?-1 Antitrypsin regulates human neutrophil chemotaxis induced by soluble immune complexes and IL-8.
ABSTRACT: Hereditary deficiency of the protein ?-1 antitrypsin (AAT) causes a chronic lung disease in humans that is characterized by excessive mobilization of neutrophils into the lung. However, the reason for the increased neutrophil burden has not been fully elucidated. In this study we have demonstrated using human neutrophils that serum AAT coordinates both CXCR1- and soluble immune complex (sIC) receptor-mediated chemotaxis by divergent pathways. We demonstrated that glycosylated AAT can bind to IL-8 (a ligand for CXCR1) and that AAT-IL-8 complex formation prevented IL-8 interaction with CXCR1. Second, AAT modulated neutrophil chemotaxis in response to sIC by controlling membrane expression of the glycosylphosphatidylinositol-anchored (GPI-anchored) Fc receptor Fc?RIIIb. This process was mediated through inhibition of ADAM-17 enzymatic activity. Neutrophils isolated from clinically stable AAT-deficient patients were characterized by low membrane expression of Fc?RIIIb and increased chemotaxis in response to IL-8 and sIC. Treatment of AAT-deficient individuals with AAT augmentation therapy resulted in increased AAT binding to IL-8, increased AAT binding to the neutrophil membrane, decreased Fc?RIIIb release from the neutrophil membrane, and normalization of chemotaxis. These results provide new insight into the mechanism underlying the effect of AAT augmentation therapy in the pulmonary disease associated with AAT deficiency.
Project description:The function of the low-affinity IgG-receptor Fc?RIIIb (CD16b), which is uniquely and abundantly expressed on human granulocytes, is not clear. Unlike the other Fc? receptors (Fc?R), it is a glycophosphatidyl inositol (GPI) -anchored molecule and does not have intracellular signaling motifs. Nevertheless, Fc?RIIIb can cooperate with other Fc?R to promote phagocytosis of antibody-opsonized microbes by human neutrophils. Here we have investigated the role of Fc?RIIIb during antibody-dependent cellular cytotoxicity (ADCC) by neutrophils toward solid cancer cells coated with either trastuzumab (anti-HER2) or cetuximab (anti-EGFR). Inhibiting Fc?RIIIb using CD16-F(ab')2 blocking antibodies resulted in substantially enhanced ADCC. ADCC was completely dependent on Fc?RIIa (CD32a) and the enhanced ADCC seen after Fc?RIIIb blockade therefore suggested that Fc?RIIIb was competing with Fc?RIIa for IgG on the opsonized target cells. Interestingly, the function of neutrophil Fc?RIIIb as a decoy receptor was further supported by using neutrophils from individuals with different gene copy numbers of FCGR3B causing different levels of surface Fc?RIIIb expression. Individuals with one copy of FCGR3B showed higher levels of ADCC compared to those with two or more copies. Finally, we show that therapeutic antibodies intended to improve Fc?RIIIa (CD16a)-dependent natural killer (NK) cell ADCC due to the lack of fucosylation on the N-linked glycan at position N297 of the IgG1 heavy chain Fc-region, show decreased ADCC as compared to regularly fucosylated antibodies. Together, these data confirm Fc?RIIIb as a negative regulator of neutrophil ADCC toward tumor cells and a potential target for enhancing tumor cell destruction by neutrophils.
Project description:Neutrophils serve as critical players in the pathogenesis of liver diseases. Chemokine receptors CXCR1 and CXCR2 are required for neutrophil chemotaxis to the site of inflammation/injury and are crucial in hepatic inflammatory response. However, key mechanism of neutrophil-mediated liver injury in acute-on-chronic liver failure (ACLF) remains highly elusive; which could be targeted for the development of new therapeutic interventions.To demonstrate the role of CXCR1/CXCR2-expressing neutrophils in hepatic injury, we investigated CXCR1/CXCR2 receptor expression in 17 hepatitis B virus-related ACLF patients in comparison to 42 chronic hepatitis B and 18 healthy controls. Mechanism of neutrophil-mediated cell death was analyzed by in vitro coculture assays and correlated with the patient data. In addition, to find out any etiological-based variations in ACLF, 19 alcohol-related ACLF patients were also included.In ACLF, neutrophils have high expression of CXCR1/CXCR2 receptors, which potentially participate in hepatocyte death through early apoptosis and necrosis in contact-dependent and -independent mechanisms. Importantly, blockade of CXCR1/CXCR2 with SCH 527123 antagonist significantly reduced cell death by targeting both the mechanisms. No etiology-based differences were seen between ACLF groups. Importantly, absolute neutrophil count was particularly higher in clinically severe ACLF patients and non-survivors (p?<?0.0001). Multivariate analysis demonstrated ANC and CXCL8/IL-8 as a predictor of mortality. Further, receiver operating characteristics curve confirmed the cutoff of ANC >73.5% (sensitivity: 76.5% and specificity: 76.5%) and CXCL8/IL-8 >27% (sensitivity: 70% and specificity: 73%) in prediction of mortality.Blockade of CXCR1/CXCR2 diminished the production of inflammatory mediators and reduced cell death; therefore, pharmacological neutralization of CXCR1/CXCR2 could provide novel therapeutic target in the management of ACLF.
