FcRγ Chain Research Articles

The Role of the Human Fc Receptor FcγRIIA in the Immune Clearance of Platelets: A Transgenic Mouse Model

AbstractIn humans, the Fc receptor for IgG, FcγRIIA, is expressed on macrophages and platelets and may play an important role in the pathophysiology of immune-mediated thrombocytopenia. Mice lack the genetic equivalent of human FcγRIIA. To better understand the role of FcγRIIA in vivo, FcγRIIA transgenic mice were generated and characterized. One transgenic mouse line expressed FcγRIIA on platelets and macrophages at levels equivalent to human cells, and cross-linking FcγRIIA on these platelets induced platelet aggregation. Immune-mediated thrombocytopenia in this transgenic line was studied using i.v. and i.p. administration of anti-mouse platelet Ab. In comparison with matched wild-type littermates that are negative for the FcγRIIA transgene, Ab-mediated thrombocytopenia was significantly more severe in the FcγRIIA transgenic mice. In contrast, FcR γ-chain knockout mice that lack functional expression of the Fc receptors FcγRI and FcγRIII on splenic macrophages did not demonstrate Ab-mediated thrombocytopenia. We generated FcγRIIA transgenic × FcR γ-chain knockout mice to examine the role of FcγRIIA in immune clearance in the absence of functional FcγRI and FcγRIII. In FcγRIIA transgenic × FcR γ-chain knockout mice, severe immune thrombocytopenia mediated by FcγRIIA was observed. These results demonstrate that FcγRIIA does not require the FcR γ-chain for expression or function in vivo. Furthermore, taken together, the data suggest that the human Fc receptor FcγRIIA plays a significant role in the immune clearance of platelets in vivo.

The alternatively spliced CD64 transcript FcγRIb2 does not specify a surface-expressed isoform

Three highly homologous genes (A, B and C) and six transcripts have been identified for the class I human IgG receptor (CD64). The hFcγRIa1 isoform encodes the prototypic high-affinity receptor for IgG. The alternatively spliced hFcγRIb2 transcript was postulated to exist as a second surface-expressed CD64 isoform on myeloid cells. In this report we assessed this proposed role for hFcγRIb2 in detail. As CD64 monoclonal antibodies might not recognize hFcγRIb2, we tagged the receptor with an hemagglutinin tag and transfected hFcγRIb2tag in the presence of FcR γ-chain into IIA1.6 cells. Both transcript and protein of hFcγRIb2tag were clearly present in transfectants. However, in contrast to the (control) hFcγRIa1tag, no surface expression of hFcγRIb2tag was detectable with a tag-specific monoclonal antibody. Confocal scan laser microscopy revealed hFcγRIb2tag to be retained in the endoplasmic reticulum, resulting in absent plasma membrane expression. These results show hFcγRIb2 neither to be surface expressed, nor to represent a separate CD64 isoform. This finding, furthermore, implicates that other FcR transcripts defined at the mRNA level may not represent true FcR isoforms either.

Fc Receptors Are Not Required for Antibody-Mediated Protection Against Lethal Malaria Challenge in a Mouse Model

The mechanisms by which Abs mediate protection during blood-stage malaria infections is controversial, with some evidence pointing to the direct effect of Abs on parasite invasion and growth, while other studies suggest that Abs act in cooperation with monocytes to achieve parasite inhibition. To determine whether the effector phase of protection in vivo to the rodent parasite Plasmodium yoelii yoelii requires Fc receptor bearing cells, we passively transferred immune sera into FcR gamma-chain knockout mice. Inflammatory macrophages from these knockout mice were unable to mediate phagocytosis or Ab-dependent cell-mediated cytotoxicity (ADCC) through Fc gamma RI, Fc gamma RII, or Fc gamma RIII. Passive transfer of either P. y. yoelii hyperimmune sera or anti-GST-PYC2 sera directed to the major merozoite surface protein (MSP-1) of this parasite enabled both BALB/cByJ mice and FcR gamma-chain-deficient mice to resist lethal P. y. yoelii 17XL (Py17XL) challenge. mAb302, a protective IgG3 Ab, also passively protected both strains of mice. Most of these samples contain Ab isotypes that would not be able to protect mice if their protective effects required Ab-dependent cell-mediated cytotoxicity. These results establish that, in this infection, protection is directly mediated by Abs and does not require the participation of Fc receptors.

