Elzbieta Pluskota

Distinct roles for the α and β subunits in the functions of integrin αMβ2

Integrin αMβ2 (Mac-1, CD11b/CD18) is a noncovalently linked heterodimer of αM and β2 subunits on the surface of leukocytes, where it plays a pivotal role in the adhesion and migration of these cells. Using HEK293 cells expressing αMβ2 or the individual constituent chains on their surface, we analyzed the contributions of the αM or β2 subunits to functional responses mediated by the integrin. In cells expressing only αM or β2, the individual subunits were not associated with the endogenous integrins of the cells, and other partners for the subunits were not detected by surface labeling and immunoprecipitation under a variety of conditions. The αM cells mediated adhesion and spreading on a series of αMβ2 ligands (fibrinogen, Factor X, iC3b, ICAM-1 (intercellular adhesion molecule-1), and denatured ovalbumin) but could not support cell migration to any of these. The spreading of the αM cells suggested an unanticipated linkage of this subunit to the cytoskeleton. The β2 cells supported migration and attachment but not spreading on a subset of the αMβ2 ligands. The heterodimeric receptor and its individual subunits were purified from the cells by affinity chromatography and recapitulated the ligand binding properties of the corresponding cell lines. These data indicate that each subunit of αMβ2 contributes distinct properties to αMβ2 and that, in most but not all cases, the response of the integrin is a composite of the functions of its individual subunits.

Distinct roles for the alpha and beta subunits in the functions of integrin αMβ2

Integrin αMβ2 (Mac-1, CD11b/CD18) is a noncovalently linked heterodimer of αM and β2 subunits on the surface of leukocytes, where it plays a pivotal role in the adhesion and migration of these cells. Using HEK293 cells expressing αMβ2 or the individual constituent chains on their surface, we analyzed the contributions of the αM or β2 subunits to functional responses mediated by the integrin. In cells expressing only αM or β2, the individual subunits were not associated with the endogenous integrins of the cells, and other partners for the subunits were not detected by surface labeling and immunoprecipitation under a variety of conditions. The αM cells mediated adhesion and spreading on a series of αMβ2 ligands (fibrinogen, Factor X, iC3b, ICAM-1 (intercellular adhesion molecule-1), and denatured ovalbumin) but could not support cell migration to any of these. The spreading of the αM cells suggested an unanticipated linkage of this subunit to the cytoskeleton. The β2 cells supported migration and attachment but not spreading on a subset of the αMβ2 ligands. The heterodimeric receptor and its individual subunits were purified from the cells by affinity chromatography and recapitulated the ligand binding properties of the corresponding cell lines. These data indicate that each subunit of αMβ2 contributes distinct properties to αMβ2 and that, in most but not all cases, the response of the integrin is a composite of the functions of its individual subunits.

Expression, activation, and function of integrin αMβ2 (Mac-1) on neutrophil-derived microparticles

Leukocyte-derived microparticles (MPs) are markers of cardiovascular diseases and contribute to pathogenesis by their interaction with various cell types. The presence and activation state of a multifunctional leukocyte receptor, integrin alpha(M)beta(2) (CD11b/18), on MPs derived from human neutrophils (PMNs) were examined. alpha(M)beta(2) expression was significantly enhanced on MPs derived from stimulated compared with resting PMNs. Furthermore, alpha(M)beta(2) on MPs from stimulated but not resting PMNs was in an activated conformation because it was capable of binding activation-specific monoclonal antibodies (CBRM1/5 and mAb24) and soluble fibrinogen. MPs expressing active alpha(M)beta(2) interacted with and were potent activators of resting platelets as assessed by induction of P-selectin expression and activation of alpha(IIb)beta(3). With the use of function-blocking antibodies and MPs obtained from alpha(M)(-/-)-deficient mice, we found that engagement of GPIbalpha on platelets by alpha(M)beta(2) on MPs plays a pivotal role in MP binding. Platelet activation by MPs occurs by a pathway dependent on Akt phosphorylation. PSGL-1/P-selectin interaction also is involved in the conjugation of MPs to platelets, and the combination of blocking reagents to both alpha(M)beta(2)/GPIbalpha and to PSGL-1/P-selectin completely abrogates MP-induced platelet activation. Thus, cooperation of these 2 receptor/counterreceptor systems regulates the prothrombotic properties of PMN-derived MPs.

