/-) mice, whereas in vitro neutrophil chemotaxis was independent of serotonin. Survival of lipopolysaccharide-induced endotoxic shock was improved in Tph1(-/-) mice. In conclusion, platelet serotonin promotes the recruitment of neutrophils in acute inflammation, supporting an important role for platelet serotonin in innate immunity.

Integrin Regulation during Leukocyte Recruitment.

Integrins are recognized as vital players in leukocyte recruitment. Integrin malfunction causes severe disease patterns characterized by the inability to fight pathogens. Although inflammatory reactions are beneficial and necessary for host defense, these reactions have to be controlled to prevent tissue destruction and harmful sequelae. In this review, we discuss the different signaling pathways leading to the change of integrin adhesiveness in neutrophils, monocytes, and lymphocytes. We thereby focus on the importance of integrin activation for the different steps of the leukocyte recruitment cascade, including rolling, adhesion, postadhesion strengthening, intravascular crawling, and transmigration, as each step necessitates the proper functioning of a distinct set of integrin molecules that has to be activated specifically. Additionally, we discuss endogenous mechanisms that balance and counteract integrin activation and limit leukocyte recruitment at the site of inflammation. Further insight into these complex mechanisms may provide new approaches for developing new anti-inflammatory therapies.

Severe impairment of leukocyte recruitment in ppGalNAcT-1-deficient mice.

P-selectin glycoprotein ligand-1 plays an important role in leukocyte recruitment. Its binding affinity to selectins is modulated by posttranslational modifications. The polypeptide N-acetylgalactosamine transferase-1 (ppGalNAcT-1) initiates core-type protein O-glycosylation. To address whether the glycosylation of P-selectin glycoprotein ligand-1 by ppGalNAcT-1 is important for leukocyte recruitment in vivo, we investigated leukocyte recruitment in untreated and TNF-α-treated cremaster muscles comparing ppGalNAcT-1-deficient mice (Galnt1(-/-)) and wild-type mice. In untreated and TNF-α-treated Galnt1(-/-) mice, leukocyte rolling, adhesion, and transmigration were significantly reduced, with markedly increased rolling velocity compared with control mice. L-selectin-dependent leukocyte rolling was completely abolished in Galnt1(-/-) mice compared with wild-type mice. Thioglycollate-induced peritonitis experiments with chimeric mice revealed that hematopoietic ppGalNAcT-1 is important for leukocyte recruitment. These data show that the loss of ppGalNAcT-1 led to reduced leukocyte rolling and recruitment and increased rolling velocity, suggesting a predominant role for ppGalNAcT-1 in attaching functionally relevant O-linked glycans to selectin ligands.