Distinct Migratory Properties of M1, M2, and Resident Macrophages Are Regulated by α\u3csub\u3ed\u3c/sub\u3eβ\u3csub\u3e2\u3c/sub\u3eand α\u3csub\u3em\u3c/sub\u3eβ\u3csub\u3e2\u3c/sub\u3eIntegrin-Mediated Adhesion

Chronic inflammation is essential mechanism during the development of cardiovascular and metabolic diseases. The outcome of diseases depends on the balance between the migration/accumulation of pro-inflammatory (M1) and anti-inflammatory (M2) macrophages in damaged tissue. The mechanism of macrophage migration and subsequent accumulation is still not fully understood. Currently, the amoeboid adhesion-independent motility is considered essential for leukocyte migration in the three-dimensional environment. We challenge this hypothesis by studying the contribution of leukocyte adhesive receptors, integrins αMβ2, and αDβ2, to three-dimensional migration of M1-polarized, M2-polarized, and resident macrophages. Both integrins have a moderate expression on M2 macrophages, while αDβ2 is upregulated on M1 and αMβ2 demonstrates high expression on resident macrophages. The level of integrin expression determines its contribution to macrophage migration. Namely, intermediate expression supports macrophage migration, while a high integrin density inhibits it. Using in vitro three-dimensional migration and in vivo tracking of adoptively-transferred fluorescently-labeled macrophages during the resolution of inflammation, we found that strong adhesion of M1-activated macrophages translates to weak 3D migration, while moderate adhesion of M2-activated macrophages generates dynamic motility. Reduced migration of M1 macrophages depends on the high expression of αDβ2, since αD-deficiency decreased M1 macrophage adhesion and improved migration in fibrin matrix and peritoneal tissue. Similarly, the high expression of αMβ2 on resident macrophages prevents their amoeboid migration, which is markedly increased in αM-deficient macrophages. In contrast, αD- and αM-knockouts decrease the migration of M2 macrophages, demonstrating that moderate integrin expression supports cell motility. The results were confirmed in a diet-induced diabetes model. αD deficiency prevents the retention of inflammatory macrophages in adipose tissue and improves metabolic parameters, while αM deficiency does not affect macrophage accumulation. Summarizing, β2 integrin-mediated adhesion may inhibit amoeboid and mesenchymal macrophage migration or support mesenchymal migration in tissue, and, therefore, represents an important target to control inflammation

Inhibition of Integrin α\u3csub\u3ed\u3c/sub\u3eβ\u3csub\u3e2\u3c/sub\u3e-Mediated Macrophage Adhesion to End Product of Docosahexaenoic Acid (DHA) Oxidation Prevents Macrophage Accumulation During Inflammation

A critical step in the development of chronic inflammatory diseases is the accumulation of proinflammatory macrophages in the extracellular matrix (ECM) of peripheral tissues. The adhesion receptor integrin αDβ2 promotes the development of atherosclerosis and diabetes by supporting macrophage retention in inflamed tissue. We recently found that the end product of docosahexaenoic acid (DHA) oxidation, 2-(ω-carboxyethyl)- pyrrole (CEP), serves as a ligand forαDβ2.CEPadduct withECM is generated during inflammation-mediated lipid peroxidation. The goal of this project was to identify a specific inhibitor for αDβ2-CEP interaction that can prevent macrophage accumulation. Using a specially designed peptide library, Biacore-detected protein-protein interaction, and adhesion of integrin-transfected HEK 293 cells, we identified a sequence (called P5 peptide) that significantly and specifically inhibited αD-CEP binding. In the model of thioglycollate-induced peritoneal inflammation, the injection of cyclic P5 peptide reduced 3-fold the macrophage accumulation in WT mice but had no effect in αD-deficient mice. The tracking of adoptively transferred, fluorescently labeled WT and αD-/- monocytes in the model of peritoneal inflammation and in vitro two-dimensional and three-dimensional migration assays demonstrated thatP5peptide does not affectmonocytetransendothelial migration or macrophage efflux from the peritoneal cavity but regulates macrophage migration through the ECM. Moreover, the injection ofP5peptide intoWTmiceona high-fat diet prevents macrophage accumulation in adipose tissue in anαDβ2-dependent manner.Takentogether, these resultsdemonstratetheimportance of αDβ2-mediated macrophage adhesion for the accumulation of infiltrating macrophages in the inflamed ECM and propose P5 peptide as a potential inhibitor of atherogenesis and diabetes

