Integrin Mac-1 Research Articles

Monocyte Dysfunction Detected by the Designed Ankyrin Repeat Protein F7 Predicts Mortality in Patients Receiving Veno-Arterial Extracorporeal Membrane Oxygenation.

Background: Veno-arterial extracorporeal membrane oxygenation (VA-ECMO) is used for critically ill patients requiring hemodynamic support but has been shown to induce an inflammatory response syndrome potentially leading to severe complications and poor outcome. Monocytes are comprised of different subsets and play a central role in the innate immune system. The unique small binding proteins, Designed Ankyrin Repeat Protein “F7” and single chain variable fragment “MAN-1,” specifically detect the activated conformation of the leukocyte integrin Mac-1 enabling the highly sensitive detection of monocyte activation status. The aim of this study was to characterize monocyte function and heterogeneity and their association with outcome in VA-ECMO patients.Methods: VA-ECMO patients were recruited from the ICUs of the University Hospital in Freiburg, Germany. Blood was sampled on day 0 and day 3 after VA-ECMO placement, after VA-ECMO explantation and from healthy controls. Monocyte subset distribution, baseline activation and stimulability were analyzed by flow cytometry using the unique small binding proteins F7 and MAN-1 and the conventional activation markers CD163, CD86, CD69, and CX3CR1. Furthermore, expression of monocyte activation markers in survivors and non-survivors on day 0 was compared. Simple logistic regression was conducted to determine the association of monocyte activation markers with mortality.Results: Twenty two patients on VA-ECMO and 15 healthy controls were recruited. Eleven patients survived until discharge from the ICU. Compared to controls, baseline monocyte activation was significantly increased, whereas stimulability was decreased. The percentage of classical monocytes increased after explantation, while the percentage of intermediate monocytes decreased. Total, classical, and intermediate monocyte counts were significantly elevated compared to controls. On day 0, baseline binding of F7 was significantly lower in non-survivors than survivors. The area under the ROC curve associated with mortality on day 0 was 0.802 (p = 0.02).Conclusions: Distribution of monocyte subsets changes during VA-ECMO and absolute classical and intermediate monocyte counts are significantly elevated compared to controls. Monocytes from VA-ECMO patients showed signs of dysfunction. Monocyte dysfunction, as determined by the unique tool F7, could be valuable for predicting mortality in patients receiving VA-ECMO and may be used as a novel biomarker guiding early clinical decision making in the future.

Heme induces significant neutrophil adhesion in vitro via an NFκB and reactive oxygen species-dependent pathway.

Intravascular hemolysis, a major manifestation of sickle cell disease (SCD) and other diseases, incurs the release of hemoglobin and heme from red blood cells, in turn triggering inflammatory processes. This study investigated the in vitro effects of heme, a major inflammatory DAMP, on the adhesive properties of isolated human neutrophils. Heme (20 and 50µM) significantly increased the adhesion of neutrophils to fibronectin and to recombinant ICAM-1, under static conditions, even more efficiently than the potent pro-inflammatory cytokine, tumor necrosis factor-α (TNF); a microfluidic assay confirmed that heme stimulated neutrophil adhesion under conditions of shear stress. Heme-induced neutrophil adhesion was associated with the increased activities, but not expressions, of the Mac-1 and LFA-1 integrin subunits, CD11b and CD11a, on the cell surface. Notably, heme (50µM) significantly induced NFκB translocation in neutrophils, and inhibition of NFκB activity with the BAY11-7082 molecule abolished heme-induced cell adhesion to fibronectin and significantly decreased CD11a activity. Flow cytometric analysis demonstrated major reactive oxygen species (ROS) generation in neutrophils following heme stimulation that could be inhibited by the antioxidant, α-tocopherol, and by BAY11-7082. Furthermore, co-incubation with α-tocopherol abrogated both heme-stimulated neutrophil adhesion and CD11a/CD11b activation. Thus, our data indicate that heme, at clinically relevant concentrations, is a potent activator of neutrophil adhesion, increasing the ligand affinity of the β2 integrins via a mechanism that may be partially mediated by an NFkB-dependent pathway and the generation of ROS. Given the fundamental role that the adhesion of neutrophils to the vascular wall plays in SCD vaso-occlusion and other vascular inflammatory processes, our findings provide further evidence that cell-free heme is a major therapeutic target in the hemolytic diseases.

