Low-affinity Fcγ Receptors Research Articles

Common polymorphisms in critical genes of innate immunity do not contribute to the risk for chronic disseminated candidiasis in adult leukemia patients

Chronic disseminated candidiasis is a serious fungal infection in immunocompromised patients, particularly those undergoing therapy for acute leukemia. Coordination between innate and adaptive immune system is critical to resistance or susceptibility to Candida infection. In order to investigate possible genetic contribution to chronic disseminated candidiasis of key molecules in the innate immune pathway, we performed a case control study using the candidate gene approach. Forty subjects with chronic disseminated candidiasis and 50 controls without chronic disseminated candidiasis but an underlying diagnosis of leukemia were enrolled in the Helsinki University Central Hospital during the period 1980-1998. Candidate genes were selected for analysis based upon the following criteria: a common polymorphism (>5% frequency) and existence a priori of clinical and biological data suggesting a role for the variant in the pathogenesis of chronic disseminated candidiasis. Six genes were selected from critical microbicidal and innate immune pathways, including three low-affinity Fcgamma receptors (FCGR2A, FCGR3A and FCGR3B), chitotriosidase (CHIT1), p22-phox NADPH oxidase (CYBA), and mannose binding lectin (MBL2). There was no statistically significant association of susceptibility to chronic disseminated candidiasis with the polymorphisms in this study. Common variants in the six studied genes most likely do not contribute to the risk for chronic disseminated candidiasis in patients with acute leukemia.

Effect of C-reactive protein on Fcgamma receptor II in cultured bovine endothelial cells.

The major CRP (C-reactive protein) receptor on leucocytes has been identified as the low-affinity IgG receptor Fcgamma receptor II (CD32). Our aim was to assess whether inflammation may modify the presence of the CD32 receptor in BAEC (bovine aortic endothelial cells). Confocal microscopy experiments showed a weak expression of the CD32 receptor in control BAEC that was slightly increased by 10 microg/ml CRP. Incubation of BAEC with TNF-alpha (tumour necrosis factor-alpha) did not modify the fluorescence signal of CD32. Addition of CRP to TNF-alpha-incubated BAEC enhanced the fluorescence signal of the CD32 receptors. The CD32 receptors showed a perinuclear cytoplasmic localization in BAEC. An alteration of the NO (nitric oxide)-dependent vasorelaxation has been defined as endothelial dysfunction. Endothelial dysfunction has been associated with the presence of superoxide anion and with a reduction in the expression of the eNOS (endothelial NO synthase). A concentration of CRP similar to that detected in patients with cardiovascular risk (10 microg/ml) failed to modify the generation of superoxide anion stimulated by TNF-alpha. Western blot experiments showed that TNF-alpha decreased the expression of the eNOS protein, which was partially protected by treatment with 10 microg/ml CRP. The protective effect of 10 microg/ml CRP on eNOS expression in TNF-alpha-incubated BAEC was prevented by an antibody against CD32 receptors. In conclusion, the present results suggest that, although CRP has been associated with inflammation, CRP may protect the expression of eNOS protein against pro-inflammatory mediators such as TNF-alpha.

A Cell-Based Artificial Antigen-Presenting Cell Coated with Anti-CD3 and CD28 Antibodies Enables Rapid Expansion and Long-Term Growth of CD4 T Lymphocytes

We compared the ability of two genetically modified myeloid cells, K562 and U937, to serve as artificial antigen-presenting cells (aAPC). Both aAPC were stably transfected with the low-affinity Fcγ receptor CD32 (K32/U32 cells). K32 cells loaded with anti-CD3 and anti-CD28 Ab (K32/CD3/28) induced more rapid CD4 T-cell expansion than CD3/28-coated beads. In contrast, U32/CD3/28 induced high levels of CD4 T-cell thymidine uptake but were unable to sustain long-term T-cell expansion. K32 cells, but not U32 cells, loaded with anti-CD3 alone also stimulated CD4 T-cell growth and IL-2 secretion, indicating the expression of additional costimulatory molecules on K32 cells. We found constitutive expression of B7-H3 and a strong upregulation of mRNA encoding for IL-15, PD-L1, and PD-L2 after coculture with CD4 T cells activated by K32/CD3/28 but not U32/CD3/28. We conclude that K32 aAPCs are a robust system for clinical scale ex vivo expansion of CD4 T cells.

