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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Arthritis Rheum. Author manuscript; available in PMC Jan 1, 2010.
Published in final edited form as:
PMCID: PMC2668824
NIHMSID: NIHMS105052

Tolerization with heat-shock protein 65 induces protection against adjuvant arthritis by modulating the antigen-directed IFN-γ, IL-17 and antibody responses

Abstract

Objective

Pre-treatment of Lewis (LEW) rats with soluble mycobacterial hsp65 (Bhsp65) affords protection against subsequently induced adjuvant arthritis (AA). This study was aimed at unraveling the mechanisms underlying the Bhsp65-induced protection against arthritis using contemporary immune parameters.

Methods

LEW rats were given 3 i.p. injections of Bhsp65 in solution prior to induction of AA with heat-killed Mycobacterium tuberculosis (Mtb). Thereafter, the Bhsp65-specific T cell proliferative, cytokine, and antibody responses were tested in tolerized rats. The roles of anergy and indoleamine 2,3 dioxygenase (IDO)-tryptophan pathway in tolerance induction were assessed along with monitoring the frequency and suppressive function of CD4+Foxp3+ T regulatory cells (Treg). Also tested was the effect of Bhsp65-tolerization on T cell responses to AA-related Bhsp70, Bhsp10 and rat hsp65 (Rhsp65).

Results

The AA-protective effect of Bhsp65-induced tolerance was associated with significantly reduced T cell proliferative response to Bhsp65, which was reversible by IL-2, indicating anergy induction. There was increased production of IFN-γ 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 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. population. Adjuvant arthritis (AA), inducible in the Lewis (LEW) (RT.1l) rat by injecting s.c. heat-inactivated Mycobacterium tuberculosis (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, 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-γ 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 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 understanding of the 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 E. coli 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 E. coli 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 Serotec (Raleigh, NC).

Induction and evaluation of AA

AA was induced in LEW rats by immunizing them s.c. at the base of the tail with 200 μL (1 mg/rat) of heat-killed M. tuberculosis H37Ra (Mtb) (Difco, Detroit, MI) suspended in mineral oil (Sigma-Aldrich). Thereafter, following the onset of AA, the severity of arthritis in each paw was evaluated on the basis of erythema and swelling, and scored as follows (26, 27): 0 = no erythema or swelling, 1 = slight erythema or swelling of the ankle or wrist, 2 = moderate erythema and swelling at the wrist or ankle, 3 = moderate erythema and swelling at the wrist and metacarpals, or ankle and metatarsals, 4 = severe erythema and swelling of the entire fore paw or hind paw. The maximum score for each paw was 4 and that per rat was 16. The ‘highest disease score’ represents the time point in the course of AA when the disease severity was maximum, and it is the sum total of scores of all 4 paws in one rat. In addition, the rats were weighed on alternate days for 4 wk after Mtb injection for monitoring general health.

Pre-treatment of naïve LEW rats with soluble antigen for the modulation of AA

LEW rats were injected i.p. on alternate days with soluble Bhsp65 or Ova (200 μg/injection each) for a total of 3 injections. After 9 d of the first injection, rats were immunized s.c. with Mtb. Thereafter, rats were observed and scored regularly for signs of arthritis (26, 27).

Lymph node cell (LNC) proliferation assay

The draining lymph nodes, (inguinal and para-aortic) of LEW rats were harvested 9 d after s.c. immunization either with Bhsp65 (100 μg/rat) emulsified in incomplete Freund’s adjuvant (IFA) or with Mtb (1 mg/rat) in oil and a single cell suspension was prepared (26, 27). These LNC were cultured for 96 h at 3 × 105 cells/well in HL-1 serum-free medium (BioWhittaker, Walkersville, MD) in the presence or absence of defined antigens (Bhsp65, Bhsp70, Bhsp10, Rhsp65 or Ova). After 3 d, the cells were pulsed with 1 μCi/well of [3H]-thymidine (MP Biomedicals, Solon, OH) and cultured for additional 16 h. The results were expressed as a stimulation index [(S.I) = ratio of cpm in the presence of antigen and cpm of cells in medium alone]. In one set of experiments, the effect of neutralization of IFN-γ in vitro on T cell proliferation was tested by using different concentrations of the anti-IFN-γ antibody (R&D Systems, Inc., Minneapolis, MN). The assay was performed as described above but with the inclusion of two additional groups of wells, one consisting of LNC cultured with Bhsp65 in the presence of different concentrations of the neutralizing antibody and the other of LNC cultured with antibody alone. LNC cultured with Bhsp65 in the absence of antibody served as the control.