Project description:Alcoholic steatohepatitis is a life-threatening condition with short-term mortality up to 40%. It features hepatic neutrophil infiltration and blood neutrophilia, and may evolve from ethanol-induced breakdown of the enteric barrier and consequent bacteraemia. Signalling through CXCR1/2 G-protein-coupled-receptors (GPCRs), the interleukin (IL)-8 receptors, is critical for the recruitment and activation of neutrophils. We have developed short lipopeptides (pepducins), which inhibit post-ligand GPCR activation precisely targeting individual GPCRs.Experimental alcoholic liver disease was induced by administering alcohol and a Lieber-DeCarli high-fat diet. CXCR1/2 GPCRs were blocked via pepducins either from onset of the experiment or after disease was fully established. Hepatic inflammatory infiltration, hepatocyte lipid accumulation and overall survival were assessed as primary outcome parameters. Neutrophil activation was assessed by myeloperoxidase activity and liver cell damage by aspartate aminotransferase and alanine aminotransferase plasma levels. Chemotaxis assays were performed to identify chemoattractant signals derived from alcohol-exposed hepatocytes.Here, we show that experimental alcoholic liver disease is driven by CXCR1/2-dependent activation of neutrophils. CXCR1/2-specific pepducins not only protected mice from liver inflammation, weight loss and mortality associated with experimental alcoholic liver disease, but therapeutic administration cured disease and prevented further mortality in fully established disease. Hepatic neutrophil infiltration and triglyceride accumulation was abrogated by CXCR1/2 blockade. Moreover, CXCL-1 plasma levels were decreased with the pepducin therapy as was the transcription of hepatic IL-1? mRNA.We propose that high circulating IL-8 in human alcoholic hepatitis may cause pathogenic overzealous neutrophil activation, and therapeutic blockade via pepducins merits clinical study.
Project description:Plasmodium falciparum malaria kills nearly a million people annually. Over 90% of these deaths occur in children under five years of age in sub-Saharan Africa. A neutrophil mediated mechanism, the antibody dependent respiratory burst (ADRB), was recently shown to correlate with protection from clinical malaria. Human neutrophils constitutively express Fc gamma receptor-Fc?RIIA and Fc?RIIIB by which they interact with immunoglobulin (Ig) G (IgG)-subclass antibodies. Polymorphisms in exon 4 of FCGR2A and exon 3 of FCGR3B genes encoding Fc?RIIA and Fc?RIIIB respectively have been described to alter the affinities of both receptors for IgG. Here, associations between specific polymorphisms, encoding Fc?RIIA p.H166R and Fc?RIIIB-NA1/NA2/SH variants with clinical malaria were investigated in a longitudinal malaria cohort study. Fc?RIIA-p.166H/R was genotyped by gene specific polymerase chain reaction followed by allele specific restriction enzyme digestion. FCGR3B-exon 3 was sequenced in 585 children, aged 1 to 12 years living in a malaria endemic region of Ghana. Multivariate logistic regression analysis found no association between Fc?RIIA-166H/R polymorphism and clinical malaria. The A-allele of FCGR3B-c.233C>A (rs5030738) was significantly associated with protection from clinical malaria under two out of three genetic models (additive: p=0.0061; recessive: p=0.097; dominant: p=0.0076) of inheritance. The Fc?RIIIB-SH allotype (CTGAAA) containing the 233A-allele (in bold) was associated with protection from malaria (p=0.049). The Fc?RIIIB-NA2*03 allotype (CTGCGA), a variant of the classical Fc?RIIIB-NA2 (CTGCAA) was associated with susceptibility to clinical malaria (p=0.0092). The present study is the first to report an association between a variant of Fc?RIIIB-NA2 and susceptibility to clinical malaria and provides justification for further functional characterization of variants of the classical Fc?RIIIB allotypes. This would be crucial to the improvement of neutrophil mediated functional assays such as the ADRB assay aimed at assessing the functionality of antibodies induced by candidate malaria vaccines.