Genomic organization of classical human low-affinity Fcgamma receptor genes.

The classical low-affinity Fcgamma receptor genes (FcgammaRIIA, B, C and FcgammaRIIIA, B) are located on chromosome 1q23, a region that shows strong linkage with human systemic lupus erythematosus (SLE) in several genome-wide scans, and family-based association between FcgammaRIIIA and SLE is now established. High homology among the Fcgamma receptor genes, however, has hampered further study of this region. We have used a human bacterial artificial chromosome (BAC) library to determine the order and orientation of these Fcgamma receptor genes and have sequenced the very highly homologous 5' region (including 3.4 kb of the promoter and the 8 kb from exon 1 to exon 3) of the FcgammaRIIB and FcgammaRIIC genes to enable study of their unique single nucleotide polymorphisms (SNP). We have utilized these data to characterize a linked set of three coding region SNPs in the FcgammaRIIC exon 3 (EC1) that includes the stop codon SNP, which provides an important insight into natural killer cell function. Together, these data provide the basis for the study of additional SNPs in FcgammaR genes in SLE disease susceptibility.

FcγRIII Mediates Neutrophil Recruitment to Immune Complexes: A Mechanism for Neutrophil Accumulation in Immune-Mediated Inflammation

Neutrophil accumulation is a hallmark of immune complex-mediated inflammatory disorders. Current models of neutrophil recruitment envision the capture of circulating neutrophils by activated endothelial cells. We now demonstrate that immobilized immune complexes alone support the rapid attachment of neutrophils, under physiologic flow conditions. Initial cell tethering requires the low-affinity Fcγ receptor IIIB (FcγRIIIB), and the β 2 integrins are additionally required for the subsequent shear-resistant adhesion. The attachment function of FcγRIIIB may be facilitated by its observed presentation on neutrophil microvilli. In vivo, in a model of acute antiglomerular basement membrane nephritis in which immune complexes are accessible to circulating neutrophils, FcγRIII-deficient mice had a significant reduction in neutrophil recruitment. Thus, the interaction of immune complexes with FcγRIII may mediate early neutrophil recruitment in immune complex-mediated inflammation.

Design and expression of polymeric immunoglobulin fusion proteins: a strategy for targeting low-affinity Fcgamma receptors.

We have developed a family of cloning vectors that direct expression of fusion proteins that mimic aggregated immunoglobulin (IgG) (AIG) and immune complex function with respect to their interactions with FcgammaR and that allow for the inclusion and targeting of a second protein domain to cells expressing FcgammaR. This was accomplished by expressing multiple linear copies of the hinge and CH2 domains (HCH2) of human IgG(1) fused to the framework region of human IgG(1). Convenient restriction sites allow for the facile introduction of additional amino-terminal domains. The resulting molecule is tripartite. The carboxyl-IgG(1) framework domain provides stability and permits dimerization, and the intervening polymer provides increased effector function and targeting to FcgammaR while the amino-terminal domain can deliver an additional signal to cells expressing FcgammaR. To demonstrate the utility of the vectors, the extracellular domain of human CD8alpha was expressed as a HCH2 polymer fusion protein. The fusion proteins were secreted in useful amounts from polyclonal cell lines established in Sf9 cells following transfection and selection with G418. The biological activity of the recombinant CD8alpha-HCH2 polymers was determined and compared to those of AIG and an anti-CD16 monoclonal antibody using an in vitro assay. The activity of the fusion proteins positively correlates to the number of HCH2 units. The largest polymer tested was severalfold more potent than AIG at similar concentrations. The HCH2 polymers described here represent a new strategy in the design of recombinant proteins for the therapeutic targeting of FcgammaR in autoimmune disorders.

Functional and selective targeting of adenovirus to high-affinity Fcgamma receptor I-positive cells by using a bispecific hybrid adapter.

