Cellular immunity to cartilage aggrecan core protein in patients with rheumatoid arthritis and non-arthritic controls.

OBJECTIVE: To identify antigen(s) among purified deglycosylated aggrecan peptides spanning the chondroitin sulphate domain that may be responsible for the initiation or perpetuation of the autoimmune responses in rheumatoid arthritis (RA). METHODS: Aggrecan was purified from human articular cartilage and deglycosylated with either bacterial glycosidases or trifluoromethanesulphonic acid (TFMS). Twelve overlapping peptides (15 residues) spanning the chondroitin sulphate domain with N-terminal residues offset by three amino acids were synthesised. T cell responses to these antigens in RA patients and age matched controls were assessed in vitro by antigen specific T cell proliferation assays. RESULTS: Enzymically deglycosylated aggrecan (EDA) stimulated proliferation of T cells isolated from the peripheral blood in a greater proportion of patients with RA than controls. In a subset (12.5%) of RA patients, the magnitude of stimulation lay outside the control range. T cell proliferative responses to TFMS treated aggrecan were greater than, but well correlated with, responses to EDA. T cells from 15 patients were also stimulated with the pooled synthetic peptides. Four of seven patients who demonstrated T cell reactivity to EDA (seven of 15) also showed enhanced T cell proliferation to synthetic peptides. CONCLUSION: These data suggest that an autoantigenic T cell epitope may lie within the chondroitin sulphate domain of aggrecan.

Cellular immunity to cartilage link protein in patients with inflammatory arthritis and non-arthritic controls.

OBJECTIVES: To determine if increased T cell responses to articular cartilage link protein have any correlation with rheumatoid arthritis (RA), and if RA patients with increased responses to link protein also respond to a 17 amino acid peptide covering the 'arthritogenic' epitope in mycobacterial hsp65 which is homologous with link protein. METHODS: The reactivity of T cells from both peripheral blood and synovial fluid, to highly purified human cartilage link protein, hsp65, the 17 amino acid peptide, and bovine type II collagen was determined in patients with RA and nonarthritic controls, by measuring the rate of mononuclear cell proliferation in the presence and absence of antigen. RESULTS: Using peripheral blood mononuclear cells (PBMC), significant reactivity (stimulation index (SI) > 1.5) to link protein was found in 12 of 46 RA patients (26%), but in only four of 44 controls (9%). A greater proportion of RA patients (eight of 17:47%) were reactive to link protein when mononuclear cells from synovial fluid were tested. SI values, however, were generally low (0.5-3.1) and only one patient showed a PBMC response above a reference range of values calculated from the logarithmic values of the normal control population. No reactivity was observed against a 17 amino acid synthetic peptide including the arthritogenic epitope from the mycobacterial hsp65 to which T cell clones isolated from rats in the adjuvant arthritis model react. However, eight of nine RA patients and all of seven controls reacted to the intact hsp65. CONCLUSION: It remains unclear if T cell responses to link protein are involved in the pathogenesis of RA, but it is unlikely that T cells specific for the sequence homologous with the arthritogenic epitope in hsp65 are present in RA patients.

Solid-phase immunoassay of serum antibodies to peptides. Covalent antigen binding to adsorbed phenylalanine-lysine copolymers.

Two small peptide antigens, glucagon and enkephalin (5-L-leucine), were covalently immobilised using either glutaraldehyde or bis-(sulphosuccinimidyl) suberate to an adsorbed layer of phenylalanine-lysine copolymer (PL) or partially acetylated PL (APL) on polystyrene. Both copolymers formed stable layers, particularly APL after adsorption from solution in distilled water. Adsorption of the copolymers under these conditions and subsequent coupling of the antigens yielded solid phases with low non-specific immunoglobulin binding characteristics in an enzyme-linked immunosorbent assay (ELISA) to detect peptide-specific antibodies in rabbit serum. The signal-to-noise ratio in this ELISA was dependent on the combination of copolymer, antigen and coupling reagent employed. Removal from the solid-phase of weakly bound antigen by washing with solutions containing Tween 20 or sodium dodecyl sulphate (SDS) increased assay sensitivity, which was 2-4-fold greater than when simple antigen adsorption was utilised. In the ELISA, the coefficient of variation was lower when covalent antigen coupling was employed.

Humoral immunity to link protein in patients with inflammatory joint disease, osteoarthritis, and in non-arthritic controls.

