Ralph C. Williams

RHEUMATOID-FACTOR-REACTIVE SITES ON CH3 ESTABLISHED BY OVERLAPPING 7-MER PEPTIDE EPITOPE ANALYSIS

Polyclonal IgM rheumatoid factors (RF) from ten patients with rheumatoid arthritis and six monoclonal IgM RF were isolated from monomeric IgG affinity columns and studied for their reactivity with the entire CH3 domain of IgG synthesized as overlapping 7-mers using a pin-ELISA assay. All ten polyclonal IgM RF showed similar profiles of reactivity which included peptides with solvent accessible residues PREPQVY (residues 343-349), PQVYTLP (residues 346-352), TLPPRSE (350-356), DGSFFLY (401-407), WQQGNVF (417-423), CSVMHEG (425-430), EGLHNHY (430-436) and KSLSLSP (439-446) of the CH3 domain. Substitution of a neutral glycine or alanine for each residue within these RF-reactive epitopes indicated that tyrosine at position 349, prolines at 343, 346 and 352, glutamine 347, valine 348, threonine 350, leucine 351, arginine 354, aspartic acid 401, tyrosine 407, serine 426, histidine 429, leucine 432, tyrosine 436 and lysine 439 represented important single amino acids within CH3 for RF reactivity. Regions of CH3 primary sequence with and without the single allotype-specific amino acid substitutions of glycine for alanine 431 (Gmx) or aspartic acid for glutamic acid (356) and leucine for methionine (358) (Gma) often showed considerable differences in reactivity with individual polyclonal and monoclonal RF. However, these differences in RF reactivity did not correlate with the individual anti-Gm RF specificity. Assays using monoclonal IgM RF produced from RA synovial B cells or peripheral blood B cells frequently showed a much more restricted spectrum of reactive CH3 epitopes. 7-mer peptides representing RF-reactive sites on CH3 preincubated with polyclonal IgM RF showed strong inhibition (55-66%) of RF binding to whole IgG on the ELISA plate. These studies indicate that it is possible to define portions of the IgG CH3 domain participating in the reaction with IgM RF using reactive epitope-mapping with sequential linear peptides derived from the primary IgG CH3 sequence.

Antigenic determinants reacting with rheumatoid factor: Epitopes with different primary sequences share similar conformation

Polyclonal or monoclonal human IgM rheumatoid factors (RF) react with eight antigenic sites on the CH3 IgG domain, four sites on CH2 and two on human beta 2-microglobulin. All 14 of these RF-reactive epitopes are linear 7-11 amino acid peptides with different primary sequence. We questioned whether RF reactivity with such a variety of epitopes showing no obvious sequence homology might result from conformational similarities shared by various RF-reactive regions. Strong support for this concept was obtained using rabbit antisera as well as mouse mAbs to individual CH3, CH2 or beta 2m RF-reactive peptides. Major cross-reactivity was demonstrated between most of the 14 different CH3, CH2, or beta 2m RF-reactive peptides using individual anti-epitope antibodies. Molecular modelling studies of these peptides showed striking similarities in three-dimensional shape among many RF-reactive peptides. Main-chain atoms rather than side chains seemed to contribute most directly to conformational similarity. Molecular simulation studies on control peptides showed no conformational similarities with RF-reactive peptides. Our studies indicate that autoantibodies such as RF recognize main-chain conformations of reactive epitopes and react with a number of antigenic determinants of quite different primary sequence but similar main chain conformations.

Distribution of anti-F(ab')2 antibodies and the 16/6 idiotype in systemic lupus erythematosus (SLE) probands and kindreds.

Levels of serum anti-F(ab′)2 antibodies and expression of the 16/6 anti-DNA idiotype were studied in 103 sera from first-degree relatives of 17 systemic lupus erythematosus (SLE) kindreds. Among healthy SLE relatives, 35.9% showed anti-F(ab′)2 elevations and 24%, Id 16/6 expression. Forty-three and two-tenths percent of healthy SLE relatives with elevated anti-F(ab′)2 also showed expression of 16/6; when Id 16/6 was positive, 16 of 25 relatives (64%) showed parallel elevations of anti-F(ab′)2. However, within individual families, distribution patterns of elevated anti-F(ab′)2 and Id 16/6 often did not coincide. Affinity-isolated anti-F(ab′)2 from four members of a single SLE kindred showed relative enrichment for Id 16/6 in only two of the four individuals studied. Moreover, none of the isolated anti-F(ab′)2 antibodies within this kindred or another kindred showing 16/6 Id expression reacted directly with 16/6 Id. Our studies suggest that whereas both anti-F(ab′)2 and Id 16/6 are increased within SLE kindreds, expression of the two does not always coincide. Furthermore, anti-F(ab′)2 antibodies do not show direct reactivity with Id 16/6. A number of anti-DNA idiotypic markers may play a role in idiotypic networks among such SLE kindreds.

