SARS-CoV-2 spike-FLIPr fusion protein plus lipidated FLIPr protects against various SARS-CoV-2 variants in hamsters.

Vaccine-induced mucosal immunity and broad protective capacity against various severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants remain inadequate. Formyl peptide receptor-like 1 inhibitory protein (FLIPr), produced by Staphylococcus aureus, can bind to various Fcγ receptor subclasses. Recombinant lipidated FLIPr (rLF) was previously found to be an effective adjuvant. In this study, we developed a vaccine candidate, the recombinant Delta SARS-CoV-2 spike (rDS)-FLIPr fusion protein (rDS-F), which employs the property of FLIPr binding to various Fcγ receptors. Our study shows that rDS-F plus rLF promotes rDS capture by dendritic cells. Intranasal vaccination of mice with rDS-F plus rLF increases persistent systemic and mucosal antibody responses and CD4/CD8 T-cell responses. Importantly, antibodies induced by rDS-F plus rLF vaccination neutralize Delta, Wuhan, Alpha, Beta, and Omicron strains. Additionally, rDS-F plus rLF provides protective effects against various SARS-CoV-2 variants in hamsters by reducing inflammation and viral loads in the lung. Therefore, rDS-F plus rLF is a potential vaccine candidate to induce broad protective responses against various SARS-CoV-2 variants.IMPORTANCEMucosal immunity is vital for combating pathogens, especially in the context of respiratory diseases like COVID-19. Despite this, most approved vaccines are administered via injection, providing systemic but limited mucosal protection. Developing vaccines that stimulate both mucosal and systemic immunity to address future coronavirus mutations is a growing trend. However, eliciting strong mucosal immune responses without adjuvants remains a challenge. In our study, we have demonstrated that using a recombinant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike-formyl peptide receptor-like 1 inhibitory protein (FLIPr) fusion protein as an antigen, in combination with recombinant lipidated FLIPr as an effective adjuvant, induced simultaneous systemic and mucosal immune responses through intranasal immunization in mice and hamster models. This approach offered protection against various SARS-CoV-2 strains, making it a promising vaccine candidate for broad protection. This finding is pivotal for future broad-spectrum vaccine development.

Structural analysis of Fc/FcγR complexes: a blueprint for antibody design.

The number of studies and the quality of the structural data of Fcγ receptors (FcγRs) has rapidly increased in the last few years. Upon critical examination of the literature, we have extracted general conclusions that could explain differences in affinity and selectivity of FcγRs for immunoglobulin G (IgG) based on structural considerations. FcγRs employ a little conserved asymmetric surface of domain D2 composed of two distinct subsites to recognize the well-conserved lower hinge region of IgG1-Fc. The extent of the contact interface with the antibody in subsite 1 of the receptor (but not in subsite 2), the geometrical complementarity between antibody and receptor, and the number of polar interactions contribute decisively toward strengthening the binding affinity of the antibody for the receptor. In addition, the uncertain role of the N-linked glycan of IgG for the binding and effector responses elicited by FcγRs is discussed. The available data suggest that not only the non-covalent interactions between IgG and FcγRs but also their dynamic features are essential for the immune response elicited through these receptors. We believe that the integration of structural, thermodynamic, and kinetic data will be critical for the design and validation of the next generation of therapeutic antibodies with enhanced effector capabilities.

Type I and type II Fc receptors regulate innate and adaptive immunity.

Antibodies produced in response to a foreign antigen are characterized by polyclonality, not only in the diverse epitopes to which their variable domains bind but also in the various effector molecules to which their constant regions (Fc domains) engage. Thus, the antibody's Fc domain mediates diverse effector activities by engaging two distinct classes of Fc receptors (type I and type II) on the basis of the two dominant conformational states that the Fc domain may adopt. These conformational states are regulated by the differences among antibody subclasses in their amino acid sequence and by the complex, biantennary Fc-associated N-linked glycan. Here we discuss the diverse downstream proinflammatory, anti-inflammatory and immunomodulatory consequences of the engagement of type I and type II Fc receptors in the context of infectious, autoimmune, and neoplastic disorders.

