Characterization of complement receptors.
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Animals↗
Biomedical subjects
H H Mussel
Publications and source records attributed to H H Mussel.
Isolation and characterization of the C3b-binding entity of C3b-receptor from human erythrocytes.
Applying 2 M KBr, membranes of Ehu were solubilized. By C3-affinity chromatography an activity could be isolated that inhibited the immune adherence reaction and C3b-dependent rosette formation. Since this material did not agglutinate EAC14oxy23b we termed it monovalent C3b receptor (mC3bR). PAGE and SDS-PAGE and staining with Coomassie brilliant blue and PAS reagent revealed a single glycoprotein band with a mol. wt. of 55,000-60,000 daltons and an electrophoretic mobility comparable to ovalbumin. This mC3bR proved to be antigenetically related to gp 205 [17]. The potential of mC3bR to react with C3b-carrying particles was not destroyed by heat and trypsin treatment but by neuraminidase or periodic acid treatment suggesting that mC3bR reacted by its carbohydrate moiety with C3b. As by mC3bR, immune adherence could be inhibited by D-glucose and D-galactose but not by their optical antipodes, L-glucose and L-galactose.
Animals↗
Differentiation of C3b receptors on human lymphocytes, phagocytes, erythrocytes and renal glomerulus cells by monoclonal antibodies.
C3b receptor protein was purified form human erythrocytes by 2 M KBr solubilization and affinity chromatography on C3-coated sepharose. This material served as antigen for raising monoclonal antibodies. To investigate the distribution and antigenetic relationship between the receptors for C3b on human erythrocytes, lymphoid and phagocytic cells, as well as kidney cells three monoclonal antibodies were selected which inhibited the binding of EAC14 degrees 23b to complement receptor-bearing cells. This could be shown for human erythrocytes by inhibiting the immune adherence reaction, for tonsil lymphocytes, Raji cells, and guinea-pig spleen cells by inhibition of rosette formation of these cells with EAC14 degrees 23b, and for human renal glomeruli by blocking of the the adherence of EAC14 degrees 23b to kidney sections. In contrast, these monoclonal antibodies were not capable of inhibiting rosette formation of human granulocytes and monocytes with EAC14 degrees 23b. The antibodies only interfered with the rosette formation, of EAC14 degrees 23bi and EAC14 degrees 23d with Raji cells and tonsil lymphocytes-if at all-at high concentrations, whereas the rosette formation of Raji cells and tonsil lymphocytes with EAC14 degrees 23b was influenced by supernatants of the selected clones up to a dilution of 1:10(3) to 1:10(5).
Animals↗
Role of beta 1H for the binding of C3b-coated particles to human lymphoid and phagocytic cells.
Coating of EAC14oxy23b with highly purified human serum beta 1H globulin (beta 1H) led to acceleration of rosette formation with human peripheral blood lymphocytes (PBL), tonsil lymphocytes, B lymphoblastoid (Raji) cells, granulocytes and monocytes. This reaction was discernible from C3bi-dependent rosette formation. Enhancement of rosette formation of C3b cells by beta 1H was most effective at limiting amounts of C3 per EAC14oxy23b. The beta 1H effect was not due to trace contamination with C3b inactivator. beta 1H-dependent rosette formation with the various lymphoid and phagocytic cells could be suppressed by the F(ab')2 fragment of anti-beta 1H suggesting beta 1H-mediated binding of beta 1H-coated particles to complement receptor-positive (CR+) cells. In turn, binding of fluid-phase beta 1H to lymphoid and phagocytic cells could be demonstrated by fluorescence and by 14C-labeled beta 1H. In addition, the functional status of these cells with respect to their receptor reactivity was altered. Treatment of normal lymphocytes (PBL, tonsil lymphocytes) and of granulocytes with beta 1H improved their rosette formation with both EAC14oxy23b and EAC14oxy23b-beta 1H. The reaction of monocytes was hardly affected. The beta 1H effect on Raji cells resulted in reduced rosette formation with EAC14oxy23b-beta 1H, while binding of EAC14oxy23b remained unchanged. These results suggest the presence of sites on CR+ cells, to which soluble and particle-bound beta 1H can bind, leading to alteration of the functional status of the cells. In all likelihood, EAC14oxy23bi can attach to the beta 1H-binding sites on CR+ cells.
Animals↗
beta 1H-hemagglutination assay: a highly sensitive test for human beta 1H and its uses.
Particle-bound human C3b (EAC14oxy23b) serves as the carrier for the detection of biologically active human beta 1 H by IgG-anti-beta 1H. This hemagglutination assay is highly sensitive (greater than or equal to 2 p of beta 1H/40 microliters), easy to perform, rapid, highly reproducible, and needs no expensive laboratory equipment. All reagents are commercially available. The correlation of the beta 1H-hemagglutination assay is applicable for the detection of beta 1H in various buffer systems and may be used for the detection of beta 1H in serum or fractions thereof. It is a very useful tool for the detection of trace contamination of beta 1H in serum fractions, particularly in complement preparations.
Animals↗
Description of a simple, specific, and sensitive test for the detection of detergent-solubilized C3b receptor.
The C3b receptor was isolated from detergent-solubilized human erythrocyte membranes by a previously described technique (1). The receptor glycoprotein was shown to enhance EAC14oxy23b rosette formation with Raji lymphoblastoid cells. This provided a specific and sensitive test to detect the solubilized C3b receptor either in crude or highly purified form. The property of the C3b receptor tested by this assay appears to be analogous to properties of beta 1H.
Burkitt Lymphoma↗
beta 1H stimulates mouse-spleen B lymphocytes as demonstrated by increased thymidine incorporation and formation of B cell blasts.
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Qualitative and quantitative assessment of C3-receptor reactivities on lymphoid and phagocytic cells.
Highly purified human C3, free of C5 and beta 1H, was used to prepare EAC14oxy23b, EAC14oxy23b' (C3b cells treated with purified C3bINA and beta 1H) and EAC14oxy23d (C3b' cells treated with trypsin). These intermediates were used to assess by rosette formation C3-receptor activity on various cells. The number of cell-bound C3 per C3b, C3b', and C3d cell was quantified by applying 14C-formaldehyde-labeled C3. Human PBL reacted to about the same degree with C3b, C3b', and C3d cells, whereas monocyte-free PBL enriched for B cells interacted preferentially with the C3b' and the C3d cells; human tonsil lymphocytes behaved similarly. The reaction of Raji cells was clearly assessable with C3b cells and was accelerated with C3b' and C3d cells. Daudi cells reacted with C3b' and C3d cells only, in comparison to Raji cells with a much lower activity. Human granulocytes reacted equally well with C3b and C3b' cells, but towards C3d cells they were almost unreactive. Human monocytes formed rosettes with C3b cells, and at a lower level, rosettes with C3b' cells. C3d cells were unreactive. Similar reaction patterns were obtained with guinea pig leukocytes, whereas mouse leukocytes were totally different, since peritoneal macrophages only formed rosettes with human C3b' cells.
Animals↗