Endoplasmic Reticulum Protein 72 Regulates Integrin Mac-1 Activity to Influence Neutrophil Recruitment.

BACKGROUND: Integrins mediate the adhesion, crawling, and migration of neutrophils during vascular inflammation. Thiol exchange is important in the regulation of integrin functions. ERp72 (endoplasmic reticulum-resident protein 72) is a member of the thiol isomerase family responsible for the catalysis of disulfide rearrangement. However, the role of ERp72 in the regulation of Mac-1 (integrin αMß2) on neutrophils remains elusive. METHODS: Intravital microscopy of the cremaster microcirculation was performed to determine in vivo neutrophil movement. Static adhesion, flow chamber, and flow cytometry were used to evaluate in vitro integrin functions. Confocal fluorescent microscopy and coimmunoprecipitation were utilized to characterize the interactions between ERp72 and Mac-1 on neutrophil surface. Cell-impermeable probes and mass spectrometry were used to label reactive thiols and identify target disulfide bonds during redox exchange. Biomembrane force probe was performed to quantitatively measure the binding affinity of Mac-1. A murine model of acute lung injury induced by lipopolysaccharide was utilized to evaluate neutrophil-associated vasculopathy. RESULTS: ERp72-deficient neutrophils exhibited increased rolling but decreased adhesion/crawling on inflamed venules in vivo and defective static adhesion in vitro. The defect was due to defective activation of integrin Mac-1 but not LFA-1 (lymphocyte function-associated antigen-1) using blocking or epitope-specific antibodies. ERp72 interacted with Mac-1 in lipid rafts on neutrophil surface leading to the reduction of the C654-C711 disulfide bond in the αM subunit that is critical for Mac-1 activation. Recombinant ERp72, via its catalytic motifs, increased the binding affinity of Mac-1 with ICAM-1 (intercellular adhesion molecule-1) and rescued the defective adhesion of ERp72-deficient neutrophils both in vitro and in vivo. Deletion of ERp72 in the bone marrow inhibited neutrophil infiltration, ameliorated tissue damage, and increased survival during murine acute lung injury. CONCLUSIONS: Extracellular ERp72 regulates integrin Mac-1 activity by catalyzing disulfide rearrangement on the αM subunit and may be a novel target for the treatment of neutrophil-associated vasculopathy.

Neutrophil attachment via Mac-1 (αMß2; CD11b/CD18; CR3) integrins induces PAD4 deimination of profilin and histone H3.

Neutrophil adhesion to endothelia, entry into tissues and chemotaxis constitute essential steps in the immune response to infections that drive inflammation. Neutrophils bind to other cells and migrate via adhesion receptors, notably the αMß2 integrin dimer (also called Mac-1, CR3 or CD11b/CD18). Here, the response of neutrophils to integrin engagement was examined by monitoring the activity of peptidylarginine deiminase 4 (PAD4). Histone H3 deimination was strongly stimulated by manganese, an integrin-activating divalent cation, even in the absence of additional inflammatory stimuli. Manganese-induced cell attachment resulted in neutrophil swarm formation that paralleled histone deimination, whereas antibodies that impair integrin binding prevented both cell adhesion and histone deimination. Manganese treatment led to putative deimination of profilin, a protein that functions as an actin-organizing hub, as detected by two-dimensional gel electrophoresis and citrulline immunoblotting. Cl-amidine, a covalent inhibitor of PAD4, and GSK484, a specific PAD4 inhibitor, blocked profilin deimination. Neutrophil migration toward leukotriene B4 and toward synovial fluid from a rheumatoid arthritis patient were inhibited by chloramidine, thus supporting the contribution of deimination to chemotaxis. The data, based on a simplified system for integrin activation, imply a mechanism whereby integrin attachment coordinates neutrophil responses to inflammation and orchestrates deimination of nuclear and cytoskeletal proteins. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

GPR55 Antagonist CID16020046 Protects against Atherosclerosis Development in Mice by Inhibiting Monocyte Adhesion and Mac-1 Expression.

