Dmitry A. Soloviev

Leukocyte engagement of fibrin(ogen) via the integrin receptor αMβ2/Mac-1 is critical for host inflammatory response in vivo

The leukocyte integrin αMβ2/Mac-1 appears to support the inflammatory response through multiple ligands, but local engagement of fibrin(ogen) may be particularly important for leukocyte function. To define the biological significance of fibrin(ogen)-αMβ2 interaction in vivo, gene-targeted mice were generated in which the αMβ2-binding motif within the fibrinogen γ chain (N390RLSIGE396) was converted to a series of alanine residues. Mice carrying the Fibγ390–396A allele maintained normal levels of fibrinogen, retained normal clotting function, supported platelet aggregation, and never developed spontaneous hemorrhagic events. However, the mutant fibrinogen failed to support αMβ2-mediated adhesion of primary neutrophils, macrophages, and αMβ2-expressing cell lines. The elimination of the αMβ2-binding motif on fibrin(ogen) severely compromised the inflammatory response in vivo as evidenced by a dramatic impediment in leukocyte clearance of Staphylococcus aureus inoculated into the peritoneal cavity. This defect in bacterial clearance was due not to diminished leukocyte trafficking but rather to a failure to fully implement antimicrobial functions. These studies definitively demonstrate that fibrin(ogen) is a physiologically relevant ligand for αMβ2, integrin engagement of fibrin(ogen) is critical to leukocyte function and innate immunity in vivo, and the biological importance of fibrinogen in regulating the inflammatory response can be appreciated outside of any alteration in clotting function.

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.

Binding of CD40L to Mac-1’s I-domain involves the EQLKKSKTL motif and mediates leukocyte recruitment and atherosclerosis – but does not affect immunity and thrombosis in mice

Rationale: CD40L figures prominently in chronic inflammatory diseases such as atherosclerosis. However, since CD40L potently regulates immune function and hemostasis by interaction with CD40 receptor and the platelet integrin GPIIb/IIIa, its global inhibition compromises host defense and generated thromboembolic complications in clinical trials. We recently reported that CD40L mediates atherogenesis independently of CD40 and proposed Mac-1 as an alternate receptor. Objective: Here, we molecularly characterized the CD40L-Mac-1 interaction and tested whether its selective inhibition by a small peptide modulates inflammation and atherogenesis in vivo. Methods and Results: CD40L concentration-dependently bound to Mac-1 I-domain in solid phase binding assays, and a high-affinity interaction was revealed by surface-plasmon-resonance analysis. We identified the motif EQLKKSKTL, an exposed loop between the &agr;1 helix and the &bgr;-sheet B, on Mac-1 as binding site for CD40L. A linear peptide mimicking this sequence, M7, specifically inhibited the interaction of CD40L and Mac-1. A cyclisized version optimized for in vivo use, cM7, decreased peritoneal inflammation and inflammatory cell recruitment in vivo. Finally, LDLr−/− mice treated with intraperitoneal injections of cM7 developed smaller, less inflamed atherosclerotic lesions featuring characteristics of stability. However, cM7 did not interfere with CD40L-CD40 binding in vitro and CD40L-GPIIb/IIIa-mediated thrombus formation in vivo. Conclusions: We present the novel finding that CD40L binds to the EQLKKSKTL motif on Mac-1 mediating leukocyte recruitment and atherogenesis. Specific inhibition of CD40L-Mac-1 binding may represent an attractive anti-inflammatory treatment strategy for atherosclerosis and other inflammatory conditions, potentially avoiding the unwanted immunologic and thrombotic effects of global inhibition of CD40L.

Regulation of Innate Immune Response to Candida albicans Infections by αMβ2-Pra1p Interaction

ABSTRACT Candida albicans is a common opportunistic fungal pathogen and is the leading cause of invasive fungal diseases in immunocompromised individuals. The induction of cell-mediated immunity to C. albicans is one of the main tasks of cells of the innate immune system, and in vitro evidence suggests that integrin αMβ2 (CR3, Mac-1, and CD11b/CD18) is the principal leukocyte receptor involved in recognition of the fungus. Using αMβ2-KO mice and mutated strains of C. albicans in two models of murine candidiasis, we demonstrate that neutrophils derived from mice deficient in αMβ2 have a reduced ability to kill C. albicans and that the deficient mice themselves exhibit increased susceptibility to fungal infection. Disruption of the PRA1 gene of C. albicans, the primary ligand for αMβ2, protects the fungus against leukocyte killing in vitro and in vivo, impedes the innate immune response to the infection, and increases fungal virulence and organ invasion in vivo. Thus, recognition of pH-regulated antigen 1 protein (Pra1p) by αMβ2 plays a pivotal role in determining fungal virulence and host response and protection against C. albicans infection.

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.