Immune-mediated Thrombocytopenia is More Severe in Human FcγRIIa Transgenic Mice than in Wild-type Mice • 782

Human FcγRIIa is the platelet receptor for IgG. FcγRIIa is also expressed in man along with the FcγRI/γ and FcγRIIa/γ complexes on spleen macrophages. Mice lack an equivalent of FcγRIIa although it is likely to be a key receptor in immune-mediated thrombocytopenias. We established transgenic mouse lines that express human platelet FcγRIIa at levels equivalent to human platelets in order to determine if immune-mediated thrombocytopenia would be more severe in these transgenic mice which more fully recapitulate the human Fc receptor endowment. We showed that the human receptor is functional on mouse platelets using platelet aggregation stimulated by antibody in vitro. To determine whether this receptor was functional in vivo, we injected mice from this transgenic line with 70 μg of 4A5 antibody, a previously characterized rat anti-mouse platelet antibody found to cause moderate thrombocytopenia in wild type mice. We compared the transgenic mice to wild type mice. We followed the time course of platelet counts in mice injected with saline or isotype control (n=6 each) versus wild type mice (n=6) and FcγRIIa transgenic mice (n=9) receiving a single intraperitoneal dose of antibody on day 0. Intravenous injection gave comparable nadir platelet counts, with more rapid kinetics as expected. The average platelet count in wild type mice decreased to a nadir of 0.60 × 106 / μL on day 6 from a pre-treatment count of 1.13 × 106/ μL following injection of the antibody, in keeping with literature results on this antibody. In FcγRIIa transgenic mice there was a greater response to 4A5. The average count reached a nadir on day 6 after injection of 0.12 × 106 / μL (n = 9; statistically significantly different from wild-type, p < 0.05), while the pre-treatment and recovery counts of the transgenic mice were similar to the controls, with platelet counts of 0.96 × 106 / μL. In FcR γ-chain knockout mice which express no FcγRI or RIII, no 4A5-induced thrombocytopenia was seen (n = 6). Human FcγRIIa transgenic mice have more severe antibody-mediated thrombocytopenia in vivo, suggesting that the human FcγRIIa receptor is functional in vivo in these transgenic mice and contributes to the increased clearance of platelets. Our human FcγRIIa transgenic mice will be a valuable model of the pathophysiology and therapy of human immune-mediated thrombocytopenia disorders, including neonatal alloimmune thrombocytopenia.

Association with FcRgamma is essential for activation signal through NKR-P1 (CD161) in natural killer (NK) cells and NK1.1+ T cells.

Natural killer (NK) cells exhibit cytotoxicity against variety of tumor cells and virus-infected cells without prior sensitization and represent unique lymphocytes involved in primary host defense. NKR-P1 is thought to be one of NK receptors mediating activation signals because cross-linking of NKR-P1 activates NK cells to exhibit cytotoxicity and IFN-gamma production. However, molecular mechanism of NK cell activation via NKR-P1 is not well elucidated. In this study, we analyzed the cell surface complex associated with NKR-P1 on NK cells and found that NKR-P1 associates with the FcRgamma chain which is an essential component of Fc receptors for IgG and IgE. The association between FcRgamma and NKR-P1 is independent of Fc receptor complexes. Furthermore, NK cells from FcRgamma-deficient mice did not show cytotoxicity or IFN-gamma production upon NKR-P1 cross-linking. Similarly, NK1.1+ T cells from FcRgamma-deficient mice did not produce IFN-gamma upon NKR-P1 crosslinking. These findings demonstrate that the FcRgamma chain plays an important role in activation of NK cells via the NKR-P1 molecule.

Development and function of T cells in T cell antigen receptor/CD3 zeta knockout mice reconstituted with Fc epsilon RI gamma.

Engagement of alpha-beta T cell receptors (TCRs) induces many events in the T cells bearing them. The proteins that transduce these signals to the inside of cells are the TCR-associated CD3 polypeptides and zeta-zeta or zeta-eta dimers. Previous experiments using knockout (KO) mice that lacked zeta (zeta KO) showed that zeta is required for good surface expression of TCRs on almost all T cells and for normal T cell development. Surprisingly, however, in zeta KO mice, a subset of T cells in the gut of both zeta KO and normal mice bore nearly normal levels of TCR on its surface. This was because zeta was replaced by the Fc epsilon RI gamma (FcR gamma). These cells were relatively nonreactive to stimuli via their TCRs. In addition, a previous report showed that zeta replacement by the FcR gamma chain also might occur on T cells in mice bearing tumors long term. Again, these T cells were nonreactive. To understand the consequences of zeta substitution by FcR gamma for T cell development and function in vivo, we produced zeta KO mice expressing FcR gamma in all of their T cells (FcR gamma TG zeta KO mice). In these mice, TCR expression on immature thymocytes was only slightly reduced compared with controls, and thymocyte selection occurred normally and gave rise to functional, mature T cells. Therefore, the nonreactivity of the FcR gamma + lymphocytes in the gut or in tumor-bearing mice must be caused by some other phenomenon. Unexpectedly, the TCR levels of mature T cells in FcR gamma TG zeta KO mice were lower than those of controls. This was particularly true for the CD4+ T cells. We conclude that FcR gamma can replace the functions of zeta in T cell development in vivo but that TCR/CD3 complexes associated with FcR gamma rather than zeta are less well expressed on cells. Also, these results revealed a difference in the regulation of expression of the TCR/CD3 complex on CD4+ and CD8+ T cells.