GPR55 recognizes several lipid molecules such as lysophosphatidylinositol. GPR55 expression was reported in human monocytes. However, its role in monocyte adhesion and atherosclerosis development has not been studied. The role of GPR55 in monocyte adhesion and atherosclerosis development was investigated in human THP-1 monocytes and ApoE-/- mice using O-1602 (a potent agonist of GPR55) and CID16020046 (a specific GPR55 antagonist). O-1602 treatment significantly increased monocyte adhesion to human umbilical vein endothelial cells, and the O-1602-induced adhesion was inhibited by treatment with CID16020046. O-1602 induced the expression of Mac-1 adhesion molecules, whereas CID16020046 inhibited this induction. Analysis of the promoter region of Mac-1 elucidated the binding sites of AP-1 and NF-κB between nucleotides -750 and -503 as GPR55 responsive elements. O-1602 induction of Mac-1 was found to be dependent on the signaling components of GPR55, that is, Gq protein, Ca2+, CaMKK, and PI3K. In Apo-/- mice, administration of CID16020046 ameliorated high-fat diet-induced atherosclerosis development. These results suggest that high-fat diet-induced GPR55 activation leads to the adhesion of monocytes to endothelial cells via induction of Mac-1, and CID16020046 blockage of GPR55 could suppress monocyte adhesion to vascular endothelial cells through suppression of Mac-1 expression, leading to protection against the development of atherosclerosis.

Complement Factor H-Related 3 Enhanced Inflammation and Complement Activation in Human RPE Cells.

Complement Factor H-Related 3 (FHR-3) is a major regulator of the complement system, which is associated with different diseases, such as age-related macular degeneration (AMD). However, the non-canonical local, cellular functions of FHR-3 remained poorly understood. Here, we report that FHR-3 bound to oxidative stress epitopes and competed with FH for interaction. Furthermore, FHR-3 was internalized by viable RPE cells and modulated time-dependently complement component (C3, FB) and receptor (C3aR, CR3) expression of human RPE cells. Independently of any external blood-derived proteins, complement activation products were detected. Anaphylatoxin C3a was visualized in treated cells and showed a translocation from the cytoplasm to the cell membrane after FHR-3 exposure. Subsequently, FHR-3 induced a RPE cell dependent pro-inflammatory microenvironment. Inflammasome NLRP3 activation and pro-inflammatory cytokine secretion of IL-1ß, IL-18, IL-6 and TNF-α were induced after FHR-3-RPE interaction. Our previously published monoclonal anti-FHR-3 antibody, which was chimerized to reduce immunogenicity, RETC-2-ximab, ameliorated the effect of FHR-3 on ARPE-19 cells. Our studies suggest FHR-3 as an exogenous trigger molecule for the RPE cell "complosome" and as a putative target for a therapeutic approach for associated degenerative diseases.

Insights on the Functional Role of Beta-Glucans in Fungal Immunity Using Receptor-Deficient Mouse Models.

Understanding the host anti-fungal immunity induced by beta-glucan has been one of the most challenging conundrums in the field of biomedical research. During the last couple of decades, insights on the role of beta-glucan in fungal disease progression, susceptibility, and resistance have been greatly augmented through the utility of various beta-glucan cognate receptor-deficient mouse models. Analysis of dectin-1 knockout mice has clarified the downstream signaling pathways and adaptive effector responses triggered by beta-glucan in anti-fungal immunity. On the other hand, assessment of CR3-deficient mice has elucidated the compelling action of beta-glucans in neutrophil-mediated fungal clearance, and the investigation of EphA2-deficient mice has highlighted its novel involvement in host sensing and defense to oral mucosal fungal infection. Based on these accounts, this review focuses on the recent discoveries made by these gene-targeted mice in beta-glucan research with particular emphasis on the multifaceted aspects of fungal immunity.

A DARPin targeting activated Mac-1 is a novel diagnostic tool and potential anti-inflammatory agent in myocarditis, sepsis and myocardial infarction.

