Intern Emerg Med

Intern Emerg Med. proliferative response to Bhsp65, which was reversible by IL-2, indicating anergy induction. There was increased production of IFN- but not IL-4/IL-10, with concurrent downregulation of IL-17 expression by Bhsp65-primed T cells. Neither the frequency nor the suppressive activity of CD4+FoxP3+ T cells changed following tolerization, but the level of serum anti-Bhsp65 antibodies was increased. However, no evidence was found for the roles of IDO or cross-tolerance to Bhsp70, Bhsp10 or Rhsp65. Conclusion Tolerization with soluble Bhsp65 leads to suppression of IL-17, anergy induction, and enhanced production of anti-Bhsp65 antibodies, which play a role in protection against AA. These results are of relevance to developing effective immunotherapeutic approaches for autoimmune Allyl methyl sulfide arthritis. INTRODUCTION The induction of antigen-specific T cell tolerance has been the cornerstone of immunological interventions aimed at the prevention and treatment of autoimmune diseases over the past several decades (1-3). Furthermore, studies pertaining to the mechanisms involved in tolerance-induced downmodulation of the autoimmune process have offered invaluable insights into the pathogenesis of autoimmunity (1-3). Rheumatoid arthritis (RA) is the most common form of inflammatory arthritis in adults (4), affecting about 1% of the U.S. Allyl methyl sulfide population. Adjuvant arthritis (AA), inducible in the Lewis (LEW) (RT.1l) rat by injecting s.c. heat-inactivated (H37Ra), shares several features with human RA (5). The 65 kD-mycobacterial heat-shock protein (Bhsp65) has been implicated in the pathogenesis of AA (6, 7) as well as RA (8, 9). Bhsp65 has been the focus of tolerogenic immune interventions aimed at the prevention and treatment of AA (10-12). However, the earlier studies on Bhsp65-induced (10-12) or BCG-induced (13) tolerance that were conducted over the past decade examined a rather limited number of immune parameters (e.g., disease severity, T cell proliferation, Allyl methyl sulfide and Th1-Th2 cytokines). In the interim, the roles of newer cytokines (e.g., IL-17 and IL-23) (14, 15), CD4+CD25+ T regulatory cells (Treg) (16, 17) and indoleamine 2, 3 dioxygenase (IDO)-tryptophan pathway in the pathogenesis of autoimmunity have been elaborated in different animal models (18). In addition, the role of antibodies in protection against AA is only beginning to be appreciated (19, 20). Furthermore, two other mycobacterial hsps namely, Bhsp70 (DnaK) (21, 22) and Bhsp10 (GroES) (23, 24), as well as self (rat) hsp65 (Rhsp65) (20) have been reported to induce protection against AA, but the inter-relationship between the T cell responses against these hsps versus Bhsp65 have not been analyzed in the context of Bhsp65-induced T cell tolerance. Considering these interesting new developments in immune regulation in the past decade, it is imperative to revisit and critically examine the roles of these immune mediators in effecting Bhsp65-induced T cell tolerance as well as downmodulation of AA. In this study, we observed that the protection against AA following tolerization with i.p. administration of soluble Bhsp65 was associated with increased IFN- secretion coupled with decreased IL-17 expression by Bhsp65-specific T cells, anergy induction (25), and enhanced antigen-specific antibody response. However, there was no significant change either in the frequency or suppressive activity of the CD4+Foxp3+ T cells (Treg). Similarly, the activity of the IDO-tryptophan pathway remained unchanged. Furthermore, there was no evidence for the Allyl methyl sulfide involvement of deviation of the cytokine response to a Th2 type, or of the cross-tolerance to three other AA-related hsps, in Bhsp65-mediated tolerance. Taken together, our results offer novel insights into a diverse array of Bhsp65-directed immune pathways in AA that are modulated by tolerance induction in the LEW rats. These results are of significance in advancing our EPHB2 understanding of the Allyl methyl sulfide pathogenesis of RA as well as for designing effective antigen-directed immunotherapeutic approaches for this debilitating autoimmune disease. MATERIALS AND METHODS Rats Inbred Lewis (LEW/SsNHsd) (RT.1l) rats (4-6 wk old, male, 130-180 g) were obtained from Harlan Sprague-Dawley (Indianapolis, IN) and housed in the vivarium of the University of Maryland School of Medicine, Baltimore (UMB). Experimental procedures were performed on these animals in compliance with the guidelines of the institutional animal care and use committee (IACUC). Antigens Recombinant Bhsp65, Bhsp70 and Bhsp10 were produced by growing cells [BL21pLysS] (Novagen, Madison, WI) transformed with pET23b-GroEL2, pET23b-dnaK and pET23b-GroES, respectively (Mycobacteria Research Laboratories, Colorado State University, Fort Collins, CO), and recombinant Rhsp65 was produced from pTrcHisA-transformed BL21 cells (20, 26). For each protein, this step was followed by elution on a nickel column and purification by dialysis. Thereafter, the proteins were passed through an endotoxin removal column (Sterogene, Carlsbad, CA) until the endotoxin level was below 0.25 EU/mL. Ovalbumin (Ova) and concanavalin A (Con A) were obtained from Sigma-Aldrich (St. Louis, MO), whereas recombinant murine IL-2 was obtained from AbD.

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