Protective effect of Tetracera scandens L. leaf extract against CCl

Objective: To investigate the protective potential of ethanolic extracts of Tetracera scandens L. (T. scandens) against CCl

p205 is a major target of autoreactive T cells in rheumatoid arthritis.

OBJECTIVE: The p205 autoantigen and interleukin-2 (IL-2) function synergistically to stimulate T lymphocytes from patients with rheumatoid arthritis (RA), and a p205-derived amino acid sequence is identical to an immunoglobulin sequence located within a domain that is reactive with rheumatoid factors (RF). This study was conducted to analyze in detail the T cell immune response against p205 and to investigate whether immunity to p205 may play a role in T cell-mediated immunopathology in active RA. METHODS: Cibachron blue, protein A-Sepharose, and gel filtration on Sephacryl were used successively to enrich p205 from synovial fluid (SF). T lymphocytes from RA patients were isolated from the peripheral blood (PB), lymph nodes, and SF, and p205 and peptides derived from known sequences were assessed by T cell proliferation assays in the presence of IL-2. RESULTS: P205-specific proliferation of T cells was observed in PB as well as in SF. When p205 was isolated from RA SF, proliferation of RA T cells peaked on day 3. With p205 purified from SF from trauma patients, there was a significant shift of the maximum T cell proliferation to day 8. T cells were of CD4 or CD8 phenotype, and B cells did not proliferate to a significant degree. The T cell response to p205 was always higher for SF mononuclear cells (SFMC) compared with PBMC (P < 0.001). In 1 RA patient who underwent repeated leukapheresis, this led to a reproducible decline in p205-specific T cell proliferation to control levels. PB T cells specifically proliferating in response to p205 were detected in 20 of 32 RA patients (63%). Of 26 patients with other inflammatory rheumatic diseases, only 1 showed a minor response to p205, while normal donors did not demonstrate a significant T cell proliferation. A synthetic p205-derived peptide, with an amino acid sequence identical to an immunoglobulin sequence located in the area where RF binds, was reactive with T cells from RA patients. CONCLUSION: P205 appears to be a major target of autoreactive T cells in RA. P205-specific T cells are primed and more abundant at the site of inflammation. As a T cell target in RA, p205 may well be an antigen involved in the initiation of RF production.

HLA class II transgenic mice: models of the human CD4+ T-cell immune response.

This review examines the field of current HLA class II transgenic mouse models and the individual approaches applied in production of these mice. The majority of these mice have been created with the objective of obtaining a disease model with clinical features mimicking human autoimmune disease. The development process of a different type of HLA class II transgenic mice, which are designed to function as a substitute for a normal human immune system in studies of human autoantigens, is described. Several HLA-DR4 transgenic lines with normally expressed HLA-DR4 molecules have been produced. To obtain adequate positive selection of the HLA-DR4-restricted CD4+ T-cell repertoire in these mice it is essential both to introduce a human CD4 transgene, and to delete the murine major histocompatibility complex (MHC) class II molecules. These HLA-DR4 transgenic mice have been used to determine the immunogenic CD4+ T-cell epitopes of several human autoantigenic proteins.

Biochemical characterization and microsequencing of a 205-kDa synovial protein stimulatory for T cells and reactive with rheumatoid factor containing sera.

Synovial fluid (SF) was found to possess stimulatory capacity for the proliferation of T cell clones derived from patients with rheumatoid arthritis (RA) when cultured together with IL-2. Using chromatography technique and gel electrophoresis, a synovial fluid protein with an apparent m.w. of 205 kDa (p205) was isolated that demonstrated a bioactivity analogous to that obtained with native synovial fluid. After electroelution, p205 dissociated into 70-kDa fragment(s). Upon IEF, it appeared as a single band with an isoelectric point of 6.5, suggesting a noncovalently bound trimer complex. Amino acid sequences of the whole protein and of tryptic peptides were determined by N terminal sequencing. The N terminal amino acid sequence of the 70-kDa fragment and of the tryptic peptides showed no identity to recently described protein sequences. One peptide matched, in 11 amino acid residues, with the human IgG1-4 constant heavy chain and rheumatoid factor (RF) binding region. The p205 induced the proliferation of peripheral blood T cells and long term T cell cultures that had been raised by alternate stimulation with IL-2 and p205. In a similar approach, synovial lining cells were shown to release a protein with biochemical characteristics similar to the synovial fluid-derived p205. Western blot analysis revealed the binding of RF-containing sera to p205, which was diminished by absorption with an RF reagent. These observations suggest that p205 is expressed by synovial cells and may be a target for T and B cells in RA.

Immunological and functional properties of the acetylcholine receptor expressed on the human cell line TE671.

In the first part of the present study we compared the antigenicity of affinity-purified acetylcholine receptors from the cell line TE671 and from human skeletal muscle. The reactivities of the two acetylcholine receptor preparations showed a strong correlation (r = 0.96) in a radioimmunoassay using sera from myasthenia gravis patients. In additional functional studies, carbamylcholine stimulated cAMP production in TE671 cells to 130%. This increase was even more pronounced when TE671 cells were grown in the presence of dexamethasone. alpha-Bungarotoxin completely blocked this carbamylcholine-induced cAMP increase. Using the Ca2+ indicator, indo-1, it was shown that intracellular Ca2+ concentrations ([Ca2+]i) were elevated in TE671 cells after stimulation with carbamylcholine. This effect was also completely blocked by alpha-bungarotoxin. To test the functional activity of autoantibodies against the acetylcholine receptor, TE671 cells were preincubated with sera from myasthenia gravis patients. In one third of sera a significant inhibition of the agonist-stimulated [Ca2+]i increase was detected, possibly caused by antibodies directed to functionally important areas of the acetylcholine receptor. There was no correlation between the inhibition rate of [Ca2+]i and anti-acetylcholine receptor antibody titres in these patient sera.

