Human FcgammaRIIb Research Articles

Lower antibody response to Porphyromonas gingivalis associated with immunoglobulin G Fcγ receptor IIB polymorphism

Human FcgammaRIIB is one of the receptors for immunoglobulin G (IgG) and suppresses the activation of B lymphocytes through cross-linking with the B cell receptor via immune complexes. This function of FcgammaRIIB is essential for the negative regulation of antibody production. Our previous study has demonstrated the gene polymorphism FcgammaRIIB-I232T to be associated with periodontitis. The polymorphism FcgammaRIIB-232T has been reported to inhibit B-cell antigen receptor signaling more effectively compared to FcgammaRIIB-232I, while other groups concluded that FcgammaRIIB-232T had no ability to inhibit activatory receptors. In this study, we examined whether FcgammaRIIB-I232T polymorphism would change the IgG antibody response to the periodontopathic bacteria Porphyromonas gingivalis. Forty-seven patients with periodontitis were genotyped with the direct sequencing of genome DNA. Serum IgG and specific IgG subclass levels for the sonicate of P. gingivalis and the recombinant 40 kDa outer membrane protein (OMP) were determined. No significant difference in the total IgG level and IgG response to P. gingivalis sonicate were observed between sera from FcgammaRIIB-232T carriers and non-carriers. The FcgammaRIIB-232T carriers revealed a significantly lower IgG(2) response to P. gingivalis 40 kDa OMP compared to non-carriers (p = 0.04, Mann-Whitney U-test). Lower responses of FcgammaRIIB-232T carriers were also observed in specific IgG and IgG(1) levels. The FcgammaRIIB-232T carriers revealed a low level of IgG(2) response to P. gingivalis 40 kDa OMP, even with a high average probing pocket depth. These results suggest that association of the FcgammaRIIB-232T allele with periodontitis might be related to the lower levels of antibody response to P. gingivalis.

Recombinant carcinoembryonic antigen as a reporter gene for molecular imaging

Reporter genes can provide a way of noninvasively assessing gene activity in vivo. However, current reporter gene strategies may be limited by the immunogenicity of foreign reporter proteins, endogenous expression, or unwanted biological activity. We have developed a reporter gene based on carcinoembryonic antigen (CEA), a human protein with limited normal tissue expression. To construct a CEA reporter gene for PET, a CEA minigene (N-A3) was fused to the extracellular and transmembrane domains of the human Fc gamma RIIb receptor. The NA3-Fc gamma RIIb recombinant gene, driven by a CMV promoter, was transfected in Jurkat (human T cell leukemia) cells. Expression was analyzed by flow cytometry, immunohistochemistry (IHC), and microPET imaging. Flow cytometry identified Jurkat clones stably expressing NA3-Fc gamma RIIb at low, medium, and high levels. High and medium NA3-Fc gamma RIIb expression could also be detected by Western blot. Reporter gene positive and negative Jurkat cells were used to establish xenografts in athymic mice. IHC showed staining of the tumor with high reporter gene expression; medium and low N-A3 expression was not detected. MicroPET imaging, using an anti-CEA (124)I-labeled single-chain Fv-Fc antibody fragment, demonstrated that only high N-A3 expression could be detected. Specific accumulation of activity was visualized at the N-A3 positive tumor as early as 4 h. MicroPET image quantitation showed tumor activity of 1.8 +/- 0.2, 15.2 +/- 1.3, and 4.6 +/- 1.2 percent injected dose per gram (%ID/g) at 4, 20, and 48 h, respectively. Biodistribution at 48 h demonstrated tumor uptake of 4.8 +/- 0.8%ID/g. The CEA N-A3 minigene has the potential to be used as a reporter gene for imaging cells in vivo.

Role of leukocyte immunoglobuin G receptors in vaccine-induced immunity to Streptococcus pneumoniae.

Members of the leukocyte immunoglobulin (Ig) G receptor (FcgammaR) family play a key role in antibody-mediated phagocytosis and can either enhance antigen presentation or down-modulate immune responses. We studied immune responses to a pneumococcal conjugate (pneumococcal polysaccharide serotype 1 [PPS1]-tetanus toxoid) and antibody-mediated protection in mice deficient for individual FcgammaRs and complement receptor 3 (CR3). FcR gamma chain-deficient (FcR gamma chain(-/-)) mice, which lack expression of both FcgammaRI and III, had significantly lower anti-PPS1 IgG2b and IgG3 responses than did wild-type mice, whereas FcgammaRII-deficient (FcgammaRII(-/-)) mice had significantly higher IgG2a and IgG3 titers. Wild-type and FcgammaRII(-/-) mice were protected against infection with pneumococcal serotype 1, whereas immunized FcR gamma chain(-/-) and FcgammaRIII-deficient mice were not. Immunized CR3-deficient mice were protected against disease, and complement depletion had little effect on protection. These data indicate that activatory leukocyte FcgammaR, but not FcgammaRII (a murine homologue of human FcgammaRIIb), contributes to IgG-mediated protection against pneumococcal disease.