Adenovirus (Ad) efficiently delivers its DNA genome into a variety of cells and tissues, provided that these cells express appropriate receptors, including the coxsackie-adenovirus receptor (CAR), which binds to the terminal knob domain of the viral capsid protein fiber. To render CAR-negative cells susceptible to Ad infection, we have produced a bispecific hybrid adapter protein consisting of the amino-terminal extracellular domain of the human CAR protein (CARex) and the Fc region of the human immunoglobulin G1 protein, comprising the hinge and the CH2 and CH3 regions. CARex-Fc was purified from COS7 cell supernatants and mixed with Ad particles, thus blocking Ad infection of CAR-positive but Fc receptor-negative cells. The functionality of the CARex domain was further confirmed by successful immunization of mice with CARex-Fc followed by selection of a monoclonal anti-human CAR antibody (E1-1), which blocked Ad infection of CAR-positive cells. When mixed with Ad expressing eGFP, CARex-Fc mediated an up to 250-fold increase of transgene expression in CAR-negative human monocytic cell lines expressing the high-affinity Fcgamma receptor I (CD64) but not in cells expressing the low-affinity Fcgamma receptor II (CD32) or III (CD16). These results open new perspectives for Ad-mediated cancer cell vaccination, including the treatment of acute myeloid leukemia.

Increased in vivo mitogenicity of anti-TCR/CD3 monoclonal antibody through reduced interaction with Fcγ receptors

Since its initial clinical use in 1980, anti-TCR/CD3 monoclonal antibody (mAb) has been shown to be a potent immunosuppressive agent in the prevention of renal allograft rejections. However, toxic side effects caused by release of cytokines, predominantly from activated CD4 + T-cells, remain a major problem with the use of these reagents. Previous work has shown that this activation is mediated via antibody binding to Fcγ receptors (FcγR) on host effector cells. In the present study, we have demonstrated in an in vivo mouse model that the anti-TCR/CD3 mouse mAb 7D6, as well as that from rat (17A2) and hamster (H57-597), induce a gradual depletion of host CD4 + T-cells without any apparent proliferative effects on the cells. In contrast, when treatment with these mAbs was combined with a mAb (2.4G2) that blocks the low-affinity Fcγ receptors (FcγRII/III), we found that the in vivo actions of the anti-TCR/CD3 mAbs resulted in a significant expansion, rather than depletion, of CD4 + cells. The ability of 2.4G2 to reduce mAb 7D6-FcγR interaction was directly demonstrated in an in vitro assay system in which 2.4G2 partially suppressed 7D6-mediated T-cell responses. Taken together, our results have shown that some so-called “nonmitogenic” anti-TCR/CD3 mAbs in fact possess potent activating properties and that their mitogenic potential can be exposed by reducing their interaction with FcγR on host effector cells.

Markedly different pathogenicity of four immunoglobulin G isotype-switch variants of an antierythrocyte autoantibody is based on their capacity to interact in vivo with the low-affinity Fcgamma receptor III.

Using three different Fcgamma receptor (FcgammaR)-deficient mouse strains, we examined the induction of autoimmune hemolytic anemia by each of the four immunoglobulin (Ig)G isotype-switch variants of a 4C8 IgM antierythrocyte autoantibody and its relation to the contributions of the two FcgammaR, FcgammaRI, and FcgammaRIII, operative in the phagocytosis of opsonized particles. We found that the four IgG isotypes of this antibody displayed striking differences in pathogenicity, which were related to their respective capacity to interact in vivo with the two phagocytic FcgammaRs, defined as follows: IgG2a > IgG2b > IgG3/IgG1 for FcgammaRI, and IgG2a > IgG1 > IgG2b > IgG3 for FcgammaRIII. Accordingly, the IgG2a autoantibody exhibited the highest pathogenicity, approximately 20-100-fold more potent than its IgG1 and IgG2b variants, respectively, while the IgG3 variant, which displays little interaction with these FcgammaRs, was not pathogenic at all. An unexpected critical role of the low-affinity FcgammaRIII was revealed by the use of two different IgG2a anti-red blood cell autoantibodies, which displayed a striking preferential utilization of FcgammaRIII, compared with the high-affinity FcgammaRI. This demonstration of the respective roles in vivo of four different IgG isotypes, and of two phagocytic FcgammaRs, in autoimmune hemolytic anemia highlights the major importance of the regulation of IgG isotype responses in autoantibody-mediated pathology and humoral immunity.