Modulation of monocyte activation in patients with rheumatoid arthritis by leukapheresis therapy.

One of the hallmarks in rheumatoid arthritis (RA) is the intense activation of the monocyte-macrophage system. In the present investigation, the modulation of blood monocyte activation was studied with regard to the secretion of cytokines and inflammatory mediators, and to the expression of cytokine receptors. Patients with severe active RA underwent repeated leukapheresis procedures that removed all circulating monocytes. Highly enriched monocyte preparations from the first and third leukapheresis were studied. There were striking differences between the two monocyte populations. Cells obtained from the first leukapheresis constitutively released large amounts of prostaglandin E2 (PGE2), neopterin, interleukin 1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha). In particular, IL-1 beta and neopterin production were further enhanced by stimulation with either interferon-gamma (IFN-gamma) or TNF-alpha without a synergistic effect. In contrast, cells derived from the third leukapheresis procedure showed a close to normal activation status with only low levels of cytokine and mediator production as well as a reduced response to cytokine stimulation. The number of the receptors for IFN-gamma and TNF-alpha was not changed between first and third leukapheresis. However, TNF-binding capacity was only detectable upon acid treatment of freshly isolated monocytes. A further upregulation was noted upon 24 h in vitro culture, suggesting occupation of membrane receptors and receptor down-regulation by endogenously produced TNF-alpha. Northern blot analysis of cytokine gene expression was in good correlation with the amount of mediators determined on the protein level. These data indicate that cells of the monocyte-macrophage system are already highly activated in the peripheral blood in RA patients with active disease. These cells can be efficiently removed by repeated leukapheresis and are replenished by monocytes that have, with respect to cytokine and mediator production, a considerably lower activation status.

Cellular immune response toward human articular chondrocytes. T cell reactivities against chondrocyte and fibroblast membranes in destructive joint diseases.

Articular cartilage is one of the major targets in destructive joint diseases in humans. We studied cellular immune reactions against cartilage cell-surface membranes, because it has recently been suggested that these represent possible antigenic structures, based upon the observation of autoantibodies with this specificity in certain joint diseases. A striking T cell reactivity toward chondrocyte membranes was found both in blood and synovial tissue from patients with rheumatoid arthritis. This reactivity was strongly dependent on the presence of monocytes and had all the characteristics of an antigen-driven process. Clonal analysis demonstrated high precursor frequencies in peripheral blood T cells that were reactive against chondrocyte membranes. This response to chondrocyte membranes greatly exceeded the T cell stimulation induced by membranes from other sources such as fibroblasts or epithelial cells. In contrast to patients with rheumatoid arthritis, individuals with osteoarthritis showed a strong peripheral blood and synovial fluid T cell response not only to chondrocyte membranes, but also to fibroblast membrane material. However, there was no reactivity to epithelial cell membranes. Normal donors generally did not show significant responses to any membrane preparation. These data indicate that there is a strong T cell reactivity toward chondrocyte membranes in destructive joint disorders, and this may significantly contribute to the pathogenetic processes that occur in these diseases.

Stimulation of rheumatoid synovial and blood T cells and lines by synovial fluid and interleukin-2: characterization of clones and recognition of a co-stimulatory effect.

Rheumatoid arthritis (RA) is characterized by the presence of interleukin-2 (Il-2) receptor-positive T cells in the peripheral blood and synovial compartments. Utilizing the limiting dilution technique, the precursor frequencies of Il-2 responsive T cells were determined in peripheral blood and synovial sites from RA patients and in the blood of normal donors. The frequencies of Il-2 responsive T cells were significantly higher in RA patients (range from 1/180 to 1/7432) compared to normal donors (range from 1/400 to 1/8163). T-cell clones raised by the addition of Il-2 alone were predominantly of the CD4-positive phenotype. Peripheral blood T cells, synovial T-cell clones and lines derived from RA patients were co-stimulated with Il-2 and synovial fluid or supernatants from cultured synovial lining cells. This co-stimulation induced a strikingly enhanced proliferative T-cell response while synovial fluid alone was without effect. This stimulatory activity was found in the high molecular weight range (approximately 150 kDa) and could not be attributed to the action of immunoglobulins or known cytokines such as Il-2 or interleukin-1 (Il-1), suggesting the activity of a material that modulates the Il-2-dependent growth of T cells. The co-stimulatory capacity of synovial fluid with Il-2 may be relevant to the activated state, especially of synovial T cells.

T cell clones in rheumatoid arthritis (RA).

The development of T cell clones has greatly enhanced our knowledge of pathogenetic mechanisms in autoimmune diseases. Thus, it has been possible to induce experimental arthritis in susceptible animals by solely injecting arthritogenic T cell lines or clones. In contrast to many animal models, in rheumatoid arthritis the inciting antigen(s) have still not been identified. However, it has been possible to raise T cell clones from the inflammatory membrane by the addition of Interleukin-2 and/or mitogens. These clones are primarilig of the CD4+ (helper/inducer) phenotype. Molecular analyses on T cell receptor genes have not clearly shown a predominant rearrangement pattern in most studies indicating a polyclonal origin of RA synovial T cells. The availability of T cell clones will clearly help in the near future to probe for the inducing antigen(s) in inflammatory rheumatic diseases thus rendering a basis for new immunotherapeutic approaches.