Real-time PCR for quantification of IFN-γ, IL-4, IL-17- and IL-23-mRNA in antigen-sensitized lymphoid cells

Appropriate primers (synthesized at the Biopolymer Core Facility, UMB, Baltimore, MD) were designed using the Primer Express 2.0 Program (Applied Biosystems, Foster City, CA) for the detection of mRNAs for IFN-γ, IL-4, IL-17, IL-23, and hypoxanthine-guanine phosphoribosyl transferase (HPRT). The draining LNC were harvested from Bhsp65- or Ova-pre-treated and Mtb immunized rats as described above and cultured for 48 h in the presence or absence of Bhsp65 or Ova. Total RNA was prepared from 1 × 106 cells and reverse-transcribed using the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA). The cDNA thus obtained was amplified using an ABI Prism 7900HT cycler (Applied Biosystems, Foster City, CA) (28). The mRNA levels of the genes of interest were normalized to the HPRT gene, and the relative gene expression levels were determined (28). ‘Fold increase’ over mRNA levels of untreated cells was then determined.

Assays for testing the roles of anergy and IDO in tolerance induction

a) Anergy: the hyporesponsive state of anergic T cells is reversible in vitro by IL-2 (25). Therefore, we tested the antigen-specific proliferative response of the LNC of Bhsp65-tolerized rats in the presence or absence of IL-2 (50 U/ml). b) IDO: the activity of IDO can be effectively inhibited by a competitive inhibitor, 1-Methyl-DL-tryptophan (1-MT) (Sigma-Aldrich, St. Louis, MO) (29). We tested the proliferative response of the Bhsp65-specific T cells in the presence of different concentrations (500 μM and 1000 μM) of 1-MT to examine the role of IDO in soluble Bhsp65-induced tolerance.

Measurement of the frequency and suppressive activity of CD4+Foxp3+ T regulatory cells (Treg)

a) Frequency estimation by flow cytometry: Peripheral blood from the test (soluble Bhsp65-pre-treated) and the control (soluble Ova-pre-treated) groups of LEW rats was collected under heparin at various time points before and after immunization with Mtb. In addition, the draining lymph nodes and spleen of tolerized or control rats were harvested at defined time point post Mtb injection. The red blood cells (RBC) were lysed with ACK lysis buffer, and the remaining cells were surface-stained with anti-rat CD4-FITC (BD Biosciences, San Jose, CA), permeabilized and stained with anti-mouse/rat Foxp3-PE (eBioscience, San Diego, CA) (16, 17). These stained cells were then analyzed using the FACS Caliber and CellQuest software (both from BD Biosciences, San Jose, CA). b) Suppressive activity testing in cell culture in vitro: Splenic cells were harvested from tolerized or control rats, and the CD4+CD25+ regulatory T cells (Treg) were enriched using a magnetic column. These Treg (2 × 105 or 1× 105) were cultured with CD4+CD25- cells (1× 105) along with irradiated APC (1× 105) and anti-rat CD3 antibody (1 μg/ml) for 4 days (16, 17, 30). Thereafter, cells were pulsed with 1 μCi/well of 3[H]-thymidine for additional 18 hr before harvesting them. Radioactivity was counted as cpm (counts per minute)

Determination of serum antibody levels by ELISA

LEW rats that had been pre-treated i.p. with soluble Bhsp65 or Ova were bled at defined time points before and after s.c. immunization with Mtb. Sera were tested in ELISA as described in detail elsewhere (20). Briefly, an ELISA plate (Greiner Bio-One, Monroe, NC) was first coated with 100 ng/well of antigen overnight at 4°C, and then following washings, the wells were saturated with 10% bovine serum albumin (BSA) (Sigma-Aldrich). Sera were added to the wells at different dilutions and then incubated for 1 h at room temperature. Following thorough washes, the plate-bound antibody was measured by horseradish peroxidase (HRP)-conjugated goat anti-rat Ig (BD Pharmingen). The absorbance was read at 450 nm using Vmax ELISA autoreader (Molecular Devices, Sunnyvale, CA).