The Interaction between Integrin Mac-1 (αMβ2, CD11b/CD18) and Sirpα Mediates Macrophage Fusion

Macrophage fusion leading to the formation of multinucleated giant cells is a hallmark of chronic inflammation. While a number of membrane proteins have been implicated in mediating cell-cell attachement during fusion, their binding partners are not yet clear. Recently, we have demonstrated that IL-4-induced fusion of mouse peritoneal macrophages depends on integrin Mac-1 (αMβ2, CD11b/CD18). Surprisingly, the genetic deficiency of ICAM-1, an established ligand of Mac-1, resulted in no decrease of macrophage fusion, suggesting the involvement of other counter-receptors for Mac-1. In the present study, we demonstrated that SIRPα, which like ICAM-1 belongs to the Ig superfamily and was previously implicated in fusion, interacts with Mac-1. Expression of Mac-1 on the surface of HEK293 cells resulted in their adhesion to recombinant ectodomain of SIRPα and this process was mediated through the αMI-domain of Mac-1. In addition, adhesion of RAW264.7 mouse macrophages to SIRPα was dependent on Mac-1. Direct interaction between the αMI-domain and SIRPα was shown by biolayer interferometry. Analyses of the interaction of αMI-domain with a peptide library spanning the ectodomain of SIRPα showed that recognition of SIRPa conforms to general principles that govern binding by Mac-1 of many of its ligands. SIRPα reportedly interacts with CD47; however, adhesion of Mac-1-expressing HEK293 cells, which express CD47, to SIRPα was not dependent on CD47 and an anti-CD47 function-blocking mAb produced only a limited inhibition of adhesion of RAW264.7 cells. Co-immunoprecipitation experiments revealed that Mac-1 forms a complex with CD47 on the surface of RAW264.7 and Mac-1-expressing HEK293 cells. Finally, co-culturing of SIPRα- and Mac-1-expressing HEK293 cells resulted in cell fusion. Collectively, these data identify SIRPα as a novel counter-receptor for Mac-1 and suggest that the Mac-1-SIRPα interaction may be involved in macrophage fusion. DisclosuresNo relevant conflicts of interest to declare.

The Cis Interaction of CD47 with Integrin Mac‐1 (α M β 2 , CD11b/CD18) Regulates Macrophage Responses

CD47 is a widely expressed cell surface receptor that has originally been identified as an integrin-associated protein. In immune cells, CD47 has been shown to associate in cis with several integrins of the β1 and β3 subfamilies and the one member of the β2 subfamily, αLβ2. Surprisingly, the association of CD47 with integrin Mac-1 (αMβ2, CD11b/CD18), the major adhesion receptor on the surface of myeloid cells has not been documented. Furthermore, while previous studies focused on the mechanism by which CD47 on various host cells prevents phagocytosis by macrophages, the question as to how CD47 on macrophages may influence the responses of these cells has not been addressed. Here, we demonstrated that CD47 modulates Mac-1-dependent macrophage functions. In particular, adhesion of macrophages isolated from CD47-/- mice to fibrinogen and ICAM-1, the established physiological ligands of Mac-1 was significantly decreased compared to wild-type counterparts. Also, spreading of CD47-deficient macrophages was decreased by four- and two-fold on fibrinogen and ICAM-1, respectively. Compared to wild-type macrophages, migration of CD47-deficient macrophages to the Mac-1 ligand, the cathelicidin peptide LL-37 was significantly reduced. The lack of CD47 on the surface of macrophages impaired their ability to fuse in the presence of IL-4. Finally, the deficiency of CD47 also reduced phagocytosis of opsonized latex beads, a process fully dependent on Mac-1. The functional link between CD47 and Mac-1 was confirmed by their physical association in cis on the surface of macrophages and Mac-1-expressing HEK293 cells using co-immunoprecipitation and the proximity ligation assay. Studies with HEK293 ceIls expressing individual αM or β2 integrin subunits of Mac-1 showed that both subunits can interact with CD47. The activation of cells with PMA, Mn2+, and mAb MEM48 increased the amount of CD47 recovered in immune complexes with Mac-1, suggesting that CD47 has increased affinity for integrin in the extended conformation. The binding site in CD47 for Mac-1 was identified in its constituent IgV domain as recombinant IgV blocked the Mac-1-CD47 complex formation and inhibited adhesion and phagocytosis. Collectively, these results indicate that Mac-1 forms a lateral complex with CD47, which regulates important macrophage functions.

