Publications (11)75.79 Total impact
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Article: Integrin α(X)β₂ is a leukocyte receptor for Candida albicans and is essential for protection against fungal infections.
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ABSTRACT: 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 α(X)β₂ in the pathogenesis of fungal infection was assessed. Both purified α(X)β₂ and α(X)β₂-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 α(M)β₂. Mice deficient in α(X)β₂ were more prone to systemic infection with the LD₅₀ fungal inoculum decreasing 3-fold in α(X)β₂-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 α(X)β₂-deficient mice within 9 d. Organs taken from α(X)β₂-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 α(X)β₂ is important for protection against systemic C. albicans infections and macrophage subsets in the liver, Kupffer cells, and in the brain, microglial cells use α(X)β₂ to control fungal invasion.The Journal of Immunology 07/2012; 189(5):2468-77. · 5.79 Impact Factor -
Article: 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.
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ABSTRACT: 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. 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. 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 α1 helix and the β-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. 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.Circulation Research 11/2011; 109(11):1269-79. · 9.49 Impact Factor -
Article: Regulation of innate immune response to Candida albicans infections by αMβ2-Pra1p interaction.
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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 α(M)β₂ (CR3, Mac-1, and CD11b/CD18) is the principal leukocyte receptor involved in recognition of the fungus. Using α(M)β₂-KO mice and mutated strains of C. albicans in two models of murine candidiasis, we demonstrate that neutrophils derived from mice deficient in α(M)β₂ 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 α(M)β₂, 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 α(M)β₂ plays a pivotal role in determining fungal virulence and host response and protection against C. albicans infection.Infection and immunity 01/2011; 79(4):1546-58. · 4.21 Impact Factor -
Article: Neutrophil apoptosis: selective regulation by different ligands of integrin alphaMbeta2.
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ABSTRACT: Neutrophils undergo spontaneous apoptosis, but their survival can be extended during inflammatory responses. alpha(M)beta(2) 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 alpha(M)beta(2) ligands, plasminogen and angiostatin, derived from plasminogen, as well as fibrinogen and its two derivative alpha(M)beta(2) 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 alpha(M)beta(2) or its individual alpha(M) or beta(2) 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.The Journal of Immunology 10/2008; 181(5):3609-19. · 5.79 Impact Factor -
Article: Expression, activation, and function of integrin alphaMbeta2 (Mac-1) on neutrophil-derived microparticles.
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ABSTRACT: 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.Blood 06/2008; 112(6):2327-35. · 9.90 Impact Factor -
Article: Identification of pH-regulated antigen 1 released from Candida albicans as the major ligand for leukocyte integrin alphaMbeta2.
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ABSTRACT: 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 alpha(M)beta(2) (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 alpha(M)beta(2)-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 alpha(M)beta(2)-dependent adhesion and migration; and 2) affinity chromatography on purified alpha(M)beta(2). 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 alpha(M)beta(2) function prevented killing of Pra1p-expressing but not Pra1p-deficient strains of C. albicans. Together, these data indicate that Pra1p is a ligand of alpha(M)beta(2) on C. albicans and that the soluble form of Pra1p may assist the fungus in escaping host surveillance.The Journal of Immunology 03/2007; 178(4):2038-46. · 5.79 Impact Factor -
Article: Cell adhesion and migration assays.
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ABSTRACT: 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.Methods in molecular medicine 02/2006; 129:267-78. -
Article: Leukocyte engagement of fibrin(ogen) via the integrin receptor alphaMbeta2/Mac-1 is critical for host inflammatory response in vivo.
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ABSTRACT: The leukocyte integrin alpha(M)beta(2)/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)-alpha(M)beta(2) interaction in vivo, gene-targeted mice were generated in which the alpha(M)beta(2)-binding motif within the fibrinogen gamma chain (N(390)RLSIGE(396)) was converted to a series of alanine residues. Mice carrying the Fibgamma(390-396A) 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 alpha(M)beta(2)-mediated adhesion of primary neutrophils, macrophages, and alpha(M)beta(2)-expressing cell lines. The elimination of the alpha(M)beta(2)-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 alpha(M)beta(2), 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.Journal of Clinical Investigation 07/2004; 113(11):1596-606. · 15.39 Impact Factor -
Article: Integrin alphaMbeta2 orchestrates and accelerates plasminogen activation and fibrinolysis by neutrophils.