RESULTS

Pre-treatment of LEW rats with soluble Bhsp65 affords protection against subsequent development of AA

Naïve LEW rats were pre-treated i.p. with soluble Bhsp65 or Ova before induction of AA and then observed and graded regularly for signs of AA. The rats that had received Bhsp65 displayed significantly lower disease severity compared to those that had received Ova (Fig. 1A & B).

Figure 1
Pre-treatment with soluble Bhsp65 but not Ova induces protection against AA

Bhsp65-pre-treatment induces antigen-specific T cell tolerance that is reversible by IL-2

We tested the T cell proliferative response to Bhsp65 of LEW rats that were pre-treated i.p. either with Bhsp65 or with Ova prior to Mtb challenge. The Bhsp65-pre-treated rats showed significantly reduced proliferative response to Bhsp65 compared to that of Ova-pre-treated rats (Fig. 2A). This reduction in proliferative response following Bhsp65 pre-treatment was also evident after s.c. challenge with Bhsp65/IFA (data not shown). Furthermore, this attenuated T cell response to Bhsp65 in Mtb-immunized rats was reversible by the addition of IL-2 and Bhsp65 (combined) to the LNC culture (Fig. 2B), which restored the proliferation in Bhsp65-pre-treated group to the same level as that of the Ova-pre-treated group, indicating reversal of the anergic state of antigen-specific T cells. The basal level proliferation induced by IL-2 alone (without Bhsp65/Ova) presumably represented the proliferation of effector T cells. The IL-2-induced reversal of the hypoproliferative Bhsp65-specific T cell response was further corroborated in Bhsp65-tolerized rats that were not immunized with Mtb (Fig. 2C).

Figure 2
IL-2 and anti-IFN-γ antibodies reverse the suppression of proliferative T cell response to Bhsp65 caused by pre-treatment of LEW rats with soluble Bhsp65

Bhsp65-induced T cell tolerance is associated with upregulation of IFN-γ secretion and concomitant downregulation of IL-17 expression

We further tested the cytokines produced by the T cells following pre-treatment i.p. of LEW rats with Bhsp65 vs. Ova prior to s.c. injection with Mtb. The draining LNC of rats pre-treated with Bhsp65 had increased secretion of IFN-γ but not IL-4 compared to that of Ova-pre-treated rats (Fig. 3A, 3B). Interestingly, this increase in IFN-γ secretion was associated with downregulation of IL-17 mRNA expression in Bhsp65-pre-treated rats (Fig. 3C). However, there was no significant change in IL-23 expression (Fig. 3D). Thus, Bhsp65-induced tolerance involves modulation of the pro-inflammatory cytokines leading to a reduced IL-17 response rather than deviation of the cytokine response to a Th2 type. We have described above that the Bhsp65-specific T cell proliferation in Bhsp65-tolerized rats was reduced significantly (Fig. 2A). In view of our cytokine results as well the suggested role of IFN-γ in inhibiting T cell proliferation, we tested whether IFN-γ also contributed to the suppression of T cell proliferation in Bhsp65-tolerized rats. Our results (Fig. 2D) revealed that neutralization of the cytokine using anti-IFN-γ antibody restored the proliferative response levels to control levels, suggesting the involvement of IFN-γ in the inhibition of T cell proliferative response to Bhsp65.