Intravenous immunoglobulin therapy: how does IgG modulate the immune system?

Intravenous immunoglobulin (IVIG) preparations comprise pooled IgG antibodies from the serum of thousands of donors and were initially used as an IgG replacement therapy in immunocompromised patients. Since the discovery, more than 30 years ago, that IVIG therapy can ameliorate immune thrombocytopenia, the use of IVIG preparations has been extended to a wide range of autoimmune and inflammatory diseases. Despite the broad efficacy of IVIG therapy, its modes of action remain unclear. In this Review, we cover the recent insights into the molecular and cellular pathways that are involved in IVIG-mediated immunosuppression, with a particular focus on IVIG as a therapy for IgG-dependent autoimmune diseases.

FcγRs in health and disease.

Genetic defects affecting the humoral immune response and especially the production of antibodies of the immunoglobulin G (IgG) isotype result in a heightened susceptibility to infections. Studies over the last years have demonstrated the crucial role of Fc-receptors for IgG (FcγRs) widely expressed on innate immune effector cells in mediating the protective function of IgG. During the last years, additional ligands interacting with FcγRs as well as additional receptors binding to IgG glycosylation variants have been identified. In this review, we discuss how the interaction of these different ligands with classical and novel Fcγ-receptors influences the immune response and which strategies microorganisms have developed to prevent them.

Dengue virus neutralization is modulated by IgG antibody subclass and Fcgamma receptor subtype.

Severe dengue virus (DENV) infection is epidemiologically linked to pre-existing anti-DENV antibodies acquired by maternal transfer or primary infection. A possible explanation is that DENV immune complexes evade neutralization by engaging Fcgamma receptors (FcgammaR) on monocytes, natural targets for DENV in humans. Using epitope-matched humanized monoclonal antibodies (mAbs) and stable FcgammaR-transfected CV-1 cells, we found that DENV neutralization by IgG1, IgG3, and IgG4 mAbs was enhanced in high-affinity FcgammaRIA transfectants and diminished in low-affinity FcgammaRIIA transfectants, whereas neutralization by IgG2 mAbs (low-affinity ligands for both FcgammaRs) was diminished equally. In FcgammaR-negative Vero cells, IgG3 mAbs exhibited the strongest neutralizing activity and IgG2, the weakest. Our results demonstrate that DENV neutralization is modulated by the Fc region in an IgG subclass manner, likely through effects on virion and FcgammaR binding. Thus, the IgG antibody subclass profile generated by DENV infection or vaccination may independently influence the magnitude of the neutralizing response.

Analysis of the Fc gamma receptor-dependent component of neutralization measured by anthrax toxin neutralization assays.

Anthrax toxin neutralization assays are used to measure functional antibody levels elicited by anthrax vaccines in both preclinical and clinical studies. In this study, we investigated the magnitude and molecular nature of Fc gamma (Fcgamma) receptor-dependent toxin neutralization observed in commonly used forms of the anthrax toxin neutralization assay. Significantly more Fcgamma receptor-dependent neutralization was observed in the J774A.1 cell-based assay than in the RAW 264.7 cell-based assay, a finding that could be due to the larger numbers of Fcgamma receptors that we found on J774A.1 cells by using flow cytometry. Thus, the extent to which Fcgamma receptor-dependent neutralization contributes to the total neutralization measured by the assay depends on the specific cell type utilized in the assay. Using Fcgamma receptor blocking monoclonal antibodies, we found that at least three murine Fcgamma receptor classes, IIB, III, and IV, can contribute to Fcgamma receptor-dependent neutralization. When antibodies elicited by immunization of rabbits with protective-antigen-based anthrax vaccines were analyzed, we found that the magnitude of Fcgamma receptor-dependent neutralization observed in the J774A.1 cell-based assay was dependent on the concentration of protective antigen utilized in the assay. Our results suggest that the characteristics of the antibodies analyzed in the assay (e.g., species of origin, isotype, and subclass), as well as the assay design (e.g., cell type and protective antigen concentration), could significantly influence the extent to which Fcgamma receptor-dependent neutralization contributes to the total neutralization measured by anthrax toxin neutralization assays. These findings should be considered when interpreting anthrax toxin neutralization assay output.