The monocyte ß2-integrin Mac-1 is crucial for leukocyte-endothelium interaction, rendering it an attractive therapeutic target for acute and chronic inflammation. Using phage display, a Designed-Ankyrin-Repeat-Protein (DARPin) was selected as a novel binding protein targeting and blocking the αM I-domain, an activation-specific epitope of Mac-1. This DARPin, named F7, specifically binds to activated Mac-1 on mouse and human monocytes as determined by flow cytometry. Homology modelling and docking studies defined distinct interaction sites which were verified by mutagenesis. Intravital microscopy showed reduced leukocyte-endothelium adhesion in mice treated with this DARPin. Using mouse models of sepsis, myocarditis and ischaemia/reperfusion injury, we demonstrate therapeutic anti-inflammatory effects. Finally, the activated Mac-1-specific DARPin is established as a tool to detect monocyte activation in patients receiving extra-corporeal membrane oxygenation, as well as suffering from sepsis and ST-elevation myocardial infarction. The activated Mac-1-specific DARPin F7 binds preferentially to activated monocytes, detects inflammation in critically ill patients, and inhibits monocyte and neutrophil function as an efficient new anti-inflammatory agent.

Dysfunction of complement receptors CR3 (CD11b/18) and CR4 (CD11c/18) in pre-eclampsia: a genetic and functional study.

OBJECTIVE: To study genetic variants and their function within genes coding for complement receptors in pre-eclampsia. DESIGN: A case-control study. SETTING: Pre-eclampsia is a common vascular disease of pregnancy. The clearance of placenta-derived material is one of the functions of the complement system in pregnancy. POPULATION: We genotyped 500 women with pre-eclamptic pregnancies and 190 pregnant women without pre-eclampsia, as controls, from the FINNPEC cohort, and 122 women with pre-eclamptic pregnancies and 1905 controls from the national FINRISK cohort. METHODS: The functional consequences of genotypes discovered by targeted exomic sequencing were explored by analysing the binding of the main ligand iC3b to mutated CR3 or CR4, which were transiently expressed on the surface of COS-1 cells. MAIN OUTCOME MEASURES: Allele frequencies were compared between pre-eclamptic pregnancies and controls in genetic studies. The functional consequences of selected variants were measured by binding assays. RESULTS: The most significantly pre-eclampsia-linked CR3 variant M441K (P = 4.27E-4, OR = 1.401, 95% CI = 1.167-1.682) displayed a trend of increased adhesion to iC3b (P = 0.051). The CR4 variant A251T was found to enhance the adhesion of CR4 to iC3b, whereas W48R resulted in a decrease of the binding of CR4 to iC3b. CONCLUSIONS: Results suggest that changes in complement-facilitated phagocytosis are associated with pre-eclampsia. Further studies are needed to ascertain whether aberrant CR3 and CR4 activity leads to altered pro- and anti-inflammatory cytokine responses in individuals carrying the associated variants, and the role of these receptors in pre-eclampsia pathogenesis. TWEETABLE ABSTRACT: Genetic variants of complement receptors CR3 and CR4 have functional consequences that are associated with pre-eclampsia.

Double-stranded RNA-induced dopaminergic neuronal loss in the substantia nigra in the presence of Mac1 receptor.

BACKGROUND: The underlying mechanism of viral infection as a risk factor for Parkinson's disease (PD), the second most common neurodegenerative disease, remains unclear. OBJECTIVE: We used Mac-1-/- and gp91phox-/- transgene animal models to investigate the mechanisms by which poly I:C, a mimic of virus double-stranded RNA, induces PD neurodegeneration. METHOD: Poly I:C was stereotaxically injected into the substantia nigra (SN) of wild-type (WT), Mac-1-knockout (Mac-1-/-) and gp91 phox-knockout (gp91 phox-/-) mice (10 µg/µl), and nigral dopaminergic neurodegeneration, α-synuclein accumulation and neuroinflammation were evaluated. RESULT: Dopaminergic neurons in the nigra and striatum were markedly reduced in WT mice after administration of poly I:C together with abundant microglial activation in the SN, and the expression of α-synuclein was also elevated. However, these pathological changes were greatly dampened in Mac-1-/- and gp91 phox-/- mice. CONCLUSIONS: Our findings demonstrated that viral infection could result in the activation of microglia as well as NADPH oxidase, which may lead to neuron loss and the development of Parkinson's-like symptoms. Mac-1 is a key receptor during this process.

CR4 Signaling Contributes to a DC-Driven Enhanced Immune Response Against Complement-Opsonized HIV-1.