Figure 3
Tolerization against Bhsp65 upregulates IFN-γ secretion but downregulates IL-17 expression by Bhsp65-primed T cells

Tolerization with Bhsp65 neither alters the frequency nor the suppressive activity of CD4+Foxp3+ T regulatory cells (Treg)

CD4+CD25+ T cells expressing Foxp3 are endowed with the ability to suppress effector T cell responses, and their role in tolerance induction and immune regulation is increasingly being realized (3, 16, 17, 31, 32). We studied the temporal profile of the frequency of Treg in peripheral blood of the test (Bhsp65-pre-treated) and the control (Ova-pre-treated or PBS-pre-treated) LEW rats during the course of AA (Fig. 4). In addition, in one group of rats (d 9 post Mtb challenge corresponding to disease onset), we also compared the Treg frequency in the draining lymph nodes and spleen in addition to the peripheral blood. Measurements of Treg frequency in peripheral blood and lymph nodes on d 9 gave similar results (Fig. 4A, 4B). Testing of spleen cells gave results similar to those obtained with LNC (data not shown). The percentage of Treg in peripheral blood neither showed a significant change during the entire course of AA in PBS-pre-treated or Ova-pre-treated groups nor was it altered by pre-treatment with soluble Bhsp65 (Fig. 4B). Determination of the suppressive function of Treg derived from Bhsp65-tolerized vs. Ova-tolerized rats revealed that the Treg of these two groups of rats had comparable levels of suppressive activity on effector T cells (Fig. 4C). Thus, Treg might not play a significant role in Bhsp65-induced suppression of AA.

Figure 4
Pretreatment with Bhsp65 is not associated with a significant change in the frequency or the suppressive activity of Treg, or in the levels of IDO activity, in Mtb-immunized LEW rats

Bhsp65-mediated tolerance is not associated with a change in the activity of indoleamine 2,3-dioxygenase (IDO)

IDO expressed in dendritic cells (DC) increases catabolism of tryptophan and leads to hyporesponsiveness and tolerization of T cells (18). Upregulation of IDO has been invoked as a mediator of tolerance induction in some experimental models (18). We tested the involvement of IDO in Bhsp65-induced tolerance in the LEW rat using 1-Methyl-DL-tryptophan (1-MT), a competitive inhibitor of IDO. Our results show that the inhibition of IDO by 1-MT failed to restore the reduced Bhsp65-specific proliferative response of LNC of Bhsp65-tolerized rats (Fig. 4D). Thus, IDO might not play a significant role in mediating Bhsp65-induced tolerance.

Bhsp65-pre-treatment leads to upregulation of the antibody response to Bhsp65

To assess the full impact of Bhsp65-pre-treatment on the antigen-directed immune responses, we tested the antibody response to Bhsp65 in tolerized LEW rats. We observed that arthritic LEW rats pre-treated with Bhsp65 generated Bhsp65-specific antibodies (total Ig) at a shorter interval of time after the pre-treatment, and their titers were enhanced much earlier (d 4) during the disease course, compared to that of the Ova-pre-treated rats (Fig. 5). On the basis of the previous reports by others and us (19, 20) on the AA-protective anti-Bhsp65 antibodies produced during the course of arthritis in LEW rats, we suggest that similar protective antibodies are generated early following tolerance induced by Bhsp65.

Figure 5
Pre-treatment of LEW rats with soluble Bhsp65 enhances the anti-Bhsp65 antibody response

Tolerization of rats against Bhsp65 does not induce cross-tolerance to other AA-related Bhsps or self(rat) mammalian hsp65 (Rhsp65)

Mtb contains multiple hsps besides Bhsp65, including Bhsp70 and Bhsp10, which have been implicated in the pathogenesis of AA (21-24). Therefore, we tested whether Bhsp65 pre-treatment had any effect on the T cell response to Bhsp70 and Bhsp10. Also included in the assay was Rhsp65, which has about 50% sequence homology with Bhsp65, and also is partially crossreactive with it. Our results show that arthritic LEW rats raised potent T cell responses to Bhsp70 and Bhsp10 (Fig. 6A). Interestingly, the T cell response to these mycobacterial hsps and Rhsp65 (Fig. 6B) as well as anti-hsp65 antibody responses (data not shown) remained unaltered following tolerization against Bhsp65. Thus, Bhsp65-induced protection against AA is not dependent on the concurrent reduction of the T cell response to Bhsp70, Bhsp10, or Rhsp65.