Structural characterization of the interaction between α M I‐domain of the integrin Mac‐1 (α M β 2 ) and the cytokine pleiotrophin

Integrin receptors are involved in numerous physiological processes, including inflammation, cell migration, extracellular matrix recognition, survival, and growth. Integrin Mac-1 (αMβ2) is a monocyte adhesion receptor that can undergo rapid activation yielding conformational changes that subsequently allow increased affinity for its ligands and enable cellular responses such as rolling, adhesion, and transmigration of phagocytes to the inflammatory sites. αMβ2 is also the most promiscuous integrin receptor because it recognizes a large number of distinct ligands that share no homologous recognition motif. In particular, αMI-domain is known to bind cationic proteins/peptides depleted in acidic residues. This contradicts the canonical ligand-binding mechanism of αI-domains, which requires an acidic amino acid in the ligand to coordinate the divalent cation within the metal ion-dependent adhesion site (MIDAS) of αI-domains. The lack of acidic amino acids in the αMI-domain-binding sequences suggests the existence of an as-yet uncharacterized interaction mechanism. In the present study, we analyzed interactions of αMI-domain with a representative Mac-1 ligand, the cationic cytokine pleiotrophin (PTN). Through nuclear magnetic resonance (NMR) chemical shift perturbations, cross saturation, and nuclear Overhauser effect spectroscopy (NOESY), we found the interaction between αMI-domain and PTN is divalent cation-independent and mediated mostly by hydrophobic contacts between the N-terminal domain of PTN and residues in the α5-β5 loop of αMI-domain. The mutation I265 in the α5-β5 region confirms this area as a binding site of αMI-domain for PTN. The observation that both increased ionic strength and mutations of acidic residues in the α5-β5 loop weaken the interaction between the proteins indicates electrostatic interaction may also play a significant role in the binding. Based on results from these experiments, we formulated a model of the interaction between αMI-domain and PTN. This unique mechanism of interaction between PTN and αMI-domain should explain ligand recognition specificity of αMβ2.

Protective Role of Activated Protein C against Viral Mimetic Poly(I:C)-Induced Inflammation.

Activated protein C (APC) is an anticoagulant plasma serine protease which exhibits potent cytoprotective and anti-inflammatory activities. Here, we studied protective effects of APC on the proinflammatory function of polyinosinic:polycytidylic acid [poly(I:C)], a synthetic analog of viral double-stranded RNA, in cellular and animal models. Poly(I:C) induced histone H3 extranuclear translocation via interaction with toll-like receptor 3 in two established endothelial cell lines. Furthermore, poly(I:C) induced histone H3 extranuclear translocation in J774A.1 macrophages and human neutrophils and formation of macrophage and neutrophil extracellular traps (ETs). Mechanistically, poly(I:C) was found to upregulate expression of peptidylarginine deiminase 4 and enhance its interaction with histone H3, thereby leading to increased histone citrullination and neutrophil ET formation. Poly(I:C) elicited proinflammatory signaling responses by inducing nuclear factor kappa B activation and disrupting endothelial cell permeability. In vivo, poly(I:C) enhanced cell surface expression of Mac-1 on neutrophils in mice and facilitated their infiltration to lung tissues. Poly(I:C) also downregulated thrombomodulin expression in mouse tissues and reduced its circulating soluble level in plasma. We demonstrate in this study that APC and a signaling-selective mutant of APC effectively inhibit proinflammatory signaling effects of poly(I:C) in both cellular and animal models. We further demonstrate that unlike the requirement for endothelial protein C receptor on endothelial cells, the integrin Mac-1 is involved in the protease-activated receptor 1-dependent APC inhibition of macrophage ET formation in J774A.1 cells. Taken together, these results support a key role for APC signaling in inhibiting the viral mimetic-induced proinflammatory signaling responses and histone translocation-associated formation of ETs by innate immune cells.

Structural Characterization of the Interaction between the αMI-Domain of the Integrin Mac-1 (αMβ2) and the Cytokine Pleiotrophin.

Integrin Mac-1 (αMβ2) is an adhesion receptor vital to many functions of myeloid leukocytes. It is also the most promiscuous member of the integrin family capable of recognizing a broad range of ligands. In particular, its ligand-binding αMI-domain is known to bind cationic proteins/peptides depleted in acidic residues. This contradicts the canonical ligand-binding mechanism of αI-domains, which requires an acidic amino acid in the ligand to coordinate the divalent cation within the metal ion-dependent adhesion site (MIDAS) of αI-domains. The lack of acidic amino acids in the αMI-domain-binding sequences suggests the existence of an as-yet uncharacterized interaction mechanism. In the present study, we analyzed interactions of the αMI-domain with a representative Mac-1 ligand, the cationic cytokine pleiotrophin (PTN). Through NMR chemical shift perturbation analysis, cross saturation, NOESY, and mutagenesis studies, we found the interaction between the αMI-domain and PTN is divalent cation-independent and mediated mostly by hydrophobic contacts between the N-terminal domain of PTN and residues in the α5-β5 loop of αMI-domain. The observation that increased ionic strength weakens the interaction between the proteins indicates electrostatic forces may also play a significant role in the binding. On the basis of the results from these experiments, we formulated a model of the interaction between the αMI-domain and PTN.