Dendritic cells (DCs) possess intrinsic cellular defense mechanisms to specifically inhibit HIV-1 replication. In turn, HIV-1 has evolved strategies to evade innate immune sensing by DCs resulting in suboptimal maturation and poor antiviral immune responses. We previously showed that complement-opsonized HIV-1 (HIV-C) was able to efficiently infect various DC subsets significantly higher than non-opsonized HIV-1 (HIV) and therefore also mediate a higher antiviral immunity. Thus, complement coating of HIV-1 might play a role with respect to viral control occurring early during infection via modulation of DCs. To determine in detail which complement receptors (CRs) expressed on DCs was responsible for infection and superior pro-inflammatory and antiviral effects, we generated stable deletion mutants for the α-chains of CR3, CD11b, and CR4, CD11c using CRISPR/Cas9 in THP1-derived DCs. We found that CD11c deletion resulted in impaired DC infection as well as antiviral and pro-inflammatory immunity upon exposure to complement-coated HIV-1. In contrast, sole expression of CD11b on DCs shifted the cells to an anti-inflammatory, regulatory DC type. We here illustrated that CR4 comprised of CD11c and CD18 is the major player with respect to DC infection associated with a potent early pro-inflammatory immune response. A more detailed characterization of CR3 and CR4 functions using our powerful tool might open novel avenues for early therapeutic intervention during HIV-1 infection.

Differential Interactions of Serum and Bronchoalveolar Lavage Fluid Complement Proteins with Conidia of Airborne Fungal Pathogen Aspergillus fumigatus.

Even though both cellular and humoral immunities contribute to host defense, the role played by humoral immunity against the airborne opportunistic fungal pathogen Aspergillus fumigatus has been underexplored. In this study, we aimed at deciphering the role of the complement system, the major humoral immune component, against A. fumigatus Mass spectrometry analysis of the proteins extracted from A. fumigatus conidial (asexual spores and infective propagules) surfaces opsonized with human serum indicated that C3 is the major complement protein involved. Flow cytometry and immunolabeling assays further confirmed C3b (activated C3) deposition on the conidial surfaces. Assays using cell wall components of conidia indicated that the hydrophobin RodAp, ß-(1,3)-glucan (BG) and galactomannan (GM) could efficiently activate C3. Using complement component-depleted sera, we showed that while RodAp activates C3 by the alternative pathway, BG and GM partially follow the classical and lectin pathways, respectively. Opsonization facilitated conidial aggregation and phagocytosis, and complement receptor (CR3 and CR4) blockage on phagocytes significantly inhibited phagocytosis, indicating that the complement system exerts a protective role against conidia by opsonizing them and facilitating their phagocytosis mainly through complement receptors. Conidial opsonization with human bronchoalveolar lavage fluid (BALF) confirmed C3 to be the major complement protein interacting with conidia. Nevertheless, complement C2 and mannose-binding lectin (MBL), the classical and lectin pathway components, respectively, were not identified, indicating that BALF activates the alternative pathway on the conidial surface. Moreover, the cytokine profiles were different upon stimulation of phagocytes with serum- and BALF-opsonized conidia, highlighting the importance of studying interaction of conidia with complement proteins in their biological niche.

Assessment of Arf6 Deletion in PLB-985 Differentiated in Neutrophil-Like Cells and in Mouse Neutrophils: Impact on Adhesion and Migration.

Chemoattractant sensing, adhesiveness, and migration are critical events underlying the recruitment of neutrophils (PMNs) to sites of inflammation or infection. Defects in leukocyte adhesion or migration result in immunodeficiency disorders characterized by recurrent infections. In this study, we evaluated the role of Arf6 on PMN adhesion in vitro and on migration to inflammatory sites using PMN-Arf6 conditional knockout (cKO) mice. In PMN-like PLB-985 silenced for Arf6 fMLP-mediated adhesion to the ß2 integrin ligands, ICAM-1 and fibrinogen or the ß1/ß2 integrin ligand fibronectin was significantly reduced. Furthermore, overexpression of wild-type Arf6 promoted basal and fMLP-induced adhesion to immobilized integrin ligands, while overexpression of the dominant-negative Arf6 has the opposite effects. Using the Elane-Cre deleting mouse strains, we report that the level of Arf6 deletion in inflammatory PMNs isolated from the dorsal air pouches was stronger when compared to naïve cells isolated from the bone marrow. In PMN-Arf6 cKO mice, the recruitment of PMNs into the dorsal air pouch injected with LPS or the chemoattractant fMLP was significantly diminished. Impaired cell migration correlated with reduced cell surface expression of CD11a and CD11b in Arf6 cKO PMNs. Our results highlight that Arf6 regulates the activity and possibly the recycling of PMN integrins, and this compromises PMN migration to inflammatory sites.