Interaction Between the Integrin Mac-1 and Signal Regulatory Protein α (SIRPα) Mediates Fusion in Heterologous Cells

Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc. Macrophage fusion leading to the formation of multinucleated giant cells is a hallmark of chronic inflammation. Several membrane proteins have been implicated in mediating cell- cell attachment during fusion, but their binding partners remain unknown. Recently, we demonstrated that interleukin-4 (IL-4)- induced fusion of mouse macrophages depends on the integrin macrophage antigen 1 (Mac-1). Surprisingly, the genetic deficiency of intercellular adhesion molecule 1 (ICAM-1), an established ligand of Mac-1, did not impair macrophage fusion, suggesting the involvement of other counter-receptors. Here, using various approaches, including signal regulatory protein α (SIRPα) knockdown, recombinant proteins, adhesion and fusion assays, biolayer interferometry, and peptide libraries, we show that SIRPα, which, similar to ICAM-1, belongs to the Ig superfamily and has previously been implicated in cell fusion, interacts with Mac-1. The following results support the conclusion that SIRPα is a ligand of Mac-1: (a) recombinant ectodomain of SIRPα supports adhesion of Mac-1-expressing cells; (b) Mac-1-SIRPα interaction is mediated through the ligand-binding αMI-domain of Mac-1; (c) recognition of SIRPα by the αMIdomain conforms to general principles governing binding of Mac-1 to many of its ligands; (d) SIRPα reportedly binds CD47; however, anti-CD47 function-blocking mAb produced only a limited inhibition of macrophage adhesion to SIRPα; and (e) co-culturing of SIRPα- and Mac-1-expressing HEK293 cells resulted in the formation of multinucleated cells. Taken together, these results identify SIRPα as a counter-receptor for Mac-1 and suggest that the Mac-1-SIRPα interaction may be involved in macrophage fusion

Pleiotrophin, a Multifunctional Cytokine and Growth Factor, Induces Leukocyte Responses Through the Integrin Mac-1

Pleiotrophin (PTN) is a multifunctional, cationic, glycosaminoglycan- binding cytokine and growth factor involved in numerous physiological and pathological processes, including tissue repair and inflammation-related diseases. PTN has been shown to promote leukocyte responses by inducing their migration and expression of inflammatory cytokines. However, the mechanisms through which PTN mediates these responses remain unclear. Here, we identified the integrin Mac-1 (αMβ2, CD11b/CD18) as the receptor mediating macrophage adhesion and migration to PTN. We also found that expression of Mac-1 on the surface of human embryonic kidney (HEK) 293 cells induced their adhesion and migration to PTN. Accordingly, PTN promoted Mac-1-dependent cell spreading and initiated intracellular signaling manifested in phosphorylation of Erk1/2. While binding to PTN, Mac-1 on Mac-1-expressing HEK293 cells appears to cooperate with cell-surface proteoglycans because both anti-Mac-1 function-blocking mAb and heparin were required to block adhesion. Moreover, biolayer interferometry andNMRindicated a direct interaction between theαMI domain, the major ligand-binding region of Mac-1, and PTN. Using peptide libraries, we found that in PTN the αMI domain bound sequences enriched in basic and hydrophobic residues, indicating thatPTNconforms to the general principle of ligandrecognition specificity of the αMI domain toward cationic proteins/ peptides. Finally, using recombinant PTN-derived fragments, we show that PTN contains two distinct Mac-1-binding sites in each of its constitutive domains. Collectively, these results identify PTN as a ligand for the integrin Mac-1 on the surface of leukocytes and suggest that this interaction may play a role in inflammatory responses