Characterization and ligand specificity of sheep IgG2 receptor.

Neutrophils and macrophages in cattle express a novel class of immunoglobulin Fc receptor, specific for bovine IgG2, termed boFcgamma2R. In cows, the ability of neutrophils to kill immunoglobulin-opsonized microorganisms appears to depend largely on this subclass. Although related to other mammalian FcgammaRs, boFcgamma2R belongs to a novel gene family that includes the human killer Ig-like receptor and FcalphaRI (CD89) proteins. In this study, we describe the presence and characterization of this novel class of FcgammaR in sheep. The comparative analysis of this novel FcgammaR has allowed us to begin an exploration of some immunological characteristic of ruminants.

Human Fc receptors: critical targets in the treatment of autoimmune diseases and transplant rejections.

The receptors for the Fc region of immunoglobulins (FcR) are members of the immunoglobulin superfamily. They are expressed on various hematopoietic cells and constitute a link between humoral and cell-mediated immunity. The activation and downmodulation of immune responses are controlled by signals from activating and inhibitory FcR, expressed on the surface of immune cells. The signaling regions, defined as immunoreceptor-tyrosine-based activation motif and immunoreceptor-tyrosine-based inhibitory motif, are contained within the cytoplasmic domain of FcR or of the adaptor proteins associated with FcR. Activating and inhibitory FcR are usually coexpressed on the surface of the same cell and coengaged by the same ligand, functioning in concert to keep a balanced immune response. Impairment of the functional balance between activating and inhibitory FcR leads either to hyperactivity to foreign and self antigens or to unresponsiveness as seen in many autoimmune diseases and infections. Pathologic conditions in which immunoglobulin-FcR interactions play a major role, as well as the outcome of treatment with intravenous immunoglobulin and monoclonal antibodies, may be influenced by targeting FcR.

Fcgamma receptors: old friends and new family members.

Although cellular receptors for immunoglobulins were first identified nearly 40 years ago, their central role in the immune response was discovered only in the last decade. They are key players in both the afferent and efferent phase of an immune response, setting thresholds for B cell activation, regulating the maturation of dendritic cells, and coupling the exquisite specificity of the antibody response to innate effector pathways, such as phagocytosis, antibody-dependent cellular cytotoxicity, and the recruitment and activation of inflammatory cells. Moreover, because of their general presence as receptor pairs consisting of activating and inhibitory molecules on the same cell, they have become a paradigm for studying the balance of positive and negative signals that ultimately determine the outcome of an immune response. This review will summarize recent results in Fc-receptor biology with an emphasis on data obtained in in vivo model systems.

Divergent immunoglobulin g subclass activity through selective Fc receptor binding.

Subclasses of immunoglobulin G (IgG) display substantial differences in their ability to mediate effector responses, contributing to variable activity of antibodies against microbes and tumors. We demonstrate that the mechanism underlying this long-standing observation of subclass dominance in function is provided by the differential affinities of IgG subclasses for specific activating IgG Fc receptors compared with their affinities for the inhibitory IgG Fc receptor. The significant differences in the ratios of activating-to-inhibitory receptor binding predicted the in vivo activity. We suggest that these highly predictable functions assigned by Fc binding will be an important consideration in the design of therapeutic antibodies and vaccines.