The Toll-Like Receptor 3 Agonist Polyriboinosinic Polyribocytidylic Acid Increases the Numbers of NK Cells with Distinct Phenotype in the Liver of B6 Mice.

One of the activating factors of the cells of the innate immune system is the agonists of toll-like receptors (TLRs). Our earlier publications detailed how poly(I:C), a TLR3 agonist, elevates the NK cell population and the associated antigen-specific CD8+ T cell responses. This study involved a single treatment of the B6 mice with poly(I:C) intraperitoneally. To perform a detailed phenotypic analysis, mononuclear cells were prepared from each of the liver, peripheral blood, and spleen. These cells were then examined for their NK cell population by flow cytometric analysis following cell staining with indicated antibodies. The findings of the study showed that the NK cell population of the liver with an NK1.1highCD11bhighCD11chigh B220+Ly6G- phenotype was elevated following the treatment with poly(I:C). In the absence of CD11b molecule (CR3-/- mice), poly(I:C) can still increase the remained numbers of NK cells with NK1.1+CD11b- and NK1.1+Ly6G- phenotypes in the liver while their numbers in the blood decrease. After the treatment with anti-AGM1 Ab, which induced depletion of NK1.1+CD11b+ cells and partial depletion of CD3+NK1.1+ and NK1.1+CD11b- cell populations, poly(I:C) normalized the partial decreases in the numbers of NK cells concomitant with increased numbers of NK1.1-CD11b+ cell population in both liver and blood. Regarding mice with a TLR3-/- phenotype, their injection with poly(I:C) resulted in the partial elevation in the NK cell population as compared to wild-type B6 mice. To summarise, the TLR3 agonist poly(I:C) results in the elevation of a subset of liver NK cells expressing the two myeloid markers CD11c and CD11b. The effect of poly(I:C) on NK cells is partially dependent on TLR3 and independent of the presence of CD11b.

Mac-1 triggers neutrophil DNA extracellular trap formation to Aspergillus fumigatus independently of PAD4 histone citrullination.

Aspergillus fumigatus (A. fumigatus) is an environmental fungus and a human pathogen. Neutrophils are critical effector cells during the fungal infections, and neutropenia is a risk factor for the development of pulmonary aspergillosis. Neutrophil extracellular traps (NETs) are released by neutrophils in response to A. fumigatus and inhibit the conidial germination. In this work, we observed that the receptors TLR2, TLR4, and Dectin-1 were dispensable for the A. fumigatus induced NET release. In contrast CD11b/CD18 was critical for the NET release in response to A. fumigatus conidia, and this required the CD11b I-domain-mediated recognition, whereas the blockade of the CD11b lectin domain did not affect the A. fumigatus induced NET release. A. fumigatus induced NET release relied on the activity of spleen tyrosine kinase (Syk), Src family kinase(s), and class IA PI3 kinase δ. Although A. fumigatus promoted histone citrullination, this process was dispensable for the NET release in response to A. fumigatus conidia. The A. fumigatus induced NET release required the reactive oxygen species generation by the NOX2 complex, in a downstream pathway requiring CD11b/CD18, Src kinase family activity, Syk and PI3K class IA δ. Our findings thus reveal the signaling pathways involved in the formation of NETs in response to A. fumigatus.

Coupling of ß2 integrins to actin by a mechanosensitive molecular clutch drives complement receptor-mediated phagocytosis.

αMß2 integrin (complement receptor 3) is a major receptor for phagocytosis in macrophages. In other contexts, integrins' activities and functions are mechanically linked to actin dynamics through focal adhesions. We asked whether mechanical coupling of αMß2 integrin to the actin cytoskeleton mediates phagocytosis. We found that particle internalization was driven by formation of Arp2/3 and formin-dependent actin protrusions that wrapped around the particle. Focal complex-like adhesions formed in the phagocytic cup that contained ß2 integrins, focal adhesion proteins and tyrosine kinases. Perturbation of talin and Syk demonstrated that a talin-dependent link between integrin and actin and Syk-mediated recruitment of vinculin enable force transmission to target particles and promote phagocytosis. Altering target mechanical properties demonstrated more efficient phagocytosis of stiffer targets. Thus, macrophages use tyrosine kinase signalling to build a mechanosensitive, talin- and vinculin-mediated, focal adhesion-like molecular clutch, which couples integrins to cytoskeletal forces to drive particle engulfment.