Structural assessment of SARS-CoV2 accessory protein ORF7a predicts LFA-1 and Mac-1 binding potential.

ORF7a is an accessory protein common to SARS-CoV1 and the recently discovered SARS-CoV2, which is causing the COVID-19 pandemic. The ORF7a protein has a structural homology with ICAM-1 which binds to the T lymphocyte integrin receptor LFA-1. As COVID-19 has a strong immune component as part of the disease, we sought to determine whether SARS-CoV2 would have a similar structural interaction with LFA-1. Using molecular docking simulations, we found that SARS-CoV2 ORF7a has the key structural determinants required to bind LFA-1 but also the related leukocyte integrin Mac-1, which is also known to be expressed by macrophages. Our study shows that SARS-CoV2 ORF7a protein has a conserved Ig immunoglobulin-like fold containing an integrin binding site that provides a mechanistic hypothesis for SARS-CoV2’s interaction with the human immune system. This suggests that experimental investigation of ORF7a-mediated effects on immune cells such as T lymphocytes and macrophages (leukocytes) could help understand the disease further and develop effective treatments.

The Functional Heterogeneity of Neutrophil-Derived Extracellular Vesicles Reflects the Status of the Parent Cell.

Similar to other cell types, neutrophilic granulocytes also release extracellular vesicles (EVs), mainly medium-sized microvesicles/microparticles. According to published data, authors have reached a consensus on the physical parameters (size, density) and chemical composition (surface proteins, proteomics) of neutrophil-derived EVs. In contrast, there is large diversity and even controversy in the reported functional properties. Part of the discrepancy may be ascribed to differences in the viability of the starting cells, in eliciting factors, in separation techniques and in storage conditions. However, the most recent data from our laboratory prove that the same population of neutrophils is able to generate EVs with different functional properties, transmitting pro-inflammatory or anti-inflammatory effects on neighboring cells. Previously we have shown that Mac-1 integrin is a key factor that switches anti-inflammatory EV generation into pro-inflammatory and antibacterial EV production. This paper reviews current knowledge on the functional alterations initiated by neutrophil-derived EVs, listing their effects according to the triggering agents and target cells. We summarize the presence of neutrophil-derived EVs in pathological processes and their perspectives in diagnostics and therapy. Finally, the functional heterogeneity of differently triggered EVs indicates that neutrophils are capable of producing a broad spectrum of EVs, depending on the environmental conditions prevailing at the time of EV genesis.

CD11b Signaling Prevents Chondrocyte Mineralization and Attenuates the Severity of Osteoarthritis.

Osteoarthritis (OA) is a progressive joint disease that is strongly associated with calcium-containing crystal formation (mineralization) by chondrocytes leading ultimately to cartilage calcification. However, this calcification process is poorly understood and treatments targeting the underlying disease mechanisms are lacking. The CD11b/CD18 integrin (Mac-1 or αMβ2), a member of the beta 2 integrin family of adhesion receptors, is critically involved in the development of several inflammatory diseases, including rheumatoid arthritis and systemic lupus erythematosus. We found that in a collagen-induced arthritis, CD11b-deficient mice exhibited increased cartilage degradation compared to WT control animals. However, the functional significance of CD11b integrin signaling in the pathophysiology of chondrocytes remains unknown. CD11b expression was found in the extracellular matrix and in chondrocytes in both healthy and damaged human and murine articular cartilage. Primary murine CD11b KO chondrocytes showed increased mineralization when induced in vitro by secondary calciprotein particles (CPP) and quantified by Alizarin Red staining. This increased propensity to mineralize was associated with an increased alkaline phosphatase (Alp) expression (measured by qRT-PCR and activity assay) and an enhanced secretion of the pro-mineralizing IL-6 cytokine compared to control wild-type cells (measured by ELISA). Accordingly, addition of an anti-IL-6 receptor antibody to CD11b KO chondrocytes reduced significantly the calcification and identified IL-6 as a pro-mineralizing factor in these cells. In the same conditions, the ratio of qRT-PCR expression of collagen X over collagen II, and that of Runx2 over Sox9 (both ratio being indexes of chondrocyte hypertrophy) were increased in CD11b-deficient cells. Conversely, the CD11b activator LA1 reduced chondrocyte mineralization, Alp expression, IL-6 production and collagen X expression. In the meniscectomy (MNX) model of murine knee osteoarthritis, deficiency of CD11b led to more severe OA (OARSI scoring of medial cartilage damage in CD11b: 5.6 ± 1.8, in WT: 1.2 ± 0.5, p < 0.05, inflammation in CD11b: 2.8 ± 0.2, in WT: 1.4 ± 0.5). In conclusion, these data demonstrate that CD11b signaling prevents chondrocyte hypertrophy and chondrocyte mineralization in vitro and has a protective role in models of OA in vivo.