Section 1C: Assessment of the functional activity and IgG Fc receptor utilisation of 64 IgG Rh monoclonal antibodies. Coordinator's report.

Sixty-four IgG Rh monoclonal antibodies (Mabs) submitted to the Fourth International Workshop on Monoclonal Antibodies Against Human Red Blood Cells and Related Antigens were characterised and tested in quantitative functional assays at five laboratories. The biological assays measured the ability of anti-D to mediate phagocytosis or extracellular lysis of RBC by IgG Fc receptor (Fc gamma R)-bearing effector cells. Interactions of RBC pre-sensitised with anti-D (EA-IgG) with monocytes in chemiluminescence (CL) assays were found proportional to the amount of IgG anti-D on the RBC. Using antibodies to inhibit Fc gamma RI, Fc gamma RII or Fc gamma RIII, the only receptor utilised in the monocyte CL and ADCC assays for interactions with EA-IgG1 was found to be Fc gamma RI. In these assays, enhanced interactions were promoted by EA-IgG3 and additional Fc gamma receptors may have contributed. IgG2 anti-D was not reactive in these assays and EA-IgG4 promoted weak reactions through Fc gamma RI. A macrophage ADCC assay showed that haemolysis of EA-IgG3 was greater than that of EA-IgG1, mediated mainly through Fc gamma RIII. In ADCC assays using lymphocytes (NK cells) as effector cells and papainised RBC target cells, only a minority of IgG1 anti-D Mabs were shown to be able to mediate haemolysis in the presence of monomeric IgG (AB serum or IVIg). These interactions were mediated solely through Fc gamma RIII. Haemolysis via Fc gamma RIII may depend on the presence of certain sugars on the oligosaccharide moiety of IgG. Most Mabs (IgG1, IgG2, IgG3 and IgG4) elicited intermediate, low or no haemolysis in these assays. Blocking studies indicated that low activity IgG1 and IgG4 anti-D utilised only Fc gamma RI. Other IgG1 and IgG3 Mabs appeared to promote haemolysis through Fc gamma RI and Fc gamma RIII while IgG2 was inhibited by Mabs to both Fc gamma RII and Fc gamma RIII, suggesting a variety of Fc gamma R are utilised for anti-D of low haemolytic activity. Excellent agreement between the results of the lymphocyte ADCC assays and antibody quantitation was observed between the participating laboratories.

The Fc gamma receptor genotype as a severity factor for chronic periodontitis in Japanese patients.

BACKGROUND: Functional polymorphisms of immunoglobulin G (IgG) Fc receptors (Fc gamma R) have been shown to be associated with generalized aggressive periodontitis (GAgP) or recurrence of chronic periodontitis (CP) in Japanese patients. The purpose of this study was to evaluate whether Fc gamma R polymorphisms are also associated with severity of CP. METHODS: Fifty Japanese non-smoking patients with severe CP and 39 Japanese non-smoking patients with moderate CP were identified according to established clinical criteria, including measurements of probing depth (PD), clinical attachment level (CAL), and alveolar bone loss (BL). Fc gamma R genotypes for 3 bi-allelic polymorphisms (Fc gamma RIIa-R/H131, Fc gamma RIIIa-158V/F, Fc gamma RIIIb-NA1/NA2) were determined in these CP patients and 64 race-matched, non-smoking healthy controls by means of allele-specific polymerase chain reactions. RESULTS: There was a significant over-representation of Fc gamma RIIIa-158V allele in severe CP patients compared to moderate CP patients (odds ratio 2.03, 95% confidence interval [CI] 1.03-4.01, chi 2 = 4.86, P = 0.028). In addition, we found a strong association between CP severity and Fc gamma R composite genotype comprising Fc gamma RIIIa-158V plus Fc gamma RIIIb-NA2 (severe CP versus moderate CP: odds ratio 4.69, 95% CI 1.52-15.10, chi 2 = 9.35, P = 0.002; severe CP versus healthy controls: odds ratio 4.10, 95% CI 1.62-10.59, chi 2 = 11.13, P = 0.0009). Moreover, CP patients positive for the composite genotype exhibited more severe signs of periodontitis than composite genotype-negative individuals (positive versus negative; mean PD: 3.8 mm versus 3.2 mm, P = 0.005; mean CAL: 4.5 mm versus 3.7 mm, P = 0.005; mean % BL: 37.6% versus 29.9%, P = 0.008). CONCLUSION: Our results document the Fc gamma RIIIa-158V allele and possibly Fc gamma RIIIb-NA2 to be associated with severity of CP in Japanese patients.

