Dynamics of mycobacterial HSP65-induced T-cell cytokine expression during oral tolerance induction in adjuvant arthritis

P. M. Cobelens, A. Kavelaars, R. van der Zee1, W. van Eden1 and C. J. Heijnen

Department of Immunology, Wilhelmina Children's Hospital of the University Medical Center Utrecht and
1 Department of Infectious Disease and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objective. To investigate whether oral administration of mycobacterial heat-shock protein 65 (HSP65) during adjuvant arthritis (AA) induces regulatory cells and cytokines.

Methods. AA was induced in Lewis rats and from the time of disease onset HSP65 in the presence of soya bean trypsin inhibitor (STI) was administered orally every other day. The number of splenic CD4+CD25+ T cells and antigen-induced cytokine mRNA expression were determined.

Results. Oral treatment with HSP65/STI reduced AA symptoms. After one feeding of HSP65/STI, the number of CD4+CD25+ splenic T cells increased and HSP65-specific T cells expressed increased levels of interferon {gamma} and interleukin 10. After two feedings, the expression of interleukin-10 mRNA remained increased, whereas there was low expression of interferon {gamma} mRNA. The number of CD4+CD25+ splenic T cells remained increased.

Conclusions. Oral treatment with HSP65/STI after AA onset reduces disease symptoms via dynamic changes in the number of CD4+CD25+ splenocytes and in antigen-induced cytokine production.

KEY WORDS: Arthritis, Tolerance, HSP65, Tr1, CD25.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Recently, we showed that ongoing adjuvant-induced arthritis (AA) in male Lewis rats can be suppressed successfully by oral administration of a low dose of mycobacterial heat-shock protein 65 (HSP65). We also demonstrated that coadministration of soya bean trypsin inhibitor (STI) is necessary for successful induction of tolerance, presumably by preventing the breakdown of HSP65 in the gastrointestinal tract [1].

It has been suggested that regulatory T cells are involved in low-dose tolerance induction via the production of regulatory cytokines such as transforming growth factor ß (TGF-ß) and interleukin (IL) 10 [2, 3]. On the other hand, high-dose tolerance is mediated mainly by the induction of anergy and/or deletion of antigen-specific T-cell clones, resulting in the absence of antigen-specific T-cell proliferation [4, 5].

During the last few years, various subsets of T cells have been defined which may play a central role in maintaining self-tolerance or in regulating autoimmune diseases [6]. One of these subsets is characterized by its expression of the IL-2 receptor {alpha} (IL-2R{alpha}); (CD25). Adoptive transfer of CD4+CD25+ T cells prevented the spontaneous development of autoimmune diseases in neonatally thymectomized mice that had been inoculated with autoaggressive T cells [7]. Interestingly, it appears that the regulatory activity of CD4+CD25+ T cells is mediated by the cytokine IL-10 [8, 9]. The functional properties of this subset of T cells resemble those of the regulatory T cells (Tr1) which were originally found after repetitive antigenic stimulation in vitro of CD4+ T cells in the presence of IL-10 [10]. Tr1 cells are characterized by their low proliferative capacity and by the secretion of high levels of IL-10 and interferon {gamma}(IFN-{gamma}). In addition it has been shown that Tr1 cells have immunosuppressive capacities both in vitro and in vivo [11].

The aim of this study was to delineate further how oral administration of low doses of HSP65 during AA suppresses disease. We therefore studied the induction of regulatory cells and antigen-induced regulatory cytokines over time.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Induction and clinical evaluation of AA
Male Lewis rats (aged 6–8 weeks) were obtained from the University of Limburg (Maastricht, The Netherlands). Rats were kept at the Utrecht University animal facility and were fed a standard diet (Hope Farms, Woerden, The Netherlands) and given water ad libitum.

AA was induced by a single intradermal injection of 100 µl of Freund's complete adjuvant [5 mg Mycobacterium tuberculosis (strain H37Ra)/ml Freund's incomplete adjuvant (Difco, Detroit, MI)] in the base of the tail. Severity of arthritis was assessed daily by standard methods in a blinded protocol [1].

Oral administration of proteins
Mycobacterial HSP65 from M. bovis was expressed in Escherichia coli and isolated as described [12].