Characterization of the human Fc gamma RIIB gene promoter: human zinc-finger proteins (ZNF140 and ZNF91) that bind to different regions function as transcription repressors.

Expression of the human low-affinity Fc receptors for IgG (human Fc gamma RII) is differentially regulated. We report here the characterization of the promoter structure of the human Fc gamma RIIB gene and the isolation of the promoter region-binding proteins by a yeast one-hybrid assay. The minimal 154-bp region upstream from the transcription start site of the human Fc gamma RIIB gene was shown to possess promoter activity in a variety of cells. An electrophoretic mobility shift assay indicated that multiple nuclear factors in cell extracts bind to the two regions [F2-3 (-110 to -93) and F4-3 (-47 to -31)] of the human Fc gamma RIIB gene promoter. Mutation analysis indicated that GGGAGGAGC (-105 to -97) and AATTTGTTTGCC (-47 to -36) sequences are responsible for binding to nuclear factors respectively. By using GGGAGGAGC and AATTTGTTTGCC as bait sequences, we cloned two zinc-finger proteins (ZNF140 and ZNF91) that bind to the F2-3 and F4-3 regions within the promoter of the human Fc gamma RIIB gene respectively. When the ZNF140 and ZNF91 were transfected with reporter plasmid, both showed repressor activity with additive effects. Thus, these results indicate that these cloned ZNF140 and ZNF91 proteins function as repressors for the human Fc gamma RIIB transcription.

Fcgamma receptor-mediated inhibition of human B cell activation: the role of SHP-2 phosphatase.

Co-clustering of the type II receptors binding the Fc part of IgG (FcgammaRIIb) and B cell receptors results in the translocation of cytosolic, negative regulatory molecules to the phosphorylated immunoreceptor tyrosine-based inhibitory motif (P-ITIM) of the FcgammaRIIb. SH2 domain-containing protein tyrosine phosphatases (SHP-1 and SHP-2), and the polyphosphoinositol 5'-phosphatase (SHIP) have been reported earlier to bind to murine FcgammaRIIb P-ITIM. However, neither the functional substrates of these enzymes, nor the mechanism of the inhibition are fully resolved. We show here that the human FcgammaRIIb binds SHP-2 when co-clustered with the B cell receptors, whereas its synthetic P-ITIM peptide bindes SHP-2 and SHIP in lysates of the Burkitt's lymphoma cell line BL41. The P-ITIM peptide binding enhances SHP-2 activity, resulting in dephosphorylation and release of P-ITIM-bound SHIP and Shc. Moreover, P-ITIM-bound SHP-2 dephosphorylates synthetic peptides corresponding to the sites of tyrosine phosphorylation on SHIP and Shc, indicating that these proteins are its potential substrates. Thus SHP-2-induced dephosphorylation may modulate the intracellular localization and/or activity of SHIP and Shc, thereby inhibiting further activation pathways which they mediate.

Expression cloning of complementary DNA encoding three distinct isoforms of guinea pig Fc receptor for IgG1 and IgG2.

Three cDNA clones encoding the guinea pig Fc receptor for IgG1 and IgG2 (Fc gamma 1/gamma 2 R) have been isolated by an expression cloning strategy using mAb directed against the receptor. When transfected into COS-7 cells, these cDNA induced cell surface expression of the receptor that bound IgG1 and IgG2 antibodies complexed with the Ag. The ligand-binding affinities of these receptors were indistinguishable. Nucleotide sequencing has indicated that one of these clones, Fc gamma 1/gamma 2R-B1, is identical to the previously isolated cDNA clone homologous to the b2 isoform of human Fc gamma RIIB and that of murine Fc gamma RII, encoding a transmembrane glycoprotein containing two Ig-like extracellular domains. Two other clones, Fc gamma 1/gamma 2R-B2 and -B3, are identical to Fc gamma 1/gamma 2R-B1 except for an inframe insertion in the cytoplasmic region. The 48-nucleotide insertion found in Fc gamma 1/gamma 2R-B2 is identical to the first 48 nucleotides of the B3 insert that comprises 132 bp. Based on the size and homology of the inserted sequence, Fc gamma 1/gamma 2R-B2 and -B3 are identified as the homologues of the b1 isoform of human Fc gamma RIIB and that of murine Fc gamma RII, respectively. Reverse transcription and polymerase chain reaction of RNA revealed that macrophages and polymorphonuclear leukocytes expressed preferentially Fc gamma 1/gamma 2R-B1. On the other hand, B lymphocytes expressed all three forms, among which Fc gamma 1/gamma 2R-B2 and -B3 were selectively expressed in LPS-activated B lymphocytes that showed a dramatic increase in the levels of cell surface expression of Fc gamma 1/gamma 2R. These results suggest that the inserted sequences of Fc gamma 1/gamma 2R-B2 and -B3 are important to generate responses specific for B lymphocytes, which may include regulation of cell activation.