Variant Genotypes of the Low-Affinity Fcγ Receptors in Two Control Populations and a Review of Low-Affinity Fcγ Receptor Polymorphisms in Control and Disease Populations

Fcγ-receptors (FcγR) provide a critical link between humoral and cellular immunity. The genes of the low-affinity receptors for IgG and their isoforms, namely, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and SH-FcγRIIIb, are located in close proximity on chromosome 1q22. Variant alleles may differ in biologic activity and a number of studies have reported the frequencies of variant FcγR alleles in both disease and control populations. No large study has evaluated the possibility of a nonrandom distribution of variant genotypes. We analyzed 395 normal individuals (172 African Americans [AA] and 223 Caucasians [CA]) at the following loci: FcγRIIa, FcγRIIIa, and FcγRIIIb, including the SH-FcγRIIIb. The genotypic distributions of FcγRIIa, FcγRIIIa, and FcγRIIIb conform to the Hardy-Weinberg law in each group. There was no strong evidence that combinations of 2-locus genotypes of the 3 loci deviated from random distributions in these healthy control populations. The distribution of SH-FcγRIIIb is underrepresented in CA compared with AA (P < .0001) controls. A previously reported variant FcγRIIb was not detected in 70 normal individuals, indicating that this allele, if it exists, is very rare (<1%). In conclusion, we present data that should serve as the foundation for the interpretation of association studies involving multiple variant alleles of the low-affinity FcγR.

Variant Genotypes of the Low-Affinity Fcγ Receptors in Two Control Populations and a Review of Low-Affinity Fcγ Receptor Polymorphisms in Control and Disease Populations

AbstractFcγ-receptors (FcγR) provide a critical link between humoral and cellular immunity. The genes of the low-affinity receptors for IgG and their isoforms, namely, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and SH-FcγRIIIb, are located in close proximity on chromosome 1q22. Variant alleles may differ in biologic activity and a number of studies have reported the frequencies of variant FcγR alleles in both disease and control populations. No large study has evaluated the possibility of a nonrandom distribution of variant genotypes. We analyzed 395 normal individuals (172 African Americans [AA] and 223 Caucasians [CA]) at the following loci: FcγRIIa, FcγRIIIa, and FcγRIIIb, including the SH-FcγRIIIb. The genotypic distributions of FcγRIIa, FcγRIIIa, and FcγRIIIb conform to the Hardy-Weinberg law in each group. There was no strong evidence that combinations of 2-locus genotypes of the 3 loci deviated from random distributions in these healthy control populations. The distribution of SH-FcγRIIIb is underrepresented in CA compared with AA (P < .0001) controls. A previously reported variant FcγRIIb was not detected in 70 normal individuals, indicating that this allele, if it exists, is very rare (<1%). In conclusion, we present data that should serve as the foundation for the interpretation of association studies involving multiple variant alleles of the low-affinity FcγR.

Low affinity Fcγ receptors on murine macrophages: mitogen-activated protein kinase activation and AP-1 DNA binding activity

Mouse macrophage cell lines such as J774 express Fc receptors for IgG2a immune complexes, which upon binding of the proper ligand, trigger several signal transduction pathways. A surface to nucleus signaling through these receptors has been demonstrated. We describe here the activation of the mitogen-activated protein kinase (MAPK) and an increase in the binding of the activator protein 1 (AP-1) to DNA upon receptor stimulation. The described effects are only partially blocked by inhibitors of the Ca 2+/diacylglycerol-dependent protein kinase (PKC), suggesting that differential signaling pathways are activated upon receptor cross-linking and that they converge at or above the MAPK level. These results pave the way to our understanding of FcγR cross-linking induced gene expression regulation.

Murine low affinity Fcγ receptors can be distinguished by anti-Ly-17 antibodies

Murine low affinity Fcγ receptors can be distinguished by anti-Ly-17 antibodies

Murine low affinity Fcγ receptors can be distinguished by anti-Ly-17 antibodies

Murine low affinity Fcγ receptors can be distinguished by anti-Ly-17 antibodies

Mitogenicity of anti-TCR/CD3 mAbs attributable to ineffective cross-linking of low-affinity Fcγ receptors

Mitogenicity of anti-TCR/CD3 mAbs attributable to ineffective cross-linking of low-affinity Fcγ receptors

Mitogenicity of anti-TCR/CD3 mAbs attributable to ineffective cross-linking of low-affinity Fc-γ receptors

Mitogenicity of anti-TCR/CD3 mAbs attributable to ineffective cross-linking of low-affinity Fc-γ receptors

