Effect of circulating immune complexes on the binding of rheumatoid factor to histones

Abstract

Objective: To determine whether the reaction of rheumatoid factor (RF) with solid phase histone is due to the simultaneous presence of circulating immune complexes (CICs) or aggregated IgG.

Methods: Serum samples from 56 patients with seropositive rheumatoid arthritis (RA) and 50 random blood bank donors were used. Binding of immunoglobulins to histone was determined by enzyme linked immunosorbent assay (ELISA) and by western blots. Aggregated IgG was obtained by heating at 61(o)C for 30 minutes.

Results: Among the RA sera tested by ELISA, 54% were positive for histone binding by IgM, IgG, or IgA and 20% by IgM only. Heating of normal sera caused a significant enhancement in the binding of IgG to histone (p<0.001). This binding had a non-cognate behaviour-that is, it was destroyed by pepsin treatment of serum and was not significantly inhibited by competition with free histone. The same behaviour was seen for IgM, IgG, and IgA binding from RA sera. However, cognate IgG antibody binding to histone was inhibited by free histone and was resistant to pepsin digestion. Addition of heat aggregated IgG to RA sera or pretreatment of histone with aggregated IgG caused a significant increase in IgM binding to histone.

Conclusion: IgM, IgG, and IgA RF bind to solid phase histone as a result of attachment to histone of immune complexes or aggregated IgG and not as a result of a cognate reaction with histone.

Figure 1

Reaction with solid phase histone of immunoglobulin from control and RA sera. Scattergram of the net A₄₅₀ values yielded by ELISA on histone coated wells, reacted with sera from 50 blood bank donors (BBD, open symbols) and 56 patients with rheumatoid arthritis (RA, solid symbols). Enzyme-conjugate was isotype-specific anti-IgM (A), anti-IgG (B), or anti-IgA (C).

Figure 2

Effect of serum heating at 61°C on the binding of IgM RF from RA sera to histone and to other nuclear antigens. Scattergram of the net A₄₅₀ yielded on ELISA by serum samples from blood bank donors (A) and patients with rheumatoid arthritis sera (B): non-heated (1, 3) or heated at 61°C for 30 minutes (2, 4, 5, 6, 7). Wells were coated with histones (1-4), ssDNA (5), nRNP/Sm (6) or were blanks (7, raw A₄₅₀). Isotype-specific enzyme-conjugate was anti-IgM.

Figure 3

Effect of the pretreatment of histone coated wells with aggregated serum IgG on the binding to histone of IgM from RA sera. A₄₅₀ yielded by blank plates (shaded bars) and by wells coated with histone (solid bars) of normal serum heated at 61°C for 30 minutes (A, B); non-heated RA serum (C, D); RA serum on wells preincubated for 30 minutes with heated normal serum (E, F) or non-heated normal serum (G, H); and by non-heated normal serum (I, J), and enzyme conjugates (K, L) as controls. RA serum was diluted 1/100 (C, E, G), or 1/1000 (D, F, H). Reactions were shown with isotype-specific enzyme-antibody conjugates: goat antihuman Fcγ chain-specific (lanes A, I, K), or rabbit antihuman Fc_5µ (lanes B, C, D, E, F, G, H, J, L) antibodies.

Figure 4

Effect of addition of heat aggregated serum IgG to RA serum on the binding to histone of IgM. Net A₄₅₀ (SD) yielded by mixtures of RA serum and non-heated normal serum (circles), and RA serum and heated normal serum (squares). The proportion of RA serum in the mix was variable (%RA serum). Binding was shown by anti-IgG enzyme-conjugate (solid symbols, solid line) or anti-IgM (open symbols, dashed line).

Figure 5

Effect of pepsin digestion on the binding of heat aggregated IgG and of antihistone antibodies to histone as determined by western blots. Proteins on the membrane were prestained molecular weight standards (lane M); a mix of all histone proteins (lane 1); H2A and H2B (lane 2); H3 and H4 (lane 3). The different panels were reacted with different primary reagents: heated normal serum (panel A); heated normal serum subjected to pepsin digestion (panel B); RA serum (specimen R27) (panel C); RA serum subjected to pepsin digestion (panel D); serum containing antihistone antibody (spiked specimen HS-1) (panel E); and serum containing antihistone antibody subjected to pepsin digestion (panel F).

Figure 6

Proportion of 40 RA serum samples with IgG that reacted with C1q or with histone, or both in ELISA. The darker zones represent discrepant specimens; the lighter overlap zones represent the samples positive or negative for both C1q and histone.