T cell-derived soluble glycoprotein GPIbα mediates PGE2 production in human monocytes activated with the vaccine adjuvant MDP.

Vaccine adjuvants containing analogs of microbial products activate pattern recognition receptors (PRRs) on antigen-presenting cells, including monocytes and macrophages, which can cause prostaglandin E2 (PGE2) release and consequently undesired inflammatory responses and fever in vaccine recipients. Here, we studied the mechanism of PGE2 production by human monocytes activated with muramyl dipeptide (MDP) adjuvant, which activates cytosolic nucleotide-binding oligomerization domain 2 (NOD2). In rabbits, administration of MDP elicited an early increase in PGE2 followed by fever. In human monocytes, MDP alone did not induce PGE2 production. However, high amounts of PGE2 and the proinflammatory cytokines IL-1ß and IL-6 were secreted by monocytes activated with MDP in the presence of conditioned medium obtained from CD3 bead-isolated T cells (Tc CM) but not from those isolated without CD3 beads. Mass spectrometry and immunoblotting revealed that the costimulatory factor in Tc CM was glycoprotein Ib α (GPIbα). Antibody-mediated blockade of GPIbα or of its receptor, Mac-1 integrin, inhibited the secretion of PGE2, IL-1ß, and IL-6 in MDP + Tc CM-activated monocytes, whereas recombinant GPIbα protein increased PGE2 production by MDP-treated monocytes. In vivo, COX2 mRNA abundance was reduced in the liver and spleen of Mac-1 KO mice after administration of MDP compared with that of treated wild-type mice. Our findings suggest that the production of PGE2 and proinflammatory cytokines by MDP-activated monocytes is mediated by cooperation between two signaling pathways: one delivered by MDP through NOD2 and a second through activation of Mac-1 by T cell-derived GPIbα.

Effects of sinomenine in LPS-associated diseases are related to inhibition of LBP, Mac-1, and L-selectin levels.

The aim of the research was to investigate the anti-endotoxin and anti-inflammatory effects of Sinomenine, an agent commonly found in Chinese herbal medicines. Endotoxin (i.e., 1 mg lipopolysaccharide (LPS)/kg)) was administered via intraperitoneal (IP) injection to piglets in high-, middle-, and low-dose sinomenine groups. Piglets were then treated with 1, 5 or 10 mg/kg sinomenine, intramuscularly (i.m.), 3 hr after LPS. Vehicle was administered, as above, to drug control group piglets followed 3 hr later by 10 mg/kg sinomenine i.m.. LPS control group piglets were challenged with 1 mg/kg LPS IP, followed by vehicle i.m., and naïve control piglets were treated with normal saline IP, followed by normal saline i.m., as above. Temperatures were measured, and blood samples were collected from the precaval veins of piglets at 12, 24, and 48 hr post-LPS or vehicle injection. Clinical signs were recorded, and index levels were analyzed via ELISA. Sinomenine was found to reduce the incidence and severity of LPS-induced toxicities, including body temperature elevation, cell adhesion, and systemic inflammation. These data suggest that sinomenine may be effective for regulating inflammatory responses and has the potential for use as an anti-endotoxin therapy.

C3- and CR3-dependent microglial clearance protects photoreceptors in retinitis pigmentosa.