Figure 6
Tolerance induction against Bhsp65 does not alter the T cell responses to other AA-related mycobacterial hsps and mammalian (rat) hsp65 (Rhsp65)

DISCUSSION

The precise autoantigen that is critical for the initiation and propagation of human RA has not yet been defined. Multiple antigens have been invoked in the immunopathogenesis of experimental arthritis as well as RA (7, 26, 33-36). Bhsp65 represents one such antigen, and both the arthritic LEW rats (6, 7, 19, 26, 37) as well as RA patients (8, 9) raise T cell responses to this antigen. Bhsp65 is a multi-determinant antigen. Considering the vast genetic heterogeneity of the human population and the critical role of the HLA haplotype on presentation of antigenic determinants to the T cells (34, 38), we believe that studying the mechanisms of tolerance induction using whole Bhsp65 protein would provide information that is relevant for eventual translational research for immunotherapy of RA patients. Most of the clinical trials that have been attempted so far in different autoimmune diseases in humans are based on whole protein antigens (e.g., myelin basic protein (MBP), type II collagen (CII), insulin, and cartilage glycoprotein 39) (35, 36). In this context, we have revisited in this study the mechanisms of tolerance induction to whole Bhsp65 employing contemporary immune parameters (e.g., IL-17, CD4+Foxp3+ regulatory T cells, IDO-tryptophan pathway and anergy induction).

We report here that the pre-treatment i.p. of LEW rats with soluble Bhsp65 imparts protection against subsequently induced AA (Fig. 1). This protection is associated with a significant reduction of proliferative response of Bhsp65-specific T cells, which was reversible by IL-2 and antigen (combination) in vitro (Fig. 2), indicating the reversal of anergy. Thus, soluble Bhsp65 induces antigen-specific T cell tolerance in part by the induction of T cell anergy, which in turn might contribute to protection against AA.

The T cells of Bhsp65-tolerized LEW rats exhibited an exaggerated IFN-γ response and reduced IL-17 expression, but without any significant alteration in IL-4 secretion (Fig. 3). Similarly, no effect on IL-10 secretion was observed (data not shown). Our results also corroborate the proposed regulatory interactions between the cytokines produced by Th1 (IFN-γ-secreting) and Th17 (IL-17-secreting) cells in the pathogenesis of autoimmunity (15, 39, 40). The role of IFN-γ in suppression of AA is supported by results of other investigators showing that LEW rats treated with anti-IFN-γ antibodies developed more severe arthritis than controls (41, 42). We suggest that the reduced expression of the pathogenic cytokine IL-17 (Fig. 3) might contribute to protection against AA in the Bhsp65-tolerized rats in part by attenuation of the pathogenicity of Mtb-primed T cells. Thus, Bhsp65-induced tolerance modulates the response to IFN-γ and IL-17 rather than deviate the cytokine response to a Th2 type.

We observed that neither the frequency nor the suppressive activity of CD4+Foxp3+ regulatory T cells changed significantly in Bhsp65-tolerized, Mtb-immunized rats (Fig. 4). Therefore, the AA-protective effect of Bhsp65 pre-treatment was apparently not attributable to a significant change in the frequency/activity of Treg owing to the expansion either of the pre-existing ‘natural’ Treg or of the antigen-induced ‘adaptive’ Treg (16, 17). Our finding contrasts with that of a few of the previous studies (30, 43, 44) that reported an increase in CD4+CD25+ T cells upon tolerance induction. This discrepancy can be explained by the fact that we stained CD4+ T cells for Foxp3, which is a specific marker for Treg, instead of CD25 alone, which also can be expressed by activated (non-Treg) effector T cells. Another explanation is the difference in the route of administration (i.p. versus oral/hepatic) of antigen/gene in these studies. However, our results are supported by those of a couple of studies in diabetes (45, 46). Furthermore, the results of studies by other investigators have shown that the frequency of Treg remains unaltered as a result of disease or immunotherapy (31, 32, 47, 48). However, in these conditions, the function of Treg is affected without a change in frequency.

