Juan Pedro López-Bote

Antibodies to dietary antigens in rheumatoid arthritis — possible molecular mimicry mechanism

Antibodies in serum from some patients with rheumatoid arthritis, recognize bovine albumin present in the milk, as determined by immunoprecipitation analysis from 125I-milk extracts. This antigen was also immunoprecipitated from bovine sera. These and ELISA studies showed that BSA is preferentially recognized over other proteins present in the milk. Panel studies demonstrated that although the average reactivity for BSA was high, only one third of the sera tested displayed a reactivity above the mean. The possibility of a molecular mimicry mechanism in RA between this food antigen and other human antigens was investigated. A sequence alignment analysis showed that the residues 141-157 of bovine albumin significantly differed from the corresponding fragment of human albumin, but were highly homologous with human collagen type I, C1q and vitamin D binding protein. In support of the immunogenicity of this fragment, we found that representative RA sera displayed a specific reactivity for a synthetic peptide containing the BSA residues responsible for the homology. Furthermore, most of the epitopes recognized on BSA by the RA sera seem to be conformationally dependent as heat denaturation or reduction followed by alkylation lead to a diminished recognition.

Synoviocytes type A bind exogenous antigens recognized by antibodies present in rheumatoid arthritis

The presence of antibodies in rheumatoid arthritis (RA) patients to antigens on the synoviocyte surface has recently been reported (Scand. J. Immunol. 27, 295, 1988). Here we have further characterized these antigens and found that they are exogenous proteins acquired from the bovine serum used in the culture medium. By immunoprecipitation and ELISA studies, we have identified bovine albumin and transferrin as the antigens recognized by the RA anybodies. These specificities were found not only in the sera but also in the synovial fluid from RA patients. A comparative study with a large panel or RA sera did not show a correlation in the antibody specificities for bovine albumin, bovine transferrin, or the 65‐kDa heat shock protein from Mycobacterium bovis. Similar experiments using rabbit and monkey sera as well as human synovial fluid and serum as a source of antigen did not reveal any reactivity with a highly positive RA serum. By sequence alignment, a high degree of homology between residues 142–156 from bovine albumin and residues 65 78 from human pro‐collagen α1 (I) was found. The capacity of the synoviocytes to bind exogenous antigens and the presence of antibodies to bovine proteins, normally present in the diet, suggest a role for these type A synoviocytes as well as a possible involvement of food antigens in the pathogenesis of RA.

Autoantibodies from Rheumatoid Arthritis Patients Recognize Antigens on the Synoviocyte Surface

We have found autoantibodies in the sera from rheumatoid arthritis (RA) patients which recognize two cell surface antigens of approximately 70 kDa and 28 kDa from synoviocyte extracts as detected by immunoprecipitation analysis. These polypeptides were immuno‐precipitated from extracts containing mainly macrophage‐like synoviocytes (type A) but not from extracts of homogeneous fibroblast‐like synoviocytes (type B). These autoantigens are not selectively expressed by RA synoviocytes, since both RA and non‐rheumatoid synovia were reactive for RA sera. From the panel of different RA sera tested, 64% immunoprecipitated the 70 kDa band, and 27% recognized the 28 kDa polypeptide. These differences in the specificity of the sera seemed to be related to the clinical state of the donor. The sera from patients suffering from other autoimmune diseases such as autoimmune thyroiditis and systemic lupus erythematosus (SLE) do not appear to be reactive for these specificities, but sera from patients with Sjögrens syndrome, psoriatic arthritis, and Crohns disease showed a weak cross‐reactivity with the 70 kDa polypeptide. This autoreactivity against synovial cells in RA supports the idea that these cells participate in the initial immune response of the disease.

Arthritis transferred by cells derived from pre-inflammatory rat synovium

Different cell populations isolated from rats during the period of latency of adjuvant arthritis were injected into the bloodstream of naive rats to test their ability to transfer articular disorders. Synovium-derived cells (synoviocytes) were able to induce arthritis in 3 out of 4 recipient animals, whereas peripheral blood leukocytes, peritoneal exudate macrophages, lymph node cells, synoviocyte lysates and synoviocytes from control animals were not able to do so. This model of cellular transferred arthritis is associated with antibody titres to hsp65 in rat sera. Our findings suggest a crucial role for synovial cells in the pathogenesis of adjuvant disease, which might be linked to their function as accessory cells.

Aggregated Human Immunoglobulins Bind to Modified Proteins

Human immunoglobulins treated at 55°C in vitro are able to interact with maleylated bovine serum albumin (mBSA), but not with unmodified BSA. Gel filtration experiments demonstrated that the mBSA binding is associated with a high molecular weight complex of aggregated IgG. This aggregated IgG with binding capacity for mBSA could also be generated in vitro by treatment of human IgG at 37°C or 40°C and by incubation with human neutrophils. Furthermore, IgG aggregates with binding activity for mBSA could be detected in untreated synovial fluids from rheumatoid arthritis patients, indicating that these complexes occur in vivo. The phenomenon of binding to aggregated IgG was extended to other modified proteins such as maleylated human serum albumin (mHSA), acetyl low density lipoprotein (Ac‐LDL) and BSA reacted with oxidized linolenic acid. Soluble forms of these modified proteins were able to compete for the interaction between aggregated IgG and surface‐bound mBSA. We also found that aggregated IgG enhanced the Ac‐LDL‐dependent foam cell formation. These findings suggest a role for aggregated IgG in the metabolism of oxidized proteins.

Decreased tubulin synthesis in synoviocytes from adjuvant-induced arthritic rats

The first microscopical alterations along adjuvant arthritis induction in rats seem to appear in the synovium. We have studied the protein synthesis pattern of the cells constitutively present in synovial membrane (synoviocytes) and have found an impairment of synthesis of some proteins when synoviocytes are derived from adjuvant arthritic rats. One of these polypeptides was identified as beta tubulin by two-dimensional gel electrophoresis, a membrane transfer assay using a specific monoclonal antibody and peptide mapping. We postulate that a repressed synthesis of tubulin may be an initial step in the triggering of the disease, since the effect was evident at pre-arthritic stages, when infiltration by inflammatory cells had not yet occurred.