Production of fever mediator PGE2 in human monocytes activated with MDP adjuvant is controlled by signaling from MAPK and p300 HAT: Key role of T cell derived factor

Fever and inflammatory responses were observed in some subjects in early clinical trials of vaccines adjuvanted with muramyl dipeptide (MDP), a NOD2 agonist. Biosynthesis of Prostaglandin E2 (PGE2) that transmits febrile signals to the brain is controlled by an inducible enzyme, Cyclooxygenase 2 (COX-2). MDP alone was not sufficient to induce expression of COX-2 and PGE2 production in vitro. Conditioned medium prepared from Peripheral Blood Mononuclear Cells (PBMCs)-derived CD3-bead purified human T cells (TCM) dramatically increased COX2 gene transcription, COX-2 protein expression, and PGE2 production in MDP-treated monocytes. We explored epigenetic changes at the COX2 promoter using Chromatin Immunoprecipitation assay (ChIP). Increase in COX2 transcription correlated with increased recruitment of RNA polymerase II (Pol II) and p300 histone acetyl transferase (HAT) to the COX2 promoter in monocytes activated with MDP and TCM. The role of p300 HAT was confirmed by using C646, an inhibitor of p300, that reduced binding of acetylated H3 and H4 histones at the COX2 promoter, COX2 transcription, and PGE2 production in monocytes. Binding of p300, Nuclear Factor Kappa B (NF-κB), and Pol II to the COX2 promoter was also sensitive to inhibitors of Mitogen-Activated Protein Kinase (MAPK) pathway and to antibodies against Macrophage-1 (Mac-1) integrin in MDP/TCM-treated monocytes. Importantly, recombinant Glycoprotein Ib alfa (GPIbα), the recently identified factor in TCM, increased binding of NF-κB, p300, and of Pol II to the COX2 promoter and COX2 transcription in MDP-treated monocytes. Our findings suggest that a second signal through Mac-1 and MAPK is triggered by a T cell derived soluble GPIbα protein leading to the assembly of the transcription machinery at the COX2 promoter and production of PGE2 in human monocytes in response to MDP/NOD2 activation.

Clinical impact of urinary CD11b and CD163 on the renal outcomes of anti-neutrophil cytoplasmic antibody-associated glomerulonephritis.

The detection of leukocyte-derived CD11b (α subunit of integrin Mac-1) and CD163 (scavenger receptor) in urine may reflect renal inflammation in antineutrophil cytoplasmic antibody-associated glomerulonephritis (ANCA-GN). The objective of this study was to evaluate the clinical significance of urinary CD11b (U-CD11b) and CD163 (U-CD163) in ANCA-GN. U-CD11b and U-CD163 were examined using enzyme-linked immunosorbent assay in ANCA-GN urine samples from our institutional cohort (n = 88) and a nationwide cohort (n = 138), and their association with renal histology was subsequently analyzed. Logistic regression analyses were performed on a nationwide ANCA cohort to determine the associations of the two urinary molecules with renal remission failure at 6 months or with yearly estimated glomerular filtration rate (eGFR) slope over a 24-month observation period. U-CD11b and U-CD163 were significantly associated with cellular crescent formation and leukocyte accumulation in glomerular crescents. With regard to interstitial inflammation, both levels of U-CD11b and U-CD163 at diagnosis remarkably increased in ANCA-GN compared with the levels observed in nonglomerular kidney disorders including nephrosclerosis, immunoglobulin G4-relateddisease and tubulointerstitial nephritis; however, the presence of U-CD11b alone was significantly correlated with tubulointerstitial leukocyte infiltrates. Although neither U-CD11b nor U-CD163 at diagnosis was associated with remission failure at 6 months, multivariate analysis demonstrated that the baseline U-CD11b levels were significantly associated with the increase in eGFR following immunosuppressive therapy. Although both U-CD11b and U-CD163 reflect renal leukocyte accumulation, U-CD11b at diagnosis provides additional clinical value by predicting the recovery rate after the treatment of ANCA-GN.