Phosphoinositide 3-Kinase and p72syk Noncovalently Associate with the Low Affinity Fcγ Receptor on Human Platelets through an Immunoreceptor Tyrosine-based Activation Motif: RECONSTITUTION WITH SYNTHETIC PHOSPHOPEPTIDES

Previously, we have demonstrated that the cytoplasmic tyrosine kinase p72syk is coupled to the platelet Fc receptor for IgG (Fc gamma RIIA) (Chacko, G. W., Duchemin, A. M., Coggeshall, K. M., Osborne, J. M., Brandt, J. T., and Anderson, C. L. (1994) J. Biol. Chem. 269, 32435-32440). Further analysis of the platelet activation by Fc gamma RIIA demonstrated that Fc gamma RIIA is also inducibly coupled to the serine/threonine and lipid kinase, phosphoinositide 3-kinase (PI 3-K). activation of platelets with anti-Fc gamma RIIA antibodies resulted in the noncovalent association of PI 3-K with Fc gamma RIIA as well as an increase in Fc gamma RIIA-associated PI 3-K activity. Binding of both p72syk and PI 3-K to Fc gamma RIIA was reconstituted with synthetic phosphopeptides corresponding to the sequence of the atypical immunoreceptor tyrosine-based activation motif (ITAM) in the cytoplasmic domain of Fc gamma RIIA. Our findings demonstrate that coupling of both p72syk and PI. 3-K activities to Fc gamma RIIA is regulated by tyrosine phosphorylation of the ITAM, and we speculate that p72syk might act as an adapter to recruit PI 3-K to activated Fc gamma RIIA.

Purification and optimization of functional reconstitution on the surface of leukemic cell lines of GPI-anchored Fcγ receptor III

Purified glycosyl phosphatidyl inositol (GPI)-anchored cell surface proteins can be reincorporated spontaneously into the cell membrane by incubating the cells with these proteins. This unique property provides a novel way of introducing cell surface receptors on live cell membranes without the use of gene transfection. Since any classical transmembrane cell surface protein can be converted to a GPI anchored protein by recombinant techniques, this method provides a means of studying ectodomain associated receptor functions on various cell types. Moreover, in some circumstances, it can be used to correct deficient cellular functions resulting from lack of cell surface protein expression. Using GPI-anchored Fcγ receptor III (CD16B), a low affinity Fcγ receptor, we have systematically studied the optimal conditions for reconstitution of a functional receptor on nucleated cells. CD16B is purified to homogeneity from neutrophil lysates by single step immunoaffinity chromatography. The purified CD16B is functionally active as evidenced by its ability to bind IgG opsonized erythrocytes. CD16B incorporation on nucleated cells is temperature dependent with an optimum of 37°C. The level of expression of incorporated CD16B is also depend on the concentration of CD16B available and the duration of incubation. The incorporated CD16B retains its ability to bind ligand and also mediates endocytosis of the bound ligand. In summary, our results demonstrate that purified, functionally active GPI-anchored receptors can be expressed on desired cells in a controlled manner and retain some functional properties.

Ligands and biological activities of soluble Fcγ receptors

Soluble forms of low-affinity Fcγ receptors (sFcγR) circulate in biologic fluids. Their plasmatic levels vary in immunological disorders or related diseases. They are produced by enzymatic cleavage of the membrane receptors or by alternative splicing. They bind IgG with the same isotype specificity as their cell surface counterparts and thus modulate Fc-dependent immune functions. Recent data suggest that they also bind non-Ig ligands present on leukocytes. Functional implications of these findings are discussed.

Fcγ receptor II stimulated formation of inositol phosphates in human platelets is blocked by tyrosine kinase inhibitors and associated with tyrosine phosphorylation of the receptor

We report that activation of phospholipase C (PLC) by cross-linking of the platelet low-affinity Fcγ receptor II (Fcγ RII) is inhibited by two structurally distinct tyrosine kinase inhibitors, staurosporine and ST271. This contrasts with PLC activation induced by thrombin and U46619, a thromboxane mimetic, whose receptors have seven transmembrane domains characteristic of G-protein coupled receptors. Several proteins undergo phosphorylation on tyrosine on FcγRII cross-linking upstream of protein kinase C (PKC), Ca 2+ and aggregation, including the FcγRII itself. The role of FcγRII phosphorylation in the regulation of PLC is discussed.