Interleukin 7 stimulates tumour necrosis factor alpha and Th1 cytokine production in joints of patients with rheumatoid arthritis.

BACKGROUND: A large number of activated T cells are found in the joints of patients with rheumatoid arthritis (RA). Interleukin 7 (IL7), a T cell growth factor and a regulator of Th1 and Th2 cytokine production, is produced by synoviocytes from patients with RA. OBJECTIVE: To investigate the effect on proinflammatory cytokine production of synovial fluid mononuclear cells (SFMC) and the mechanism by which IL7 influences CD4+ T cell activity in patients with RA. METHODS: In a cross sectional group of patients with RA, IL7 levels were compared with those of healthy controls and related to disease activity. The effect of IL7 on cytokine production was tested by RA SFMC and on SF CD4+ T cells in the presence of mononuclear cells (MC). Production of tumour necrosis factor alpha (TNF alpha), IL1 beta, interferon gamma (IFN gamma), and IL4 was measured by enzyme linked immunosorbent assay (ELISA) and by single cell FACS analysis. Expression of the IL7 receptor alpha chain on CD4+ T cells (essential for IL7 signalling) was assessed. Direct effects of IL7 on isolated synovial fluid (SF) CD4+ T cells were studied by cytokine analysis. By neutralisation of IL12 in MC cultures, indirect effects of IL7 on T cells through accessory cells were studied. RESULTS: IL7 serum levels were higher in patients with RA than in healthy controls and correlated positively with C reactive protein levels. IL7 stimulated TNFalpha production by SFMC and very potently stimulated IFN gamma and TNF alpha production by SF CD4+ T cells. These effects were probably mediated through the IL7 receptor alpha chain, which was abundantly expressed on SF CD4+ T cells. Besides the direct stimulation of T cell cytokine production by IL7, its action was partly dependent on IL12, indicating that IL7 also stimulates accessory cell function, leading to T cell activation. CONCLUSION: IL7 stimulates proinflammatory cytokine production of intra-articular CD4+ T cells and accessory cells from patients with RA. The correlation with measures of disease activity indicates that IL7 might substantially contribute to the perpetuation of Th1 and TNF alpha mediated proinflammatory responses in patients with RA.

Experimentally challenged reactivity of the hypothalamic pituitary adrenal axis in patients with recently diagnosed rheumatoid arthritis.

OBJECTIVE: There is evidence that the hypothalamic pituitary adrenal (HPA) axis is subresponsive in patients with rheumatoid arthritis (RA). We assessed HPA axis responses to experimental stressors mimicking daily life challenges in patients with RA to determine whether HPA axis activity is associated with Th1 and Th2 activity. METHODS: ACTH and cortisol responses in reaction to the succession of a bicycle ergometer task, a cold pressor task, and a computerized Stroop Color-Word interference test, as well as basal Th1 and Th2 cell activity, were assessed in 29 patients (21 female, 8 male) with recently diagnosed RA (mean disease duration 29 wks, range 5-69), mean age 55.7 years, none receiving glucocorticoid treatment, and 30 (20 female, 10 male) healthy age and sex matched controls (mean age 54.1 yrs). RESULTS: Mean ACTH and cortisol levels did not differ between the groups (p > 0.10). Patients tended to have a less pronounced ACTH response (F2.50 = 2.7, p = 0.08) and had a significantly smaller cortisol response (P F2.50 = 6.1, p < 0.01) than healthy controls in reaction to the stressors. This difference in cortisol response was reduced, but remained significant when ACTH responsiveness was accounted for by entering it as a covariate (P F2.49 = 3.7, p = 0.03). ACTH and cortisol levels and responses were not associated (all p > 0.19) with basal interferon-gamma and interleukin 4 as reflections of Th1 and Th2 cell activity, respectively. HPA axis activity was not linked to current disease activity. CONCLUSION: Our findings show reduced HPA axis responsiveness in RA patients with recent diagnosis receiving longterm medication that is suggested to be located both at a hypothalamic/pituitary and at an adrenal level. It appears that common HPA axis activity accomplishes low amounts of cortisol release, which makes it difficult to determine an influence of endogenous cortisol changes on the Th1/Th2 balance.

Limitations of the semisynthetic library approach for obtaining human monoclonal autoantibodies to the thyrotropin receptor of Graves' disease.

Graves' disease (GD) is characterized by the presence of autoantibodies against the TSH-receptor (TSH-R) which are pathogenic and, upon binding to the receptor, trigger intracellular signal transduction. The autoantibodies are oligoclonal and as they are responsible for disease activity, their characterization would lead to a better understanding of the development of GD. Attempts to isolate anti-TSH-R antibodies from patients have proved to be difficult due to the exceedingly low serum levels due to rarity of these B cells, together with difficulties in obtaining purified TSH-R capable of interacting with patients autoantibodies. We employed phage antibody display technology and performed selection with a previously characterized semisynthetic antibody library on the purified extracellular ectodomain of the TSH-R. We report the isolation of six different anti-TSH-R monoclonal phage antibodies (moPhabs) from this library. All the moPhabs recognized TSH-R and its recombinant fragments by Western blotting, but failed to recognize the native TSH-R by flow cytometry. Consequently, the moPhabs did not lead to TSH-R activation. As these were the first moPhabs to TSH-R, they were analysed in terms of nucleotide and amino acid sequence and epitope specificity on the receptor. The moPhabs used immunoglobulin VH1 and VH3 germ line genes, all associated with Vlambda3 genes. Interestingly, the CDR3 regions of all moPhabs were remarkably similar, though not identical. In light of the common CDR3 usage, the epitopes recognized on TSH-R appeared to be restricted to amino acids residues 405-411 and 357-364. In summary, our results show that semisynthetic libraries may be limited in isolating human monoclonal antibodies that resemble pathogenic antithyrotropin receptor autoantibodies present in patients with GD. It is likely that until preparations of purified TSH-R that can be recognized by patients autoantibodies become available, similar to the recently described glycosylphosphatidylinositol (GPI) anchored TSH-R ectodomain, monoclonal antibodies from phage antibody display to TSH-R will be limited for isolating the rare, pathogenic antibodies of GD.