The Interaction of Integrin α IIb β 3 with Fibrin Occurs through Multiple Binding Sites in the α IIb β-Propeller Domain

The currently available antithrombotic agents target the interaction of platelet integrin αIIbβ3 (GPIIb-IIIa) with fibrinogen during platelet aggregation. Platelets also bind fibrin formed early during thrombus growth. It was proposed that inhibition of platelet-fibrin interactions may be a necessary and important property of αIIbβ3 antagonists; however, the mechanisms by which αIIbβ3 binds fibrin are uncertain. We have previously identified the γ370–381 sequence (P3) in the γC domain of fibrinogen as the fibrin-specific binding site for αIIbβ3 involved in platelet adhesion and platelet-mediated fibrin clot retraction. In the present study, we have demonstrated that P3 can bind to several discontinuous segments within the αIIb β-propeller domain of αIIbβ3 enriched with negatively charged and aromatic residues. By screening peptide libraries spanning the sequence of the αIIb β-propeller, several sequences were identified as candidate contact sites for P3. Synthetic peptides duplicating these segments inhibited platelet adhesion and clot retraction but not platelet aggregation, supporting the role of these regions in fibrin recognition. Mutant αIIbβ3 receptors in which residues identified as critical for P3 binding were substituted for homologous residues in the I-less integrin αMβ2 exhibited reduced cell adhesion and clot retraction. These residues are different from those that are involved in the coordination of the fibrinogen γ404–411 sequence and from auxiliary sites implicated in binding of soluble fibrinogen. These results map the binding of fibrin to multiple sites in the αIIb β-propeller and further indicate that recognition specificity of αIIbβ3 for fibrin differs from that for soluble fibrinogen

Platelet factor 4 improves survival in a murine model of antibiotic-susceptible and methicillin-resistant Staphylococcus aureus peritonitis

The complement receptor CR3, also known as integrin Mac-1 (CD11b/CD18), is one of the major phagocytic receptors on the surface of neutrophils and macrophages. We previously demonstrated that in its protein ligands, Mac-1 binds sequences enriched in basic and hydrophobic residues and strongly disfavors negatively charged sequences. The avoidance by Mac-1 of negatively charged surfaces suggests that the bacterial wall and bacterial capsule possessing net negative electrostatic charge may repel Mac-1 and that the cationic Mac-1 ligands can overcome this evasion by acting as opsonins. Indeed, we previously showed that opsonization of Gram-negative Escherichia coli with several cationic peptides, including PF4 (Platelet Factor 4), strongly augmented phagocytosis by macrophages. Here, we investigated the effect of recombinant PF4 (rPF4) on phagocytosis of Gram-positive Staphylococcus aureus in vitro and examined its impact in a mouse model of S. aureus peritonitis. Characterization of the interaction of rPF4 with nonencapsulated and encapsulated S. aureus showed that rPF4 localizes on the bacterial surface, thus making it available for Mac-1. Furthermore, rPF4 did not have direct bactericidal and bacteriostatic activity and was not toxic to host cells. rPF4 enhanced phagocytosis of S. aureus bioparticles by various primary and cultured Mac-1-expressing leukocytes by several folds. It also increased phagocytosis of live nonencapsulated and encapsulated bacteria. Notably, the augmentation of phagocytosis by rPF4 did not compromise the intracellular killing of S. aureus by macrophages. Using a murine S. aureus peritonitis model, we showed that treatment of infected mice with rPF4 caused a significant increase in the clearance of antibiotic-susceptible S. aureus and its methicillin-resistant (MRSA) variant and markedly improved survival. These findings indicate that rPF4 binding to the bacterial surface circumvents its antiphagocytic properties, improving host defense against antibiotic-susceptible and antibiotic-resistant bacteria

The Assembly of Nonadhesive Fibrinogen Matrices Depends on the αC Regions of the Fibrinogen Molecule