SUN-380 CLINICAL IMPACTS OF URINARY CD11B AND CD163 ON PATIENT OUTCOMES IN ANCA-ASSOCIATED GLOMERULONEPHRITIS

The leukocyte-derived molecules, CD11b (α subunit of leukocyte integrin Mac-1) and scavenger receptor CD163, undergo ectodomain cleavage upon inflammation-related cell activation; therefore, the presence of these molecules in urine may reflect the renal inflammation with glomerular crescents containing robust extra-capillary leukocytes in anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis (ANCA-GN). This study was undertaken to evaluate the clinical significance of urinary CD11b (U-CD11b) and CD163 (U-CD163) in ANCA-GN. We examined ANCA-GN urine samples by enzyme-linked immunosorbent assay in our institutional cohort (88 patients at diagnosis) and in a nation-wide ANCA vasculitis (AAV) cohort (138 patients at diagnosis; 56 patients after 6 months of treatment) and analyzed the associations of the results with renal histology related to ANCA-GN, and with remission status and patient outcome evaluated by the renal Vasculitis Damage Index (VDI) score at 6 months after remission induction therapy. Both U-CD11b and U-CD163 at diagnosis were significantly elevated in patients with ANCA-GN compared to those in with nephrosclerosis (n=20) and IgG4-related disease (n=12). Those in tubulointerstitial nephritis (n=15) demonstrated a mild increase of U-CD11b, but not of U-CD163, at levels statistically comparable to that of AAV. Histological analysis in tubulointerstitial area demonstrated moderate correlation of U-CD11b with interstitial CD11b+ infiltrates. When focusing on glomerular injury, U-CD11b and U-CD163 levels were increased particularly in patients with ANCA-GN in the crescentic category according to the European Vasculitis Study Group classification. Detailed histological analyses evidenced the significant associations of U-CD11b and U-CD163 levels with cellular crescent formation and accumulation of leukocytes expressing the surface markers in glomerular crescents. Although the levels of U-CD163, but not of U-CD11b, in identical patients at diagnosis were significantly reduced after the 6-month follow-up period, patients with unachieved remission at 6 months showed significant elevation of U-CD163 at diagnosis, but not at 6 months. In contrast, baseline U-CD11b levels did not affect the remission status after 6 months of treatment, but the levels at 6 months tended to be decreased in patients with unachieved remission. Analyses of U-CD11b and U-CD163 levels according to the renal VDI score revealed significant elevation of U-CD11b at 6 months and U-CD163 at diagnosis in ANCA-GN patients with a renal VDI score ≥ 2 compared to those with score of 0 at 6 months. Receiver operating characteristic curves for U-CD11b and U-CD163 at diagnosis demonstrated the superiority of U-CD163 to U-CD11b as a biomarker to predict the remission status (AUC; U-CD11b 0.517, U-CD163 0.723) and renal VDI score ≥ 2 (AUC; U-CD11b 0.647, U-CD163 0.785) at 6 months after remission induction therapy. U-CD11b and U-CD163 have different clinical values: U-CD11b reflects the ANCA-GN inflammatory status in the glomerulus and tubulointerstitium, and is associated with persistent renal damages, whereas U-CD163 specifically reflects glomerular inflammation and is associated with susceptibility to immunosuppressive treatment.

Mechanisms of allergy and adult asthma.

Allergic asthma reflects the interplay between inflammatory mediators and immune, airway epithelial, and other cells. This review summarizes key insights in these areas over the past year. Key findings over the past year demonstrate that epithelial cells mediate tight junction breakdown to facilitate the development of asthma-like disease in mice. Innate lymph lymphoid cells (ILC), while previously shown to promote allergic airway disease, have now been shown to inhibit the development of severe allergic disease in mice. Fibrinogen cleavage products (previously shown to mediate allergic airway disease and macrophage fungistatic immunity by signaling through Toll-like receptor 4) have now been shown to first bind to the integrin Mac-1 (CD11c/CD18). Therapeutically, recent discoveries include the development of the antiasthma drug PM-43I that inhibits the allergy-related transcription factors STAT5 and STAT6 in mice, and confirmatory evidence of the efficacy of the antifungal agent voriconazole in human asthma. Studies over the past year provide critical new insight into the mechanisms by which epithelial cells, ILC, and coagulation factors contribute to the expression of asthma-like disease and further support the development antiasthma drugs that block STAT factors and inhibit fungal growth in the airways.

Increased Adhesive Potential of Antiphospholipid Syndrome Neutrophils Mediated by β2 Integrin Mac-1.

