Analysis of human C4A and C4B binding to an immune complex in serum

Abstract

Previous studies using isolated complement proteins have shown that more C4A than C4B binds to certain types of immune complexes. However, the in vivo binding of the C4 isoforms to an immune complex has not been investigated in detail and may differ from events when measured with the isolated proteins. We report here the binding of C4A and C4B to an immune complex of bovine serum albumin (BSA) anti-BSA as it occurs in serum. We found that when using the isolated C4 proteins more C4A than C4B bound to the complex, but in serum similar amounts of C4A and C4B were found to bind. Furthermore, these results were not explainable by a difference in activity between isoforms. In an attempt to explain these results a number of unexpected observations were noted. First C4A, but not C4B, bound specifically to a yet unidentified 38-kD serum protein. Second, when both covalent and non-covalent binding was assessed, we found that as serum concentration increased there followed a concomitant decrease in covalent binding and C4B was more affected than C4A. The potential biological significance of these findings is discussed.

Fig. 1

Binding of C4 to bovine serum albumin (BSA) beads treated with anti-BSA, C1, and increasing concentrations of C4A-deficient or C4B-deficient serum. C4 binding was detected by flow cytometry as described in Materials and Methods. The mean ± s.e.m. are shown for three experiments. Controls included beads only; BSA-coated beads; and immune complex-coated beads without C1. Less than 2% binding to the controls occurred (data not shown). ▪, C4A; □, C4B.

Fig. 2

Representative fluorescence profile of the results presented in Fig. 1. Results are depicted as histograms with log fluorescence on the ordinate and relative cell number on the abscissa. The number indicates the percentage of beads staining positively for C4. (a) Binding results of C4 at a serum concentration of 50% to controls consisting of immune complex-coated beads without C1. (b–d) Representative of the binding of C4 to the immune complex-coated beads treated with C1 at serum concentrations of 20%, 35%, and 50%, respectively.

Fig. 3

Binding of C4 to bovine serum albumin (BSA) anti-BSA beads treated with human C1 and purified C4A3B1, C4A3, and C4B1 as measured by flow cytometry. C4 activity was determined by ¹⁴C-methylamine and equivalent units of active C4AB, C4A and C4B were used. The controls were identical to those described in Fig. 1. The mean ± s.e.m. are shown for three experiments.

Fig. 4

Binding of purified C4A3 and C4B1 to the immune complex-coated beads plus C1 in the presence of C4-depleted serum. C4 activity was determined by ¹⁴C-methylamine and equivalent units of active C4A (▪) and C4B (□) were added to C4-depleted serum. The final concentration of serum in the reaction mixture was 50%. The controls were identical to those described in Fig. 1. The mean ± s.e.m. are shown for three experiments. C4dgp, C4-deficient guinea pig.

Fig. 5

SDS–PAGE and Western blot analysis of C4 binding to immune complexes and serum proteins. Lane 1 contains serum depleted of C4. Lanes 2 and 3 contain purified C4A and C4B, respectively. Lanes 4 and 5 contain immune complex-coated beads without C1 plus serum from individuals deficient in either C4B or C4A, respectively. In the absence of C1, C4 has not been activated, as evidenced by the absence of a C4-immunoglobulin adduct. Lanes 6 and 7 contain the immune complex-coated beads with C1 plus the serum from individuals deficient in either C4B or C4A, respectively. In this case C4 has been activated, as evidenced by the presence of the C4-immunoglobulin adduct. The top-most band seen in lane 7 is probably an artefact, as it did not appear in subsequent experiments. An additional 130-kD band is seen in lane 6, but not lane 7.

Fig. 6

SDS–PAGE and Western blot analysis of C4 binding to serum proteins. Lanes 1 and 2 contain serum deficient in either C4B or C4A, respectively, after activation by the C1-containing immune complex-coated beads. For these experiments the C4 immune complex-coated beads were removed by centrifugation prior to analysis and the blots were over-developed to rule out a quantitative difference. Similar to the results shown in Fig. 5, lanes 6 and 7, lane 1 shows a distinct 130-kD band not seen in lane 2.

Fig. 7

Densitometric analysis of the covalent binding of C4A (▪) and C4B (□) to the immune complex-coated beads. Immune complex-coated beads were added to serum containing C4A or C4B and analysed for the amount of covalently bound C4 in an SDS-resistant fashion by SDS–PAGE and densitometry. The proportion of C4 α′-chain that formed adducts with the IgG was determined using NIH Image 1.54 software, as described in Materials and Methods.

Fig. 8

Representative experiment of the results presented in Fig. 7. Lanes 1–4 denote serum concentrations of 15%, 20%, 35% and 50%, respectively. The results presented in (a) were obtained using serum deficient in C4B, whereas the results in (b) were obtained using serum deficient in C4A. To maximize separation of the α′-chain and the immunoglobulin-C4 adduct the γ-chain was allowed to run off during electrophoresis. The experiment was monitored using molecular weight rainbow markers from BioRad.