Project description:We have identified a rare healthy Fc?RIIIB (CD16B)-null donor completely lacking FCGR3B RNA and protein expression and dissected the role of the different neutrophil Fc? receptors in the response to therapeutic anti-CD20 monoclonal antibodies. We observed that polymorphonuclear neutrophils (PMNs) from Fc?RIIIB wild-type (WT) individuals or the null donor were more effectively activated by chronic lymphocytic leukemia (CLL) B-cell targets opsonized with glycoengineered anti-CD20 antibodies compared with fully core-fucosylated anti-CD20 antibodies, suggesting the presence and role of Fc?RIIIA (CD16A) on PMNs. Indeed, we demonstrated by reverse-transcription polymerase chain reaction, flow cytometry, and western blot analysis that PMNs from Fc?RIIIB WT donors and the null individual express low levels of Fc?RIIIA on their surfaces. Fc?RIIIA is a functional and activating molecule on these cells, because anti-CD16 F(ab')<sub>2</sub> antibodies alone were able to activate highly purified PMNs from the Fc?RIIIB-null donor. Use of blocking anti-CD16 and anti-CD32 antibodies showed that Fc?RIIIA is also a major mediator of phagocytosis of CD20-opsonized beads by Fc?RIIIB WT and null PMNs. In contrast, trogocytosis of antibody-opsonized CLL B cells by PMNs was mediated primarily by Fc?RIIIB in WT PMNs and by Fc?RIIA in null PMNs. We conclude that Fc?RIIIA is an important player in PMN functions, whereas Fc?RIIIB is dispensable for activation and phagocytosis. We discuss the clinical implications of these findings.
Project description:The Fc? receptor IIIb (Fc?RIIIb) is a low-affinity receptor of IgG and is essential in neutrophil-mediated effector functions. Different allelic forms of Fc?RIIIb carrying human neutrophil antigen (HNA-1a, -1b, -1c, and -1d) have been identified. Here, we have generated stable transfected HEK293 cell lines expressing HNA-1aa, -1bb, and -1bc. Of these, cells expressing HNA-1bc interacted significantly stronger (binding affinities, 2.277 versus 0.743) with human IgG than cells expressing the HNA-1aa or -1bb alloforms. The higher affinity of IgG toward the HNA-1c alloform was confirmed using neutrophils derived from German blood donors. Neutrophils from HNA-1abc-phenotyped individuals bound IgG significantly stronger (1.825 versus 0.903) than did neutrophils from HNA-1ab-typed individuals. These findings were confirmed by surface plasmon resonance (SPR) analysis demonstrating that recombinant HNA-1bc had a higher affinity (dissociation constant [Kd ], 7.24 × 10-6 M) than recombinant HNA-1bb (Kd , 1.15 × 10-5 M) against normal IgG. Finally, we demonstrated that Plasmodium falciparum merozoites opsonized with human IgG affinity purified against P. falciparum glutamate-rich protein (GLURP) enhanced stronger reactive oxygen species (ROS) emission in neutrophils obtained from HNA-1abc donors than in neutrophils from HNA-1ab donors. Collectively, these results indicate that the amino acid substitution Ala78Asp resulting in the HNA-1c allotype leads to higher affinity toward human IgG, enhancement of neutrophil activation, and possibly effective clearance of malaria by intracellular ROS.
Project description:Neutrophils (PMN) are the most abundant leukocytes in the blood. PMN migrate from the circulation to sites of infection, where they are responsible for antimicrobial functions. PMN use phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes. NETs are fibers composed of chromatin and neutrophil-granule proteins. Several pathogens, including bacteria, fungi, and parasites, and also some pharmacological stimuli such as phorbol 12-myristate 13-acetate (PMA) are efficient inducers of NETs. Antigen-antibody complexes are also capable of inducing NET formation. However the particular Fc? receptor involved in triggering this function is a matter of controversy. In order to provide some insight into what Fc? receptor is responsible for NET formation, each of the two human Fc? receptors was stimulated individually by specific monoclonal antibodies and NET formation was evaluated. Fc?RIIa cross-linking did not promote NET formation. Cross-linking other receptors such as integrins also did not promote NET formation. In contrast Fc?RIIIb cross-linking induced NET formation similarly to PMA stimulation. NET formation was dependent on NADPH-oxidase, PKC, and ERK activation. These data show that cross-linking Fc?RIIIb is responsible for NET formation by the human neutrophil.