While the role of antiphospholipid antibodies in activating endothelial cells has been extensively studied, the impact of these antibodies on the adhesive potential of leukocytes has received less attention. This study was undertaken to investigate the extent to which antiphospholipid syndrome (APS) neutrophils adhere to resting endothelial cells under physiologic flow conditions and the surface molecules required for that adhesion. Patients with primary APS (n = 43), patients with a history of venous thrombosis but negative test results for antiphospholipid antibodies (n = 11), and healthy controls (n = 38) were studied. Cells were introduced into a flow chamber and perfused across resting human umbilical vein endothelial cells (HUVECs). Surface adhesion molecules were quantified by flow cytometry. Neutrophil extracellular trap release (NETosis) was assessed in neutrophil-HUVEC cocultures. Upon perfusion of anticoagulated blood through the flow chamber, APS neutrophils demonstrated increased adhesion as compared to control neutrophils under conditions representative of either venous (n = 8; P < 0.05) or arterial (n = 15; P < 0.0001) flow. At the same time, APS neutrophils were characterized by up-regulation of CD64, CEACAM1, β2 -glycoprotein I, and activated Mac-1 on their surface (n = 12-18; P < 0.05 for all markers). Exposing control neutrophils to APS plasma or APS IgG resulted in increased neutrophil adhesion (n = 10-11; P < 0.0001) and surface marker up-regulation as compared to controls. A monoclonal antibody specific for activated Mac-1 reduced the adhesion of APS neutrophils in the flow-chamber assay (P < 0.01). The same monoclonal antibody reduced NETosis in neutrophil-HUVEC cocultures (P < 0.01). APS neutrophils demonstrate increased adhesive potential, which is dependent upon the activated form of Mac-1. In patients, this could lower the threshold for neutrophil-endothelium interactions, NETosis, and possibly thrombotic events.

Different Calcium and Src Family Kinase Signaling in Mac-1 Dependent Phagocytosis and Extracellular Vesicle Generation.

Encountering opsonized particles by neutrophils results in phagocytosis of the particle and generation of extracellular vesicles with antibacterial property (aEV). The aim of the present study is to compare the involvement of different receptors and receptor-proximal signaling pathways in these two parallel processes. Investigating human neutrophils from peripheral blood, we show that complement receptors are decisive for both processes whereas immunoglobulin binding Fc receptors (FcR) only participate moderately in phagocytosis and pattern recognition receptors induce mild EV production but only minimal phagocytosis. Studying bone marrow derived neutrophils of genetically modified animals we verify that the involved complement receptor is CR3, also known as the β2 integrin Mac-1. We show that genetic deletion of the adaptor molecules FcRγ chain or DAP12 does not influence either process, suggesting potential redundant function. Combined absence of the Src family kinases Hck, Fgr, and Lyn drastically impairs phagocytosis but does not influence aEV production. In contrast, deletion of PLCγ2 has no influence on phagocytosis, but reduces aEV formation. In accord with the essential role of PLCγ2, aEV biogenesis both from murine and from human neutrophils is dependent on presence of extracellular calcium. Absence of external calcium prevented the generation of antibacterial EVs, whereas the spontaneous EV formation was not influenced. We thus show that phagocytosis and biogenesis of antibacterial EVs are independent processes and proceed on different signaling pathways although the same receptor plays the critical role in both. Our data reveal the possibility in neutrophilic granulocytes to modulate aEV production without disturbing the phagocytic process.

Role of Mac-1 integrin in generation of extracellular vesicles with antibacterial capacity from neutrophilic granulocytes

ABSTRACT Production of extracellular vesicles (EVs) involved in intercellular communication is a common capacity of most cell types. Upon encountering opsonized microorganisms, neutrophilic granulocytes release EVs that compromise bacterial growth. We carried out a systematic investigation of the involvement of potential opsonin receptors in EV-generation from human and murine neutrophils. Applying flow cytometric, proteomic and functional analysis as well as using genetically modified mice, we demonstrate that formation of antibacterial EVs depends upon stimulation of the multifunctional Mac-1 integrin complex, also called as complement receptor 3 (CR3), whereas activation of immunoglobulin binding Fc receptors or pattern recognition receptors alone or in combination is ineffective. Mac-1/CR3 stimulation and downstream tyrosine kinase signalling affect both the numbers, the cargo content and the antibacterial capacity of the produced vesicles. In contrast, Mac-1/CR3 signalling is not required for spontaneous EV formation, clearly indicating the existence of separate molecular pathways in EV biogenesis. We propose that EVs are “tailor-made” with different composition and functional properties depending on the environmental circumstances.

Combination Therapy Targeting Platelet Activation and Virus Replication Protects Mice against Lethal Influenza Pneumonia.