IDO-expressing DCs exert tolerizing effect on the T cells by local depletion of tryptophan, by generating metabolites of tryptophan that are cytotoxic, or by upregulation of inhibitory costimulation molecules on their surface (18). These changes in turn lead to growth arrest, anergy induction or deletion of T cells. IFN-γ is known to induce IDO activity in DC (18). Since soluble Bhsp65-mediated tolerance led to anergy induction as well as increased IFN-γ production by the T cells, we examined the role of IDO in this tolerance model. Our results (Fig. 4D) suggest that IDO might not play a significant role in Bhsp65-mediated tolerance. However, at present we cannot exclude the possibility that IDO might be more effective in modulating the activity of pathogenic T cell responses within the milieu of the target site (the joints) than that within the draining lymph nodes in the periphery.

LEW rats pre-treated with soluble Bhsp65 raised a robust anti-Bhsp65 antibody response during the incubation phase of AA, and it corresponded well with the observed protection against AA (Fig. 5). We and others (19, 20) have previously shown that arthritic LEW rats develop antibodies to Bhsp65, and that the adoptive transfer of these antibodies into naïve LEW rats offer protection against subsequently induced AA. Furthermore, the AA-resistant rat strains generate high levels of anti-Bhsp65 antibodies early on following Mtb injection (19, 20). In this context, we suggest that the protection against AA induced by soluble Bhsp65-induced tolerance occurs in part through the induction of an early and high level of AA-protective anti-Bhsp65 antibody response.

Studies by other investigators have shown that the pre-treatment of LEW rats with Bhsp70 (21, 22) or Bhsp10 (23, 24) leads to protection against AA, demonstrating that mycobacterial hsps other than Bhsp65 also possess AA-regulatory attributes. However, the interplay between the T cell responses to these 3 mycobacterial hsps and self hsp65 (Rhsp65) during AA under tolerogenic conditions has not been fully elucidated. Our results showed that the T cell responses to Bhsp70, Bhsp10, and Rhsp65 in Mtb-immunized rats were unaffected by tolerization against Bhsp65 (Fig. 6). Thus, the Bhsp65-based tolerization regimen failed to induce cross-tolerance or bystander suppression of the T cell proliferative response to Bhsp70, Bhsp10, or Rhsp65, and it offered protection against AA despite the presence of unabated T cell responses against these two additional arthritis-related mycobacterial and self hsps. Our results differ from that of another study (37) in which DNA vaccination with mammalian (human) hsp70 was shown to induce T cell response to human hsp60, and vice versa, indicating the influence of immune response to one hsp on that directed to another hsp. The fine epitope differences between mycobacterial and human homologous hsps as well as a difference in the immunization regimen (tolerogenic vs. immunogenic) might account for the observed differences in the two studies. We (49) and others (50) have described earlier the role of mammalian (rat/human) hsp65 in the regulation of AA. In these studies, self hsp65 was injected into LEW rats either with an adjuvant (s.c.) (49) or as a DNA vaccine (i.m.) (50). In contrast, the present study is focused on tolerance induction by soluble mycobacterial hsp65 (Bhsp65) injected i.p. The precise immunological changes that are uniquely associated with protection against arthritis induced by Bhsp65 vs. mammalian hsp65 remain to be elucidated.

In summary, the results of our study in the AA model provide a comprehensive assessment of diverse and contemporary tolerogenic and regulatory pathways involved in the Bhsp65-induced protection against autoimmune arthritis. These insights are relevant both for further understanding of the pathogenesis of RA as well as for developing and refining the antigen-directed immunotherapeutic approaches for autoimmune arthritis.

ACKNOWLEDGEMENTS

We wish to thank Ranjani Prabhakara and Yinghua Yang for helping with experiments and Ricardo Feldman, Agnes Azimzadeh, Wendy Davidson and David Scott for their helpful discussions. This work was supported by grants from the Arthritis Foundation (Maryland Chapter) & the Maryland Arthritis Research Center (MARRC), Baltimore, MD.

This work was supported by grants from the Arthritis Foundation (Maryland Chapter) & the Maryland Arthritis Research Center (MARRC), Baltimore, MD, and the National Institutes of Health, Bethesda, MD.

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