Overexpression of a Minimal Domain of Calpastatin Suppresses IL-6 Production and Th17 Development via Reduced NF-κB and Increased STAT5 Signals

Calpain, a calcium-dependent cysteine protease, is reportedly involved in the pathophysiology of autoimmune diseases such as rheumatoid arthritis (RA). In addition, autoantibodies against calpastatin, a natural and specific inhibitor of calpain, are widely observed in RA. We previously reported that E-64-d, a membrane-permeable cysteine protease inhibitor, is effective in treating experimental arthritis. However, the exact role of the calpastatin-calpain balance in primary inflammatory cells remains unclear. Here we investigated the effect of calpain-specific inhibition by overexpressing a minimal functional domain of calpastatin in primary helper T (Th) cells, primary fibroblasts from RA patients, and fibroblast cell lines. We found that the calpastatin-calpain balance varied during Th1, Th2, and Th17 development, and that overexpression of a minimal domain of calpastatin (by retroviral gene transduction) or the inhibition of calpain by E-64-d suppressed the production of IL-6 and IL-17 by Th cells and the production of IL-6 by fibroblasts. These suppressions were associated with reductions in RORγt expression and STAT3 phosphorylation. Furthermore, inhibiting calpain by silencing its small regulatory subunit (CPNS) suppressed Th17 development. We also confirmed that overexpressing a minimal domain of calpastatin suppressed IL-6 by reducing NF-κB signaling via the stabilization of IκBα, without affecting the upstream signal. Moreover, our findings indicated that calpastatin overexpression suppressed IL-17 production by Th cells by up-regulating the STAT5 signal. Finally, overexpression of a minimal domain of calpastatin suppressed IL-6 production efficiently in primary fibroblasts derived from the RA synovium. These findings suggest that inhibiting calpain by overexpressing a minimal domain of calpastatin could coordinately suppress proinflammatory activities, not only those of Th cells but also of synovial fibroblasts. Thus, this strategy may prove viable as a candidate treatment for inflammatory diseases such as RA

Serum Adenosine Deaminase Level Is High But Not Related with Disease Activity Parameters in Patients with Rheumatoid Arthritis

Serum adenosine deaminase (ADA) has been previously proposed to predict disease activity in patients with rheumatoid arthritis (RA). The aim of this study was to investigate the level of serum ADA, and the relationship between ADA and disease activity markers, in a group of patients with RA., A hundred and 10 patients with a diagnosis of RA were recruited from outpatient clinic of Rheumatology Unit. Demographic properties comprising age, gender, disease duration and drugs were recorded. Disease activity based on disease activity score (DAS)28-erythrocyte sedimentation rate (ESR) and DAS28- C reactive protein (CRP,) ESR, CRP levels, as well as pain by visual analog scale and rheumatoid factor (RF) were recorded. Serum ADA levels (IU/L) were determined in all RA patients and in 55 age and sex similar healthy control subjects., Ninety-six female and 14 male RA patients with a mean age of 54.32±11.51, and with a mean disease duration of 11.5±9.13 years were included to the study. The control group comprised of 48 female and 7 male healthy subjects. 35.5% of the patients were on methotrexate (MTX) and 64.5% of patients were on combined DMARDs or combined MTX and anti-TNF therapies. The mean serum ADA level was statistically higher in RA patients than in control subjects (27.01±10.6 IU/L vs 21.8 ±9.9 IU/L). The mean values of ESR (23.2±14.8 mm/h), CRP (1.71±1.11mg/dL), pain by VAS (37.2±27.1), DAS28-ESR (2.72±0.77), DAS28 CRP (1.37±0.5) were not correlated with ADA levels (p>0.05)., Our results have shown that serum ADA levels are higher in RA patients than in controls but were not related with any of the disease activity markers. We conclude that ADA in the serum may not be a reliable biochemical marker to predict disease activity in patients with RA.PubMedScopu