Excessive neutrophils recruited during influenza pneumonia contribute to severe lung pathology through induction of neutrophil extracellular traps (NETs) and release of extracellular histones. We have recently shown that activation of platelets during influenza enhances pulmonary microvascular thrombosis, leading to vascular injury and hemorrhage. Emerging evidence indicates that activated platelets also interact with neutrophils, forming neutrophil-platelet aggregates (NPAs) that contribute to tissue injury. Here, we examined neutrophil-platelet interactions and evaluated the formation of NPAs during influenza pneumonia. We also evaluated the efficacy of clopidogrel (CLP), an antagonist of the ADP-P2Y12 platelet receptor, alone or in combination with an antiviral agent (oseltamivir) against influenza infection in mice. Our studies demonstrated increased platelet activation and induction of NPAs in influenza-infected lungs, and that these NPAs led to NET release both in vitro and in vivo. Furthermore, neutrophil integrin Mac-1 (macrophage-1 antigen)-mediated platelet binding was critical for NPA formation and NET release. Administration of CLP reduced platelet activation and NPA formation but did not protect the mice against lethal influenza challenge. However, administration of CLP together with oseltamivir improved survival rates in mice compared with oseltamivir alone. The combination treatment reduced lung pathology, neutrophil influx, NPAs, NET release, and inflammatory cytokine release in infected lungs. Taken together, these results provide the first evidence that NPAs formed during influenza contribute to acute lung injury. Targeting both platelet activation and virus replication could represent an effective therapeutic option for severe influenza pneumonia.

A critical role for microglia in maintaining vascular integrity in the hypoxic spinal cord

Hypoxic preconditioning reduces disease severity in a mouse model of multiple sclerosis (MS), in part by enhancing the barrier properties of spinal cord blood vessels. Because other studies have shown that similar levels of hypoxia transiently increase permeability of central nervous system (CNS) blood vessels, the goal of this study was to define the impact of chronic mild hypoxia (CMH, 8% O2) on the integrity of spinal cord blood vessels and the responses of neighboring glial cells. Using extravascular fibrinogen as a marker of vascular disruption, we found that CMH triggered transient vascular leak in spinal cord blood vessels, particularly in white matter, which was associated with clustering and activation of Mac-1-positive microglia around disrupted vessels. Microglial depletion with the colony stimulating factor-1 receptor (CSF-1R) inhibitor PLX5622, while having no effect under normoxic conditions, profoundly increased vascular leak in both white and gray matter during CMH, and this was associated with disruption of astrocyte-vascular coupling and enhanced loss of tight junction proteins. Microglial repair of leaky blood vessels was blocked by a peptide that inhibits the interaction between fibrinogen and its Mac-1 integrin receptor. These findings highlight an important role for microglia in maintaining vascular integrity in the hypoxic spinal cord and suggest that a fibrinogen-Mac-1 interaction underpins this response. As relative hypoxia is experienced in many situations including high altitude, lung disease, obstructive sleep apnea, and age-related CNS ischemia/hypoxia, our findings have important implications regarding the critical role of microglia in maintaining vascular integrity in the CNS.

Mice expressing the variant rs1143679 allele of ITGAM (CD11b) show impaired DC-mediated T cell proliferation

Genome-wide association studies (GWAS) and functional genomic analyses have implicated several ITGAM (CD11b) single-nucleotide polymorphisms (SNPs) in the development of SLE and other disorders. ITGAM encodes the αM chain of the β2 integrin Mac-1, a receptor that plays important roles in myeloid cell functions. The ITGAM SNP rs1143679, which results in an arginine to histidine change at amino acid position 77 of the CD11b protein, has been shown to reduce binding to several ligands and to alter Mac-1-mediated cellular response in vitro. Importantly, however, the potential contribution of this SNP variant to the initiation and/or progression of immune and inflammatory processes in vivo remains unexplored. Herein, we describe for the first time the generation and characterization of a mouse line expressing the 77His variant of CD11b. Surprisingly, we found that 77His did not significantly affect Mac-1-mediated leukocyte migration and activation as assessed using thioglycollate-induced peritonitis and LPS/TNF-α-induced dermal inflammation models. In contrast, expression of this variant did alter T cell immunity, as evidenced by significantly reduced proliferation of ovalbumin (OVA)-specific transgenic T cells in 77His mice immunized with OVA. Reduced antigen-specific T cell proliferation was also observed when either 77His splenic dendritic cells (DCs) or bone marrow-derived DCs were used as antigen-presenting cells (APCs). Although more work is necessary to determine how this alteration might influence the development of SLE or other diseases, these in vivo findings suggest that the 77His variant of CD11b can compromise the ability of DCs to induce antigen-driven T cell proliferation.