Fcgamma receptor-mediated immune phagocytosis depends on the class of FcgammaR and on the immunoglobulin-coated target cell.

BACKGROUND AND OBJECTIVES: Three human Fcgamma receptors (FcgammaR) are known to mediate immune phagocytosis. A variety of different phagocytic assays have been described, but their comparability is complicated by the use of different effector cells and different antibody-coated target cells. The aim of this study was to determine the influence of these variable components on the FcgammaR-mediated phagocytosis. MATERIALS AND METHODS: We sensitized human red blood cells (RBC) with polyclonal human anti-D (huaD), or with human monoclonal anti-D of the isotypes IgG1 (huIgG1) or IgG3 (huIgG3). Sheep RBC coated with rabbit immunoglobulin (RBC-RAS) were also used. Monocytes or polymorphonuclear neutrophils (PMN) were incubated with different FcgammaR-specific antibodies or their F(ab')2 fragments to determine the contribution of the different FcgammaRs on these effector cells in the phagocytic process of different antibody-coated target cells. RESULTS: huaD-RBC and huIgG1-RBC were preferentially ingested via the FcgammaRI on monocytes and, to a minor extent, also by the FcgammaRII. PMN ingested these target cells only after induction of the FcgammaRI by interferon-gamma (IFN-gamma). huIgG3-RBC extensively formed rosettes with monocytes but were seldom ingested. RAS-RBC phagocytosis was induced primarily via the FcgammaRI on monocytes and was mediated by the FcgammaRII on PMN. CONCLUSION: When performing phagocytosis assays with different effector and target cells, one has to take into account that phagocytosis is mediated by different FcgammaR, making comparability of these assays more difficult.

A novel PCR-based method for direct Fc gamma receptor IIIa (CD16) allotyping.

Leukocyte IgG receptors (Fc gamma R) are important immune-response modulating molecules. Fc gamma RIIIa is expressed on macrophages, NK-cells and gamma delta-T cells and exhibits a genetically determined, functional polymorphism at nucleotide 559. This allelic difference predicts either a phenylalanine (F158) or valine (V158) at amino acid 158 in the membrane-proximal extracellular domain, and has been shown to be associated with autoimmune and infectious diseases. Published methods to determine Fc gamma RIIIa genotypes are cumbersome. Therefore, we developed a novel, rapid and reliable PCR-based method to determine Fc gamma RIIIa genotypes. Comparison of genotyping results with direct Fc gamma RIIIa sequencing of 60 blood donors showed 100% accuracy of this new method. Since genotype frequencies of Fc gamma R polymorphisms depend strongly on race and ethnicity, we compared Fc gamma RIIIa genotype frequencies of 176 Caucasian Dutch and 104 Japanese blood donors. Interestingly, these frequencies were not significantly different (P>0.1), in contrast to the Fc gamma RIIa and Fc gamma RIIIb genotype frequencies (P<0.001).

Structural basis of the interaction between IgG and Fcgamma receptors.