Urinary loss of immunoglobulin G anti-F(ab′)2 and anti-DNA antibody in systemic lupus erythematosus nephritis

The objective of this study was to determine whether the low levels of serum immunoglobulin G (IgG) anti-F(ab)2 seen in some patients with active systemic lupus erythematosus (SLE) were directly related to the deposition of antibody with this specificity in the kidney or alternatively to the urinary loss of IgG anti-F(ab)2. Serum Levels of IgG anti-F(ab)2, anti-tetanus toxoid, and anti-ds DNA antibody were measured in parallel with urinary excretion of these same 3 antibodies in 28 patients with SLE nephritis and in 28 control patients with other forms of chronic kidney disease. Low levels of both serum IgG anti-F(ab)2 or anti-tetanus antibody appeared to correlate with increased levels of urinary loss of these same antibodies in some patients with SLE and in control subjects with kidney disease. However, urinary loss could not account for low serum levels of either IgG antibody in many subjects. Quantitative 24-hour urinary losses of IgG anti-F(ab)2 and anti-DNA were much higher in patients with SLE than in control subjects with kidney disease (P < .05), whereas amounts of IgG urinary loss of anti-tetanus were similar in patients with SLE and in control subjects. In nearly 1 third of SLE nephritis patients, 13% to 53% of total excreted urinary IgG showed anti-DNA enzyme-linked-immunosorbent assay reactivity. Urinary IgG in many patients with SLE showed both anti-DNA and anti-F(ab)2 reactivity, but dual anti-DNA/F(ab)2 specificity was more pronounced in affinity-isolated serum IgG anti-DNA or anti-F(ab)2 than in excreted urinary IgG molecules. The affinity of urinary IgG for either DNA or F(ab)2 was much lower than the same antibody activities measured either in serum or in kidney biopsy eluates. When the relative affinity of anti-DNA antibody in serum, urine, and kidney biopsy eluate was measured in parallel, the highest affinity antibody was found in kidney biopsy eluates, followed by serum antibody with urine antibody affinity showing the lowest values. These findings suggest a relative concentration of the highest affinity, doubly reactive IgG anti-DNA/F(ab)2 in SLE kidney tissues during SLE nephritis and implicate this process as an important factor in ongoing tissue damage.

Molecular localization of human IgG anti-F(ab′)2 reactivity with variable- and constant-region λ light-chain epitopes

Human IgG antibodies reacting with antigenic determinants on F(ab′)2 fragments represent generic antiidiotypic antibodies present in the serum of normal individuals. Additionally, the titers of these antibodies in the sera of patients with systemic lupus erythematosus (SLE) are inversely related to disease activity. Because these autoantibodies recognize predominantly light chain-related epitopes, especially λ type, we synthesized constant (C)λ- and variable (V)λ-related overlapping 7-mer peptides on polypropylene pins to determine anti-F(ab′)2-reactive epitopes on humanλ light chains. ELISA reactivity of affinity-purified anti-F(ab′)2 antibodies obtained from normal individuals and from patients with SLE, as well as murine anti-human light-chain monoclonal antibodies specific for Cλ and Vλ subgroup-related determinants, was tested using the overlapping 7-mers of humanλ light-chain sequence. The patterns of reactivity against Cλ-related peptides were similar in both normal and SLE-derived anti-F(ab′)2 antibodies. However, reactivity profiles against Vλ-related peptides were distinctively different between the normal and the SLE-associated anti-F(ab′)2 autoantibodies. A decrease in reactivity among the SLE IgG anti-F(ab′)2 antibodies was noted for particular amino acid Vλ complementarity-determining region (CDR) residues, including glycine at positions 27 and 54, alanine at 16 and 37, and tyrosine at 28 and 91. This different pattern of reactivity from normal may indicate that in SLE there is a failure of antiidiotypic control mechanisms, as reflected by a defect in production of antibody to immunodominant Vλ CDR residues.

Monoclonal IgM Rheumatoid Factors Generated from Synovial B Cells of Rheumatoid Arthritis Patients React with β2-Microglobulin Monoclonal RF React with β2m

Four of 15 monoclonal human IgM rheumatoid factors (RF) derived from synovial B cells of patients with rheumatoid arthritis showed positive ELISA reactions with human β2-microglobulin. These findings were different from those previously noted using IgM RF derived from monoclonal Waldenstroms paraproteins or the IgM components of mixed cryoglobulins, and resembled the anti-β2microglobulin specificity of polyclonal IgM RF from patients with rheumatoid arthritis. Reactions of monoclonal IgM synovial RF with overlapping 7-mers of β2m sequence indicated major regions of positive reactivity at positions 57–64 and 89–95 which were maintained in the presence of high salt (300 mM NaCl) conditions. Glycine substitution of each residue within RF-reactive β2m regions indicated that tryptophanes at position 60 and 95, lysine at 58, phenylalanine at 62, valine at 93 and arginine at 97 constituted important single amino acids for the reactive epitopes. These findings indicate that clonally restricted human IgM RF deriv...