Cartilage link protein of high purity was prepared and used in an enzyme linked immunosorbent assay (ELISA). Antibodies to link protein were sought in the sera of 98 patients with rheumatic disorders; 38 with rheumatoid arthritis (RA), 29 with osteoarthritis (OA), 13 with psoriatic arthritis (PA), nine with ankylosing spondylitis (AS), nine with systemic lupus erythematosus (SLE), and in 83 healthy controls. Antibodies were detected in all groups with the following prevalences: 21/83 normals, 9/38 RA, 7/29 OA, 7/13 PA, 3/9 AS, and 4/9 SLE. No statistically significant differences existed between the groups with regard to either prevalence or mean titre of anti-link antibodies. Serum antibodies to proteoglycan link protein appear to be no more common in patients with rheumatic disorders than in healthy controls.

Antibodies to the five histones and poly(adenosine diphosphate-ribose) in drug induced lupus: implications for pathogenesis.

Certain drugs are a frequent source of antinuclear antibody (ANA) induction, and ANA is invariably present in the few patients who progress to the drug induced lupus syndrome. This report concerns the fine specificity of the ANA response to hydralazine, penicillamine, and sulphasalazine therapy. Using highly purified individual histones in fluorimetric assays, antihistone antibodies are always detectable, often in large amounts, but the pattern of response to individual histones is variable and not drug specific. In addition to the response to the three histones H1, H2B, and H3 reminiscent of idiopathic systemic lupus erythematosus, antibody to histone H2A predominates in some drug induced cases. Contrary to previous thought, histones are not the sole target of the antinuclear response: we also demonstrate a significant correlation between ANA titre and antibody to poly(adenosine diphosphate-ribose). Like the histones, this is a macromolecule that can bind to deoxyribonucleic acid (DNA). It is proposed that drug induced damage to chromatin leads to ANA production, while drug induced impairment of complement activity may then enable these autoantibodies to mediate the lupus syndrome.

Patterns of antihistone antibody specificity in systemic rheumatic disease. I Systemic lupus erythematosus, mixed connective tissue disease, primary sicca syndrome, and rheumatoid arthritis with vasculitis.

A new fluorimetric assay was used to measure the relative amounts of antibodies to individual nuclear histones in sera from 102 patients with systemic lupus erythematosus (SLE), mixed connective tissue disease, primary sicca syndrome, and rheumatoid arthritis with vasculitis. In SLE sera, the predominant responses were to histones H-1, H-2B, and H-3, with marked elevations of binding to H-1 and H-2B in one-third of the patients, and to H-3 in one-fourth; antibodies of both the IgG and IgM classes were also detected. In a few SLE sera, the pattern of histone response differed or was restricted to 1 immunoglobulin class. In mixed connective tissue disease, only 2 of 9 sera showed elevated histone binding activity, the response being predominantly to H-3 in 1 patient and to H-1 and H-2B in the other. Binding to H-2B was also prominent in 2 of 3 patients with primary sicca syndrome. The highest antihistone reactivity and the most heterogeneous response patterns were observed in patients who had rheumatoid arthritis with vasculitis; 6 of 8 of those sera had elevated histone reactivity. In SLE, the highest histone binding results were found among patients with a history of photosensitivity. Histones are closely associated with DNA in the nucleosome, and we speculate that antihistone antibodies could arise as a result of damage to DNA, induced by drugs or irradiation.

A fluorimetric assay for human antibodies to all the histones.

We describe a fluorescence immunoassay for anti-histone antibodies in human sera. Histones are bound to immobilised tyrosine-glutamic acid copolymer on a polystyrene cuvette. With mixed histones as antigen normal sera showed low levels of antibody binding. Much higher values were obtained with some sera from rheumatoid arthritis (RA) patients positive for antinuclear antibodies, and from patients with vasculitic RA, systemic lupus erythematosus (SLE) and drug induced LE. Antibodies to all 5 individual histones were elevated in SLE and vasculitic RA patients. Preliminary results suggest that differences in response patterns may be disease related.

Binding of isolated rheumatoid factors to histone proteins and basic polycations.

A fluorimetric immunoassay has been used to assess reactivity of rheumatoid factor (RF) with both histone proteins and other basic polycations (poly-L-lysine, poly-L-ornithine, and protamine) bound to an immobilised tyrosine-glutamic acid polyanionic copolymer. Isolated RF preparations can bind to histone proteins in this assay, notably to H3 and H4 histones, and this activity was always masked in the original whole seropositive sera. Binding of isolated RF was often noted also to the other large-molecular-weight basic polycations.