The binding of multivalent antigen-antibody complexes to receptors for the Fc portion of IgG (FcgammaR) induces the clustering of the FcgammaR and triggers cell activation leading to defence reactions against pathogens. The Fc portion of IgG consists of two identical polypeptide chains which are related to each other by a 2-fold axis and are folded in two structural domains, the C(H)2 domain, near the flexible hinge region of the IgG molecule, and the C(H)3 domain. We studied the interaction in solution between the Fc fragment of mouse IgG2b and the extracellular region of mouse FcgammaRII. We find that one Fc molecule binds one FcgammaRII molecule only. Using NMR spectroscopy, we show that FcgammaRII binds to a negatively charged area of the C(H)2 domain, corresponding to the lower hinge region, and that the binding of FcgammaRII onto one of the two symmetrically related sites on the Fc induces a conformational change in the other site. We therefore propose a model that explains why IgG molecules are unable to trigger FcgammaR-mediated cellular responses spontaneously in the absence of crosslinking by multivalent antigens.

Macrophage Fcgamma receptors expression is altered by treatment with dopaminergic drugs.

Macrophage Fcgamma receptors have an important role in host defense and the pathophysiology of immune mediated disorders. Alteration of splenic macrophage Fcgamma receptors expression predisposes to severe infection. Inhibition or blockade of splenic macrophage Fcgamma receptors is one of the mechanisms by which immune cytopenias improve. Dopaminergic drugs have clinically significant regulatory functions on the immune response. Using an experimental model in the guinea pig we assessed the effect of commonly used dopaminergic drugs on the expression of macrophage Fcgamma receptors. Three dopa-antagonists, bromocryptine, leuprolide, and pergolide, and seven dopa-antagonists, chlorpromazine, SCH 23390, metochlopramide, sulpiride, veralipride, alizapride, and cisapride, were studied. Following guinea pig treatment with dopaminergic drugs, the clearance of IgG-sensitized RBCs in vivo, the in vitro binding of IgG-sensitized RBCs by isolated splenic macrophages and flow cytometry with monoclonal antibodies were performed. Treatment with dopa-agonists enhanced the clearance of IgG-sensitized RBCs, the in vitro binding of IgG-sensitized RBCs by isolated splenic macrophages, and the cell surface expression of both macrophage Fcgamma receptors, and vice versa, dopa-antagonists impaired macrophage Fcgamma receptors expression. Macrophage FcgammaR1,2 was more sensitive than FcgammaR2 to such dopaminergic effect. These alterations of macrophage Fcgamma receptors expression are mediated by both D1 and D2 dopamine receptors, with a major participation of D2 receptors. Dopaminergic drugs alter the clearance of IgG-coated cells by an effect at the expression of splenic macrophage Fcgamma receptors.

Fc gamma receptor-mediated activation of phospholipase D regulates macrophage phagocytosis of IgG-opsonized particles.

Receptors for the Fc portion of IgG (Fc gamma Rs) integrate the innate and acquired components of immunity by coupling the specific recognition of IgG Abs to the activation of phagocytic leukocytes. Knowledge of the molecular mechanisms that regulate phagocyte stimulation by Fc gamma Rs may permit therapeutic modulation to augment immunoprotective aspects and minimize damage to host tissues in diverse inflammatory diseases. Since phospholipase D (PLD) has been linked to the stimulation of cytotoxic leukocyte responses, we characterized Fc gamma R-dependent activation of PLD in human macrophages. IgG-coated SRBCs (EIgG) stimulated a 9.4-fold increase in PLD activity compared with SRBCs treated with control Ab (p < 0. 001), determined by formation of the PLD-specific product phosphatidylethanol in the presence of 0.5% ethanol. Levels of phosphatidic acid, the physiologic product of PLD-mediated catalysis, were significantly increased in the absence of ethanol (6.4-fold, p < 0.001). PLD activity was also stimulated by immune complex-coated latex beads or cross-linking of Abs specific for Fc gamma RI, Fc gamma RII, or Fc gamma RIII. Phagocytosis of EIgG was reduced by two inhibitors of PLD-mediated signaling, 2,3-diphosphoglycerate or 1-butanol. Addition of purified PLD restored control levels of phagocytosis in cells in which endogenous PLD was inhibited. The tyrosine kinase inhibitors genistein and herbimycin A caused concordant reductions in Fc gamma R-stimulated PLD activity and phagocytosis. These studies demonstrate that Fc gamma R-mediated phagocytosis is accompanied by tyrosine kinase-dependent activation of PLD and support the hypothesis that stimulation of PLD functions to regulate the ingestion of IgG-opsonized particles.