Expression and physical association of FcαR and FcRγ chain in human mesangial cells

Expression and physical association of FcαR and FcRγ chain in human mesangial cells

FcR γ-Chain Is Essential for Both Surface Expression and Function of Human FcγRI (CD64) In Vivo

Most Ig receptors exist as hetero-oligomeric complexes with separate ligand binding (alpha) and signal transducing (beta, gamma, or zeta) subunits. For Fc gamma RIIIa and Fc epsilon RI, association with the FcR gamma-chain is essential for surface expression. However, the human high affinity IgG receptor, hFc gamma RI, was found to be surface-expressed by itself in transient transfection models. We have now analyzed the integrity of hFc gamma RI expression in more detail in stable transfectants. In vitro we noted that, in the absence of FcR gamma-chain, surface expression of hFc gamma RI rapidly declined to background levels, in both IIA1.6 B cells and NIH3T3 fibroblasts. The effect of FcR gamma-chain on hFc gamma RI surface expression in vivo was evaluated by using two newly generated transgenic mouse lines, selectively expressing hFc gamma RI on myeloid cells. These transgenic mice were crossed with FcR gamma-chain-deficient mice. Analysis of blood monocytes and peritoneal macrophages showed that surface expression of hFc gamma RI was reduced by approximately 80%. The remaining approximately 20% of receptors were still capable of binding IgG-opsonized RBC, suggesting FcR gamma-chain not to be critical for hFc gamma RI ligand-binding capacity. Importantly, however, hFc gamma RI signaling capacity was lost in FcR gamma-chain-deficient cells. No phagocytosis could be observed using either ligand sensitized (EA-IgG2a) or CD64-targeted erythrocytes (using a bispecific antibody) in both hFc gamma RI transgenic lines. This documents the FcR gamma-chain to be indispensable for both surface membrane expression and function of human Fc gamma RI in vivo.

Physical association of Fc receptor γ chain homodimer with IgA receptor

The receptor for IgA, FcαR, consists of one IgA-binding α chain and a signal-transducing dimeric FcRγ chain. Immunoprecipitation with an anti-FcαRα chain monoclonal antibody from the lysates of U937 cells (human monocytic cell line) revealed an association of 20 kd (unreduced) and 10 kd (reduced) molecules to FcαRα chain on sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). These molecules were confirmed to be FcRγ chain by immunoblotting probed with anti-FcRγ chain antibody. A serial immunoprecipitation with both antibodies further ascertained the FcRγ association with FcαRα. The lysates precleared with anti-FcRγ antibody were subjected to the second immunoprecipitation with an anti-FcαRα monoclonal antibody. By this preclearance, FcRγ disappeared, and the FcαRα appeared to be significantly decreased on SDS-PAGE, suggesting that a part of FcαRα was co-absorbed with FcRγ. Therefore, it may be likely that FcαR is expressed in two forms, namely, with or without FcRγ. We next reconstituted the FcαRα-FcRγ association by introducing both chains into host cells. The expression of FcαRα was achieved by introducing FcαRα alone, and the co-introduction of FcRγ did not enhance FcαRα expression on the cell surface, suggesting again the occurrence of the two forms of FcαR. (J A LLERGY C LIN I MMUNOL 1995;96:1152-60.)

Differential contribution of the FcR gamma chain to the surface expression of the T cell receptor among T cells localized in epithelia: analysis of FcR gamma-deficient mice.

The function of the Fc receptors gamma chain (FcR gamma) for the expression of the T cell receptor (TCR) complex and for T cell development, especially for T cells localized in epithelia, was investigated by analyzing FcR gamma-deficient mice. In wild-type mice, CD8 alpha alpha + beta -TCR alpha beta + T cells of intestinal intraepithelial lymphocytes (i-IEL) utilized CD3 zeta homodimers and zeta-FcR gamma heterodimers, whereas CD8 alpha alpha + beta -TCR gamma delta + i-IEL used zeta-FcR gamma and FcR gamma homodimers in the TCR complex. On the other hand, these T cells in FcR gamma-deficient mice contained only zeta homodimers. The surface expression of the TCR complex was reduced in CD8 alpha alpha + beta -i-IEL and dendritic epidermal T cells (DETC) in these mice, whereas the development of these T cells was normal. The degree of reduction appeared to depend on the expression level of FcR gamma. In contrast to these populations, TCR gamma delta + intraepithelial T cells in reproductive organs (r-IEL) were dramatically decreased, suggesting that the development of r-IEL is FcR gamma-dependent, probably due to the predominant usage of FcR gamma homodimers in the TCR complex. These results indicate that the FcR gamma chain contributes differently to the TCR expression and to the development of T cells localized in epithelia.

Colour perception in pseudophakia.

Minor differences in colour perception between pseudophakic, phakic, and spectacle aphakic eyes were identified by the Pickford-Nicholson anomaloscope and the Farnsworth-Munsell 100-hue test. The results suggest that pseudophakic eyes are more sensitive to red and less sensitive to blue than aphakic eyes corrected with spectacles. Spectrophotometer measurements reveal that the Rayner-Pearce posterior chamber intraocular lens used in this study transmits an evenly balanced colour spectrum, whereas an aphakic spectacle lens exhibits significant colour distortion, reducing the red and enhancing the blue transmission. This distortion may possibly be attributed to the increased chromatic aberration in the spectacle lens compared with the intraocular lens.