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ABSTRACT: Plasmin, the pivotal thrombolytic enzyme, is generated on the surface of many cell types, where urokinase receptor (uPAR)-bound urokinase (uPA) activates cell-bound plasminogen (Plg). It has been reported that neutrophils mediate endogenous thrombolysis involving a uPA-dependent mechanism, and we previously demonstrated that both uPAR and integrin alpha(M)beta(2) recognize uPA to control cell migration and adhesion. In the present study, we report that the alpha(M)beta(2) regulates neutrophil-dependent fibrinolysis. Phorbol 12-myristate 13-acetate (PMA)-stimulated but not resting neutrophils dissolved fibrin clots, and this activity was not only uPA- and Plg-dependent but also alpha(M)beta(2)-dependent. Purified alpha(M)beta(2) directly bound uPA (K(d) = 40 nm) and Plg (K(d) = 1 microm) in a dose-dependent and saturable manner. In Plg activation assays, addition of purified alpha(M)beta(2), but not a control protein, to a single chain uPA (sc-uPA)/Plg mixture, decreased the K(m) from 2 to 0.1 microm, thereby augmenting the overall reaction efficiency by 50-fold. The binding of sc-uPA to alpha(M)beta(2) was critical for the alpha(M)beta(2)-mediated enhancement of plasmin (Plm) generation, because this effect was lost when WT-sc-uPA was replaced with a kringle-less mutant (DeltaK-sc-uPA), which does not bind to alpha(M)beta(2). Plm inactivation by alpha(2)-antiplasmin was significantly delayed when Plm was preincubated with purified, soluble alpha(M)beta(2). When Plg was added to PMA-stimulated neutrophils, both uPA and Plg were co-immunoprecipitated with alpha(M)beta(2.) Thus, assembly of Plg and uPA on integrin alpha(M)beta(2) regulates Plm activity and, thereby, plays a crucial role in neutrophil-mediated thrombolysis.Journal of Biological Chemistry 05/2004; 279(17):18063-72. · 4.77 Impact Factor -
Article: Integrin αMβ2 Orchestrates and Accelerates Plasminogen Activation and Fibrinolysis by Neutrophils
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ABSTRACT: Plasmin, the pivotal thrombolytic enzyme, is generated on the surface of many cell types, where urokinase receptor (uPAR)-bound urokinase (uPA) activates cell-bound plasminogen (Plg). It has been reported that neutrophils mediate endogenous thrombolysis involving a uPA-dependent mechanism, and we previously demonstrated that both uPAR and integrin αMβ2 recognize uPA to control cell migration and adhesion. In the present study, we report that the αMβ2 regulates neutrophil-dependent fibrinolysis. Phorbol 12-myristate 13-acetate (PMA)-stimulated but not resting neutrophils dissolved fibrin clots, and this activity was not only uPA- and Plg-dependent but also αMβ2-dependent. Purified αMβ2 directly bound uPA (Kd = 40 nm) and Plg (Kd = 1 μm) in a dose-dependent and saturable manner. In Plg activation assays, addition of purified αMβ2, but not a control protein, to a single chain uPA (sc-uPA)/Plg mixture, decreased the Km from 2 to 0.1 μm, thereby augmenting the overall reaction efficiency by 50-fold. The binding of sc-uPA to αMβ2 was critical for the αMβ2-mediated enhancement of plasmin (Plm) generation, because this effect was lost when WT-sc-uPA was replaced with a kringle-less mutant (ΔK-sc-uPA), which does not bind to αMβ2. Plm inactivation by α2-antiplasmin was significantly delayed when Plm was preincubated with purified, soluble αMβ2. When Plg was added to PMA-stimulated neutrophils, both uPA and Plg were co-immunoprecipitated with αMβ2. Thus, assembly of Plg and uPA on integrin αMβ2 regulates Plm activity and, thereby, plays a crucial role in neutrophil-mediated thrombolysis.Journal of Biological Chemistry 04/2004; 279(17):18063-18072. · 4.77 Impact Factor -
Article: Convergence of the adhesive and fibrinolytic systems: recognition of urokinase by integrin alpha Mbeta 2 as well as by the urokinase receptor regulates cell adhesion and migration.
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ABSTRACT: Previous studies demonstrated that integrin alpha(M)beta(2) (CD11b/18, Mac-1) forms a physical complex with the urokinase-type plasminogen activator receptor (uPAR/CD87) on leukocytes. In this study, we used human peripheral blood neutrophils and transfected cells expressing alpha(M)beta(2), uPAR, or both receptors to show that the integrin can directly interact with urokinase (uPA). We demonstrate that alpha(M)beta(2) supported adhesion and migration of these cells to uPA, and, in each case, blockade of alpha(M)beta(2) suppressed the response. Within uPA, both the kringle and proteolytic domains are recognized by alpha(M)beta(2), which are distinct from the growth factor domain that binds to uPAR. Within the alpha(M) subunit of the integrin, the I domain interacts with uPA, which is distinct from the region that interacts with uPAR. On cells expressing uPAR and alpha(M)beta(2), both receptors mediated adhesion and migration. This cooperation was particularly apparent in the responses of neutrophils to uPA, where blockade of alpha(M)beta(2) reduced uPAR-mediated responses and engagement of uPAR enhanced recognition of uPA by alpha(M)beta(2). Thus, recognition of uPA by alpha(M)beta(2) allows for formation of a multicontact trimolecular complex, in which a single uPA ligand may bind simultaneously to both uPAR and alpha(M)beta(2). This complex may play an important role in the control of inflammatory cell migration and vascular homeostasis.Blood 03/2003; 101(4):1582-90. · 9.90 Impact Factor