Antineutrophil cytoplasmic antibodies preferentially engage Fc gammaRIIIb on human neutrophils.

Antineutrophil cytoplasmic Abs (ANCA) are found in the circulation of many patients with systemic vasculitis. ANCA bind to ANCA target, such as proteinase 3 and myeloperoxidase, and activate neutrophils in an Fc gammaR-dependent manner. Human neutrophils constitutively express Fc gammaRIIa (CD32) and Fc gammaRIIIb (CD16), and there is clear in vitro experimental evidence of ANCA-mediated engagement of Fc gammaRIIa. However, direct experimental evidence of ANCA engagement of neutrophil Fc gammaRIIIb has been obscured by technical problems related to activation-induced receptor shedding and activation-induced expression of receptor on the surface of neutrophils. In this study, by blocking receptor shedding and using appropriate reporter anti-Fc gammaR mAb, we show that human cANCA and pANCA, and murine mAb with corresponding reactivities, can indeed engage Fc gammaRIIIb. Furthermore, our data suggest that Fc gammaRIIIb is preferentially engaged by ANCA relative to Fc gammaRIIa presumably due to the nearly 10-fold excess of Fc gammaRIIIb expression relative to Fc gammaRIIa expression. These results clearly demonstrate that the Fc region of ANCA bound to an ANCA target on the neutrophil surface engage Fc gammaRIIIb and indicate that Fc gammaRIIIb and Fc gammaRIIa may both be active participants in ANCA-induced neutrophil activation. However, given the low levels of ANCA target expression on neutrophils from patients with systemic vasculitis, Fc gammaRIIIb is likely to play a critical role in initiating and perpetuating ANCA-induced neutrophil activation.

NA1/NA2 antisera inhibit FcgammaRI- but not FcgammaRII- mediated phagocytosis.

BACKGROUND AND OBJECTIVES: Alloantibodies against the granulocyte-specific NA antigens play an important role in alloimmune neonatal neutropenia. As the NA system is located on the FcgammaRIIIb, the influence of NA-specific antibodies on granulocyte function is of special interest. MATERIALS AND METHODS: We tested alloantisera specific for NA1 and NA2 for their ability to influence FcgammaR-mediated phagocytosis of polymorphonuclear neutrophils by use of different FcgammaR-specific targets. Red blood cells coated with human IgG anti-D served as specific targets for FcgammaRI-mediated phagocytosis while mouse IgG1 anti-glycophorin A was used to coat red blood cells (RBCs) to obtain FcgammaRII specific targets. To test for a hypothetical induction of phagocytosis by FcgammaRIIIb we used D-- RBCs coated with human monoclonal anti-D as target cells for unprimed neutrophils. RESULTS: Granulocyte phagocytosis was directly induced by FcgammaRI and FcgammaRII but not by FcgammaRIIIb. NA1 alloantisera significantly inhibited FcgammaRI-mediated phagocytosis of IFN-gamma-stimulated neutrophils if the corresponding antigen was expressed. Conversely, NA2 alloantisera inhibited FcgammaRI-mediated phagocytosis in NA2-positive individuals. There was no effect of NA1- and NA2-specific alloantibodies on FcgammaRII-mediated phagocytosis. CONCLUSION: NA-specific alloantisera inhibit the FcgammaRI-induced phagocytosis in primed neutrophils, but they do not significantly inhibit their FcgammaRIIa-specific phagocytosis of mIgG1-coated RBCs.