The C3d receptor is a specific marker of B lymphocytes. Recently we have shown that C3d receptor activity is carried by a gp 140 membrane antigen. A polyclonal antibody has been prepared by immunizing a rabbit with highly purified gp 140 molecule isolated from membranes of the human B lymphoblastoid cell line Raji and its high specificity was previously demonstrated. We tested the effect of this antibody to the C3d receptor on the B cell proliferative response. Purified B cells from human blood were induced to proliferate by a B cell growth factor (BCGF)-containing partially purified supernatant from activated T cells. The anti-C3d receptor F(ab')2 enhanced the BCGF-dependent B cell proliferation. This effect was dose dependent, was observed in the presence of different concentrations of BCGF and did not correspond to a change in the time course of the response. The anti-C3d receptor F(ab')2 had no mitogenic effect in the absence of T cell supernatant. In contrast the undigested anti-C3d receptor IgG suppressed the BCGF-dependent B cell proliferation. These results emphasize the potentialities of anti-gp 140 F(ab')2 to explore the involvement of the C3d receptor in the regulation of B cell response to T cell products.
gp140, previously identified as a 140-kDa C3b-binding membrane glycoprotein present on Raji cell surface, was shown to be the C3dg/C3d receptor of B lymphocytes (CR2). Specific polyclonal anti-gp140, prepared by immunizing rabbits with this highly purified C3 receptor, blocked Raji cell rosettes with EC3b, EC3bi, EC3dg and EC3d, and also blocked normal lymphocyte rosettes with EC3dg and EC3d without affecting CR1 or CR3 activity. Moreover, a monoclonal anti-C3 (C3b/#130), described by others as reacting with the d region highly expressed on EC3bi, EC3dg and EC3d and poorly exposed on EC3b, completely inhibited EC3bi, EC3dg and EC3d rosettes with Raji cells, but had no effect on EC3b rosettes. Treatment of Raji cells with rabbit anti-gp140 blocked the uptake of three 125I-labeled monoclonal antibodies anti-B2, HB-5 and OKB7 reported to react with C3d-binding proteins, indicating that each of these monoclonal antibodies recognizes epitopes present on gp140. The neutrophil C3dg receptor was examined to determine its relationship to lymphocyte CR2. While neutrophil rosettes with EC3d were undetectable, a specificity for C3d was suggested by the inhibition of EC3dg rosettes by fluid phase C3d-complexes bearing no detectable C3dg. However, such neutrophil EC3dg and EC3bi rosettes were not inhibited by rabbit anti-gp140 nor an excess of anti-CR1, anti-CR2, and anti-CR3. In addition, neutrophils did not bind 125I-labeled anti-gp140, anti-B2, or HB-5. Thus, the neutrophil C3dg receptor is distinct from gp140, the lymphocyte CR2, and should be designated CR4.
In a previous report (M. Barel et al. FEBS Lett., 1981. 136: 111) using radiolabeling methods, we characterized from the membrane of the human B lymphoblastoid cell line Raji, a 140 000-Mr glycoprotein (gp140) carrying a C3b-binding activity with 125I-labeled C3b or Sepharose-bound C3b. The facts of absence on Raji cells of CR1, the C3b receptor purified from human erythrocytes, the observations made by others that H-like activity (the 150 000 Mr C3b binding serum protein) was present in Raji cells and the same molecular weight range of H and gp140, led us to investigate the relationship between both antigens. A rabbit antibody anti-5.4 was prepared against gp140, highly purified from Raji cells. However, anti-H specificities were detected in crude anti-5.4 IgG, while anti-serum H IgG did not react with gp140 antigen. The crude anti-5.4 IgG fraction, anti-gp 140 IgG or F(ab')2 and anti-H specificities present in anti-5.4 IgG, separated by absorption on Sepharose-bound H, and anti-serum H IgG were tested on Raji cells by immunofluorescence techniques, by measuring the inhibition of specific cytotoxic assays and the inhibition of specific binding of soluble or particle-bound C3b to the cell surface and on solubilized antigens by immunoblotting techniques. All the data obtained support that: (a) anti-H specificities are not shared by antibodies bearing anti-gp 140 specificities and their presence in crude anti-5.4 IgG is more likely due to a contamination by H antigen of gp 140 antigen used in the immunization process, and (b) gp 140 antigen is highly expressed on Raji cell surface, whereas H antigen can not be detected under the same conditions. Molecular analysis of gp140 and H antigens confirmed differences between both antigens in molecular weight, trypsin sensitivity and charge properties. All the results presented herein support the notion that gp140 is not identical with the H molecule and that C3b binding to gp140 is not mediated by H. The relationship between gp140 and C3 receptors described by others is discussed.
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