Project description:Soluble immune complexes (ICs) are abundant in autoimmune diseases, yet neutrophil responses to these soluble humoral factors remain uncharacterized. Moreover, the individual role of the uniquely human Fc?RIIA and glycophosphatidylinositol (GPI)-linked Fc?RIIIB in IC-mediated inflammation is still debated. Here we exploited mice and cell lines expressing these human neutrophil Fc?Rs to demonstrate that Fc?RIIIB alone, in the absence of its known signaling partners Fc?RIIA and the integrin Mac-1, internalizes soluble ICs through a mechanism used by GPI-anchored receptors and fluid-phase endocytosis. Fc?RIIA also uses this pathway. As shown by intravital microscopy, Fc?RIIA but not Fc?RIIIB-mediated neutrophil interactions with extravascular soluble ICs results in the formation of neutrophil extracellular traps (NETs) in tissues. Unexpectedly, in wild-type mice, IC-induced NETosis does not rely on the NADPH oxidase, myeloperoxidase, or neutrophil elastase. In the context of soluble ICs present primarily within vessels, Fc?RIIIB-mediated neutrophil recruitment requires Mac-1 and is associated with the removal of intravascular IC deposits. Collectively, our studies assign a new role for Fc?RIIIB in the removal of soluble ICs within the vasculature that may serve to maintain homeostasis, whereas Fc?RIIA engagement of tissue soluble ICs generates NETs, a proinflammatory process linked to autoimmunity.
Project description:Administration of therapeutic monoclonal antibodies (mAbs) is frequently accompanied by severe first infusion reactions (FIR). The mechanism driving FIR is still unclear. This study aimed to investigate the cellular and molecular mechanisms causing FIR in humanized mouse models and their potential for evaluating FIR risk in patients.Mice humanized for Fc gamma receptors (Fc?Rs) were generated by recombination-mediated genomic replacement. Body temperature, cytokine release and reactive oxygen species (ROS) were measured to assess FIR to mAbs.Infusion of human mAb specific for mouse transferrin receptor (HamTfR) into Fc?R-humanized mice, produced marked transient hypothermia accompanied by an increase in inflammatory cytokines KC and MIP-2, and ROS. FIR were dependent on administration route and Fc-triggered effector functions mediated by neutrophils. Human neutrophils also induced FIR in wild type mice infused with HamTfR. Specific knock-in mice demonstrated that human Fc?RIIIb on neutrophils was both necessary and sufficient to cause FIR. Fc?RIIIb-mediated FIR was abolished by depleting neutrophils or blocking Fc?RIIIb with CD11b antibodies.Human Fc?RIIIb and neutrophils are primarily responsible for triggering FIR. Clinical strategies to prevent FIR in patients should focus on this pathway and may include transient depletion of neutrophils or blocking Fc?RIIIb with specific mAbs.
Project description:BACKGROUND AND PURPOSE: Neutrophil migration into tissues is involved in the genesis of inflammatory pain. Here, we addressed the hypothesis that the effect of CXC chemokines on CXCR1/2 is important to induce neutrophil migration and inflammatory hypernociception. EXPERIMENTAL APPROACH: Mice were treated with a non-competitive allosteric inhibitor of CXCR1/2, DF 2162, and neutrophil influx and inflammatory hypernociception were assessed by myeloperoxidase assay and electronic pressure meter test, respectively, in various models of inflammation. KEY RESULTS: DF 2162 inhibited neutrophil chemotaxis induced by CXCR1/2 ligands but had no effect on CXCL8 binding to neutrophils. A single mutation of the allosteric site at CXCR1 abrogated the inhibitory effect of DF 2162 on CXCL-8-induced chemotaxis. Treatment with DF 2162 prevented influx of neutrophils and inflammatory hypernociception induced by CXCL1 in a dose-dependent manner. The compound inhibited neutrophil influx and inflammatory hypernociception induced by carrageenan, lipopolysaccharide and zymosan, but not hypernociception induced by dopamine and PGE(2). DF 2162 had a synergistic effect with indomethacin or the absence of TNFR1 to abrogate carrageenan-induced hypernociception. Treatment with DF 2162 diminished neutrophil influx, oedema formation, disease score and hypernociception in collagen-induced arthritis. CONCLUSIONS AND IMPLICATIONS: CXCR1/2 mediates neutrophil migration and is involved in the cascade of events leading to inflammatory hypernociception. In addition to modifying fundamental pathological processes, non-competitive allosteric inhibitors of CXCR1/2 may have the additional benefit of providing partial relief for pain and, hence, may be a valid therapeutic target for further studies aimed at the development of new drugs for the treatment of rheumatoid arthritis.