Complement activation has been implicated as contributing to neurodegeneration in retinal and brain pathologies, but its role in retinitis pigmentosa (RP), an inherited and largely incurable photoreceptor degenerative disease, is unclear. We found that multiple complement components were markedly up-regulated in retinas with human RP and the rd10 mouse model, coinciding spatiotemporally with photoreceptor degeneration, with increased C3 expression and activation localizing to activated retinal microglia. Genetic ablation of C3 accelerated structural and functional photoreceptor degeneration and altered retinal inflammatory gene expression. These phenotypes were recapitulated by genetic deletion of CR3, a microglia-expressed receptor for the C3 activation product iC3b, implicating C3-CR3 signaling as a regulator of microglia-photoreceptor interactions. Deficiency of C3 or CR3 decreased microglial phagocytosis of apoptotic photoreceptors and increased microglial neurotoxicity to photoreceptors, demonstrating a novel adaptive role for complement-mediated microglial clearance of apoptotic photoreceptors in RP. These homeostatic neuroinflammatory mechanisms are relevant to the design and interpretation of immunomodulatory therapeutic approaches to retinal degenerative disease.

Structural basis of the leukocyte integrin Mac-1 I-domain interactions with the platelet glycoprotein Ib.

Cell-surface receptor interactions between leukocyte integrin macrophage-1 antigen (Mac-1, also known as CR3, αMß2, CD11b/CD18) and platelet glycoprotein Ibα (GPIbα) are critical to vascular inflammation. To define the key residues at the binding interface, we used nuclear magnetic resonance (NMR) to assign the spectra of the mouse Mac-1 I-domain and mapped the residues contacting the mouse GPIbα N-terminal domain (GPIbαN) to the locality of the integrin metal ion-dependant adhesion site (MIDAS) surface. We next determined the crystal structures of the mouse GPIbαN and Mac-1 I-domain to 2 Å and 2.5 Å resolution, respectively. The mouse Mac-1 I-domain crystal structure reveals an active conformation that is stabilized by a crystal contact from the α7-helix with a glutamate side chain completing the octahedral coordination sphere of the MIDAS Mg2+ ion. The amino acid sequence of the α7-helix and disposition of the glutamic acid matches the C-terminal capping region α-helix of GPIbα effectively acting as a ligand mimetic. Using these crystal structures in combination with NMR measurements and docking analysis, we developed a model whereby an acidic residue from the GPIbα leucine-rich repeat (LRR) capping α-helix coordinates directly to the Mac-1 MIDAS Mg2+ ion. The Mac-1:GPIbαN complex involves additional interactions consolidated by an elongated pocket flanking the GPIbαN LRR capping α-helix. The GPIbαN α-helix has an HxxxE motif, which is equivalent by homology to RxxxD from the human GPIbαN. Subsequent mutagenesis of residues at this interface, coupled with surface plasmon resonance studies, confirmed the importance of GPIbαN residues H218, E222, and the Mac-1 MIDAS residue T209 to formation of the complex.

LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) distinctly regulate neutrophil extravasation through hotspots I and II.

Precise spatiotemporal regulation of leukocyte extravasation is key for generating an efficient immune response to injury or infection. The integrins LFA-1(CD11a/CD18) and Mac-1(CD11b/CD18) play overlapping roles in neutrophil migration because they bind the same as well as different ligands in response to extracellular signaling. Using two-photon intravital imaging and transmission electron microscopy, we observed the existence of preferred sites for neutrophil entrance into the endothelial cell monolayer and exit from the basement membrane and pericyte sheath during neutrophil extravasation, namely, hotspots I and II, by elucidating distinctive roles of LFA-1 and Mac-1. To penetrate the vascular endothelium, neutrophils must first penetrate the endothelial cell layer through hotspot I (i.e., the point of entry into the endothelium). Neutrophils frequently remain in the space between the endothelial cell layer and the basement membrane for a prolonged period (>20 min). Subsequently, neutrophils penetrate the basement membrane and pericyte sheath at hotspot II, which is the final stage of exiting the vascular endothelium. To further investigate the roles of LFA-1 and Mac-1, we newly generated LFA-1 FRET (CD11a-YFP/CD18-CFP) mice and Mac-1 FRET (CD11b-YFP/CD18-CFP) mice. Using both FRET mice, we were able to determine that LFA-1 and Mac-1 distinctly regulate the neutrophil extravasation cascade. Our data suggest that the vascular endothelium functions as a double-layered barrier in the steps of neutrophil extravasation. We propose that the harmonized regulation of neutrophil penetration through the endothelium via hotspots I and II may be critical for vascular homeostasis during inflammation.

Interaction between the integrin Mac-1 and signal regulatory protein α (SIRPα) mediates fusion in heterologous cells.

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 αMI-domain 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.