Identification of pH-Regulated Antigen 1 Released from Candida albicans as the Major Ligand for Leukocyte Integrin αMβ2

Candida albicans is a common opportunistic fungal pathogen and is the leading cause of invasive fungal disease in immunocompromised individuals. The induction of cell-mediated immunity to C. albicans is of critical importance in host defense and the prime task of cells of the innate immune system. We previously demonstrated that the integrin αMβ2 (CD11b/CD18) is the major leukocyte receptor involved in C. albicans recognition, mediating both adhesive and migratory responses to the fungus. In the present study, we demonstrate that various C. albicans strains release a protease-sensitive activity into their conditioned medium that supports αMβ2-mediated cell adhesion and migration. The isolation and characterization of this protein was undertaken by two independent approaches: 1) immunoaffinity purification on a mAb raised to conditioned medium which blocked αMβ2-dependent adhesion and migration; and 2) affinity chromatography on purified αMβ2. Each approach led to the isolation of the same protein, which was unequivocally identified as pH-regulated Ag 1 (Pra1p), based on mass spectrometry and amino acid sequence analyses. C. albicans mutant strains lacking Pra1p were unable to support leukocyte adhesion or migration. In a neutrophil-mediated fungal killing assay, such mutant strains were resistant to killing and/or phagocytosis. Addition of purified Pra1p or reagents that block αMβ2 function prevented killing of Pra1p-expressing but not Pra1p-deficient strains of C. albicans. Together, these data indicate that Pra1p is a ligand of αMβ2 on C. albicans and that the soluble form of Pra1p may assist the fungus in escaping host surveillance.

Src Homology Domain 2-containing Tyrosine Phosphatase 2 Associates with Intercellular Adhesion Molecule 1 to Regulate Cell Survival

Intercellular adhesion molecule-1 (ICAM-1) binds to the plasma protein fibrinogen (Fg) to mediate leukocyte/endothelial cell interactions. In our studies, the ligation of Fg to ICAM-1 on tumor necrosis factor-α-stimulated endothelial cells resulted in the tyrosine phosphorylation of Src homology domain 2 (SH2)-containing phosphatase-2 (SHP-2). The ICAM-1 cytoplasmic sequence IKKYRLQ conforms poorly to the concensus immunoreceptor tyrosine-based inhibition motifs found in receptors that bind SHP-2. Nevertheless, the tyrosine phosphorylated sequence (IKKpYRLQ) bound specifically to the SH2 domain proximal to the NH2-terminal of SHP-2 (SHP-2-N) but not to the SH2 domain proximal on the COOH-terminal side (SHP-2-C). Phosphorylated ICAM-1 bound SHP-2-N. In immunoprecipitation experiments, SHP-2 associated with phosphorylated ICAM-1. Cells expressing truncated ICAM-1 that lacked the cytoplasmic sequence (ICAM-1(TR)) failed to associate with SHP-2. ICAM-1 containing the tyrosine to alanine substitution at position 485 (ICAM-1(Y485A)) associated weakly with SHP-2. Cells expressing ICAM-1(TR) and ICAM-1(Y485A) underwent apoptosis upon adhesion to Fg, whereas the wild type ICAM-1 maintained cell survival. These results indicate that ICAM-1 interactions with SHP-2 allow better cellular survival mediated through Fg-ICAM-1 ligation.

Thrombospondin-4 Regulates Vascular Inflammation and Atherogenesis

Rationale: Thrombospondin (TSP)-4 is an extracellular protein that has been linked to several cardiovascular pathologies. However, a role for TSP-4 in vascular wall biology remains unknown. Objective: We have examined the effects of TSP-4 gene (Thbs4) knockout on the development of atherosclerotic lesions in ApoE−/− mice. Methods and Results: Deficiency in TSP-4 reduced atherosclerotic lesions: at 20 weeks of age, the size of the aortic root lesions in Thbs4−/−/ApoE−/− mice was decreased by 48% in females and by 39% in males on chow diets; in mice on Western diets, lesions in the descending aorta were reduced by 30% in females and 33% in males. In ApoE−/− mice, TSP-4 was abundant in vessel areas prone to lesion development and in the matrix of the lesions themselves. TSP-4 deficiency reduced the number of macrophages in lesions in all groups by ≥2-fold. In addition, TSP-4 deficiency reduced endothelial cell activation (expression of surface adhesion molecules) and other markers of inflammation in the vascular wall (decreased production of monocyte chemoattractant protein-1 and activation of p38). In vitro, both the adhesion and migration of wild-type macrophages increased in the presence of purified recombinant TSP-4 in a dose-dependent manner (up to 7- and 4.7-fold, respectively). These responses led to p38-MAPkinase activation and were dependent on &bgr;2 and &bgr;3 integrins, which recognize TSP-4 as a ligand. Conclusions: TSP-4 is abundant in atherosclerotic lesions and in areas prone to development of lesions and may influence the recruitment of macrophages by activating endothelial cells and directly interacting with macrophages to increase their adhesion and migration. Our observations suggest an important role for this matricellular protein in the local regulation of inflammation associated with atherogenesis.