Integrin αXβ2 Is a Leukocyte Receptor for Candida albicans and Is Essential for Protection against Fungal Infections

The opportunistic fungus Candida albicans is one of the leading causes of infections in immunocompromised patients, and innate immunity provides a principal mechanism for protection from the pathogen. In the present work, the role of integrin αXβ2 in the pathogenesis of fungal infection was assessed. Both purified αXβ2 and αXβ2-expressing human epithelial kidney 293 cells recognized and bound to the fungal hyphae of SC5314 strain of C. albicans but not to the yeast form or to hyphae of a strain deficient in the fungal mannoprotein, Pra1. The binding of the integrin to the fungus was inhibited by β-glucans but not by mannans, implicating a lectin-like activity in recognition but distinct in specificity from that of αMβ2. Mice deficient in αXβ2 were more prone to systemic infection with the LD50 fungal inoculum decreasing 3-fold in αXβ2-deficient mice compared with wild-type mice. After challenging i.v. with 1.5 × 104 cell/g, 60% of control C57BL/6 mice died within 14 d compared with 100% mortality of αXβ2-deficient mice within 9 d. Organs taken from αXβ2-deficient mice 16 h postinfection revealed a 10-fold increase in fungal invasion into the brain and a 2-fold increase into the liver. These data indicate that αXβ2 is important for protection against systemic C. albicans infections and macrophage subsets in the liver, Kupffer cells, and in the brain, microglial cells use αXβ2 to control fungal invasion.

Leukocyte integrin Mac-1 regulates thrombosis via interaction with platelet GPIbα

Inflammation and thrombosis occur together in many diseases. The leukocyte integrin Mac-1 (also known as integrin αMβ2, or CD11b/CD18) is crucial for leukocyte recruitment to the endothelium, and Mac-1 engagement of platelet GPIbα is required for injury responses in diverse disease models. However, the role of Mac-1 in thrombosis is undefined. Here we report that mice with Mac-1 deficiency (Mac-1−/−) or mutation of the Mac-1-binding site for GPIbα have delayed thrombosis after carotid artery and cremaster microvascular injury without affecting parameters of haemostasis. Adoptive wild-type leukocyte transfer rescues the thrombosis defect in Mac-1−/− mice, and Mac-1-dependent regulation of the transcription factor Foxp1 contributes to thrombosis as evidenced by delayed thrombosis in mice with monocyte-/macrophage-specific overexpression of Foxp1. Antibody and small-molecule targeting of Mac-1:GPIbα inhibits thrombosis. Our data identify a new pathway of thrombosis involving leukocyte Mac-1 and platelet GPIbα, and suggest that targeting this interaction has anti-thrombotic therapeutic potential with reduced bleeding risk.

Dual Role of the Leukocyte Integrin αMβ2 in Angiogenesis

Polymorphonuclear neutrophils (PMNs) and macrophages are crucial contributors to neovascularization, serving as a source of chemokines, growth factors, and proteases. αMβ2(CD11b/CD18) and αLβ2(CD11a/CD18) are expressed prominently and have been implicated in various responses of these cell types. Thus, we investigated the role of these β2 integrins in angiogenesis. Angiogenesis was analyzed in wild-type (WT), αM-knockout (αM−/−), and αL-deficient (αL−/−) mice using B16F10 melanoma, RM1 prostate cancer, and Matrigel implants. In all models, vascular area was decreased by 50–70% in αM−/− mice, resulting in stunted tumor growth as compared with WT mice. In contrast, αL deficiency did not impair angiogenesis and tumor growth. The neovessels in αM−/− mice were leaky and immature because they lacked smooth muscle cell and pericytes. Defective angiogenesis in the αM−/− mice was associated with attenuated PMN and macrophage recruitment into tumors. In contrast to WT or the αL−/− leukocytes, the αM−/− myeloid cells showed impaired plasmin (Plm)-dependent extracellular matrix invasion, resulting from 50–75% decrease in plasminogen (Plg) binding and pericellular Plm activity. Surface plasmon resonance verified direct interaction of the αMI-domain, the major ligand binding site in the β2 integrins, with Plg. However, the αLI-domain failed to bind Plg. In addition, endothelial cells failed to form tubes in the presence of conditioned medium collected from TNF-α–stimulated PMNs derived from the αM−/− mice because of severely impaired degranulation and secretion of VEGF. Thus, αMβ2 plays a dual role in angiogenesis, supporting not only Plm-dependent recruitment of myeloid cells to angiogenic niches, but also secretion of VEGF by these cells.

Cell Adhesion and Migration Assays

Adhesion and migration are basic responses of living cells to environmental stimuli. Such responses are central to a broad range of physiological processes, such as the immune response, repair of injured tissues, and prevention of excessive bleeding. Cell adhesion and migration also contributes to pathologies, including vascular and inflammatory diseases, as well as tumor growth and metastasis. These cellular responses depend on engagement of adhesion receptors by components of the extracellular matrix or molecules present on the surface of other cells. Hence, cell adhesion and migration assays are crucial methods in cell biology. In this chapter, several detailed protocols describing cell adhesion and migration assays are presented, and advantages and disadvantages of each method are discussed.