Patients With Scleritis Are Deficient in Their Ability to Regulate Activities of Pro–inflammatory Cytokines

Abstract

Abstract: : Purpose: Ocular inflammatory diseases are characterized by production of a plethora of proinflammatory cytokines and the extent of pathology depends to a large degree on the ability of the host to limit the destructive effects of the cytokine by endogenous feedback regulators of cytokine activity. A family of proteins called suppressor of cytokine signaling (SOCS) has recently been identified as important negative regulators of the intensity and duration of cytokine activities. In this study, we have analyzed PBMC from patients with scleritis to investigate whether inflammation in this disease derives from inability to control actions of proinflammatory cytokines. Methods: We have cloned and sequenced human SOCS1, SOCS3 and CIS (cytokine–induced SH2 protein) cDNAs and developed a sensitive real–time RT–PCR assay for quantifying SOCS expression in clinical specimen. Human peripheral blood mononuclear cells (PBMC) were isolated from six patients and four normal healthy blood donors (10 ml) by using "Isolymph", a one–step method for isolation of pure lymphocytes. PBMC was stimulated with human recombinant IL–2 (10ng/ml) for 3 days in RPMI medium containing glutamine and ß–mercaptoethanol. Total RNA was isolated from freshly isolated cells and real–time PCR was performed in triplicates using labeled probes in an ABI 7700 sequence detection system. Results: We show here that activation of PBMC by IL–2 induces significant upregulation of the expression of SOCS1 and CIS, indicating existence of an intact negative feedback regulatory mechanism in these individuals. In contrast, 80% of patients are unable to induce expression of either protein. Further more the inability to activate this negative regulatory pathway correlates with disease severity Conclusions: Scleritis is an inflammatory disease of largely unknown etiology. Our data suggest that the inability to inhibit the intensity and duration of the activities of proinflammatory cytokines may in part contribute to the pathogenic mechanisms of scleritis.

Keywords: cytokines/chemokines • sclera • inflammation