Adsorption of fibrinogen on fibrin clots and other surfaces strongly reduces integrin-mediated adhesion of platelets and leukocytes with implications for the surface-mediated control of thrombus growth and blood compatibility of biomaterials. The underlying mechanism of this process is surface-induced aggregation of fibrinogen, resulting in the assembly of a nanoscale multilayered matrix. The matrix is extensible, which makes it incapable of transducing strong mechanical forces via cellular integrins, resulting in insufficient intracellular signaling and weak cell adhesion. To determine the mechanism of the multilayer formation, the physical and adhesive properties of fibrinogen matrices prepared from human plasma fibrinogen (hFg), recombinant normal (rFg), and fibrinogen with the truncated αC regions (FgAα251) were compared. Using atomic force microscopy and force spectroscopy, we show that whereas hFg and rFg generated the matrices with a thickness of ∼8 nm consisting of 7–8 molecular layers, the deposition of FgAα251 was terminated at two layers, indicating that the αC regions are essential for the multilayer formation. The extensibility of the matrix prepared from FgAα251 was 2-fold lower than that formed from hFg and rFg. In agreement with previous findings that cell adhesion inversely correlates with the extensibility of the fibrinogen matrix, the less extensible FgAα251 matrix and matrices generated from human fibrinogen variants lacking the αC regions supported sustained adhesion of leukocytes and platelets. The persistent adhesiveness of matrices formed from fibrinogen derivatives without the αC regions may have implications for conditions in which elevated levels of these molecules are found, including vascular pathologies, diabetes, thrombolytic therapy, and dysfibrinogenemia

Fibrin and D-dimer bind to monomeric GPVI

Fibrin has recently been shown to activate platelets through the immunoglobulin receptor glycoprotein VI (GPVI). In the present study, we show that spreading of human platelets on fibrin is abolished in patients deficient in GPVI, confirming that fibrin activates human platelets through the immunoglobulin receptor. Using a series of proteolytic fragments, we show that D-dimer, but not the E fragment of fibrin, binds to GPVI and that immobilized D-dimer induces platelet spreading through activation of Src and Syk tyrosine kinases. In contrast, when platelets are activated in suspension, soluble D-dimer inhibits platelet aggregation induced by fibrin and collagen, but not by a collagen-related peptide composed of a repeat GPO sequence or by thrombin. Using surface plasmon resonance, we demonstrate that fibrin binds selectively to monomeric GPVI with a KD of 302 nM, in contrast to collagen, which binds primarily to dimeric GPVI. These results establish GPVI as the major signaling receptor for fibrin in human platelets and provide evidence that fibrin binds to a distinct configuration of GPVI. This indicates that it may be possible to develop agents that selectively block the interaction of fibrin but not collagen with the immunoglobulin receptor. Such agents are required to establish whether selective targeting of either interaction has the potential to lead to development of an antithrombotic agent with a reduced effect on bleeding relative to current antiplatelet drugs

β2 integrins as regulators of dendritic cell, monocyte, and macrophage function

Emerging evidence suggests that the β2 integrin family of adhesion molecules have an important role in suppressing immune activation and inflammation. β2 integrins are important adhesion and signaling molecules that are exclusively expressed on leukocytes. The four β2 integrins (CD11a, CD11b, CD11c, and CD11d paired with the β2 chain CD18) play important roles in regulating three key aspects of immune cell function: recruitment to sites of inflammation; cell–cell contact formation; and downstream effects on cellular signaling. Through these three processes, β2 integrins both contribute to and regulate immune responses. This review explores the pro- and anti-inflammatory effects of β2 integrins in monocytes, macrophages, and dendritic cells and how they influence the outcome of immune responses. We furthermore discuss how imbalances in β2 integrin function can have far-reaching effects on mounting appropriate immune responses, potentially influencing the development and progression of autoimmune and inflammatory diseases. Therapeutic targeting of β2 integrins, therefore, holds enormous potential in exploring treatment options for a variety of inflammatory conditions