The integrin coactivator Kindlin-2 plays a critical role in angiogenesis in mice and zebrafish

Kindlin-2, a widely distributed cytoskeletal protein, has been implicated in integrin activation, and its absence is embryonically lethal in mice and causes severe developmental defects in zebrafish. Knockdown of kindlin-2 levels in endothelial cells resulted in defective adhesive and migratory responses, suggesting that angiogenesis might be aberrant even with partial reduction of kindlin-2. This hypothesis has now been tested in the kindlin-2(+/-) mice. RM1 prostate tumors grown in kindlin-2(+/-) mice had fewer blood vessels, which were thinner and shorter and supported less tumor growth compared with wild-type littermates. The vessels that did form in the kindlin-2(+/-) mice lacked smooth muscle cells and pericytes and had thinner basement membranes, indicative of immature vessels. VEGF-induced angiogenesis in matrigel implants was also abnormal in the kindlin-2(+/-) mice. Vessels in the kindlin-2(+/-) mice were leaky, and BM transplantation from kindlin-2(+/-) to WT mice did not correct this defect. Endothelial cells derived from kindlin-2(+/-) mice had integrin expression levels similar to WT mice but reduced αVβ3-dependent signaling, migration, adhesion, spreading, and tube formation. Developmental angiogenesis was markedly impaired by kindlin-2 morpholinos in zebrafish. Taken together, kindlin-2 plays an important role in pathologic and developmental angiogenesis, which arises from defective activation of integrin αVβ3.

Neutrophil Apoptosis: Selective Regulation by Different Ligands of Integrin αMβ2

Neutrophils undergo spontaneous apoptosis, but their survival can be extended during inflammatory responses. αMβ2 is reported either to delay or accelerate neutrophil apoptosis, but the mechanisms by which this integrin can support such diametrically opposed responses are poorly understood. The abilities of closely related αMβ2 ligands, plasminogen and angiostatin, derived from plasminogen, as well as fibrinogen and its two derivative αMβ2 recognition peptides, P1 and P2-C, differed markedly in their effects on neutrophil apoptosis. Plasminogen, fibrinogen, and P2-C suppressed apoptosis via activation of Akt and ERK1/2 kinases, while angiostatin and P1 failed to activate these prosurvival pathways and did not prevent neutrophil apoptosis. Using cells transfected with αMβ2 or its individual αM or β2 subunits, and purified receptors and its constituent chains, we show that engagement of both subunits with prosurvival ligands is essential for induction of the prosurvival response. Hence, engagement of a single integrin by closely related ligands can induce distinct signaling pathways, which can elicit distinct cellular responses.

αMβ2 Integrin Activation Prevents Alternative Activation of Human and Murine Macrophages and Impedes Foam Cell Formation

Rationale: The alternative activation of monocytes by interleukin (IL)-13 and IL-4 is a significant component of the inflammatory response. The consequences of alternative activation in inflammatory diseases remain to be determined. Objective: In this report, we explored how integrins, receptors important for monocyte migration to inflammatory sites, regulate IL-13–mediated monocyte activation. We focused on the analysis of 2 proteins, which are upregulated during the alternative activation and are important for the development of atherosclerosis, an oxidative enzyme 15-lipoxygenase (15-LO) and a scavenger receptor CD36. Methods and Results: We found that adhesion of resting monocytes through &bgr;2 integrins and inside-out activation of &bgr;2 integrins by monocyte chemoattractant protein-1 did not change IL-13–stimulated 15-LO upregulation; however, preincubation of monocytes with the antibody MEM48, which generates full activation of &bgr;2 integrins, significantly inhibited 15-LO mRNA and protein expression. In contrast, activation of &bgr;1 integrins had no effect on 15-LO expression. Analysis of integrin clustering through &agr;M, &agr;L, &agr;X, and &agr;D subunits demonstrated the pivotal role for integrin &agr;M&bgr;2 in inhibiting 15-LO expression. IL-13 treatment upregulates 15-LO–dependent CD36 expression on human monocytes; our studies showed that &bgr;2 integrin activation and &agr;M integrin clustering significantly inhibited IL-13–dependent CD36 mRNA and protein expression, as well as CD36-related foam cell formation. Moreover, IL-13 stimulation of &agr;M-deficient peritoneal macrophages demonstrated an upregulated level of 15-LO induction, CD36 expression, and lipid accumulation as compared with wild-type controls. Conclusions: The adhesion of monocytes/macrophages through activated integrin &agr;M&bgr;2 has a regulatory and potential atheroprotective function during the alternative activation of macrophages.

Plasminogen and Its Receptors as Regulators of Cardiovascular Inflammatory Responses

In addition to its role in fibrinolysis, plasminogen (Plg) influences inflammatory cell migration and thereby plays a prominent role in cardiovascular pathology. The contribution of Plg to inflammatory cell recruitment depends on its tethering to the surface of responding cells. Plasminogen receptors (Plg-Rs) are heterogeneous and can be classified as tailless, lacking cytoplasmic tails, or tailed (having cytoplasmic tails). In vivo observations implicate several tailless Plg-Rs in inflammatory responses. Tailed Plg-Rs on leukocytes include several integrins, which have also been implicated in Plg-dependent responses. Surface expression of both tailless and tailed Plg-Rs can be modulated in number and/or function. A common mechanism involving intracellular calcium mobilization and calcium channels regulates expression of both classes of Plg-Rs. Data are emerging to indicate that targeting Plg and Plg-Rs may limit inflammation and cardiovascular pathology.