Rats were treated orally with 30 µg HSP65 or E. coli-derived superoxide dismutase (SOD) (Sigma, St Louis, MO, USA) in 1 ml of phosphate-buffered saline using an 18-gauge animal-feeding needle. As described, rats were deprived of food but not water for 14–16 h prior to antigen feeding and antigen was co-administered with 10 mg STI (Sigma) [1]. Treatment was started when most of the animals lost weight, that is, at the onset of clinical arthritis (day 11). Oral administration of HSP65 was repeated every other day for an additional three doses.

Flow cytometry and cytokine analysis
At the indicated time points, rats were killed by decapitation and spleen and mesenteric lymph nodes (MLN) were harvested. Cell suspensions were obtained as described [1].

For flow cytometric analysis, splenocytes and MLN cells were stained with anti-CD4/anti-CD25 (Ox-38-phycoerythrin/OX-39-fluorescein isothiocyanate (FITC); Pharmingen, San Diego, CA, USA). Flow cytometric analysis was performed with a fluorescence-activated cell sorter (FACS) (FACS-Calibur; Becton Dickinson, San Jose, CA, USA). Fluorescence data are expressed as the percentage of positive cells.

For analysis of cytokine profiles, splenocytes were cultured in 24-well flat-bottomed wells (Costar, Cambridge, MA, USA) at 2x106 cells/well in the presence of mycobacterial HSP65 (10 µg/ml), M. tuberculosis (Mt; 10 µg/ml) or concanavalin A (Con A) (1 µg/ml) for 24 h. RNA was isolated using RNAzol B (Cinna/Tel-Test, Friendswood, TX, USA) and cytokine RNA expression was analysed with the RNase Protection kit (Pharmingen). RNA expression was analysed by phosphorimaging and quantitated using the software Molecular Analyst, version 1.5 (Bio-Rad, Richmond, CA, USA). Results are expressed as the percentage of household gene (L32) expression.

Statistical analysis
All data were analysed by Student's t-test for unpaired data. P values less than 0.05 were considered statistically significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Clinical effect of oral administration of mycobacterial HSP65 after induction of disease
AA was induced in Lewis rats and treatment was started at the onset of clinical signs of arthritis (on day 11). From this time point, rats received 30 µg HSP65 orally every other day for a total of five doses. Control rats received 30 µg SOD as an irrelevant antigen. To prevent the protein from early breakdown in the gastrointestinal tract, the proteinase inhibitor STI was always co-administered with HSP65 or SOD. Figure 1Go shows that oral administration of mycobacterial HSP65 resulted in an immediate reduction in disease severity. In addition, a significant reduction in the mean cumulative disease score was observed, as determined by summing the scores on days 11–21 (mean±S.E.M. score, 45.2±5.5 in controls vs 28.3±3.5 in HSP65/STI-treated animals; P<0.03).



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FIG. 1.  Effect of oral administration of mycobacterial HSP65 after disease induction on score for AA. AA was induced in Lewis rats by intradermal injection of 0.5 mg M. tuberculosis (Mt) in Freund's incomplete adjuvant into the base of the tail. On day 11, treatment was started with oral administration of 30 µg HSP65 in combination with 10 mg STI. Control rats received 30 µg SOD in combination with STI. Values are mean and S.E.M. (n=8 rats per group).

 

CD25 expression on CD4+ T cells after oral administration of HSP65/STI
To evaluate whether the decreased disease activity after oral administration of HSP65/STI was associated with alterations in the percentage of CD4+CD25+ T cells, splenic cells and MLN cells were examined by FACS analysis. Oral administration of HSP65/STI resulted in more splenic and MLN CD4+ T cells expressing CD25 after both one and two feedings, compared with SOD/STI-treated animals. After three feedings, the percentage of CD4+CD25+ cells in spleen and MLN returned to that observed in SOD/STI-treated rats. (Fig. 2AGo and BGo).



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FIG. 2.  Expression of CD25 and antigen-induced cytokines by cells from rats treated with SOD/STI ({blacksquare}) and HSP65/STI ({square}). Splenocytes and MLN cells were investigated for the expression of CD4/CD25 (A, B) on days 13, 15 and 17 after immunization, i.e. 48 h after one, two or three feedings of antigen. In addition, spleen cells obtained on days 13, 15 and 17 after immunization from both SOD/STI- and HSP65/STI-treated animals were cultured in the presence of 10 µg/ml of mycobacterial HSP65 (C, D), 10 µg/ml of Mt (E, F) or 1 µg/ml Con A (G, H) for 24 h. After isolation of mRNA, an RNase protection assay was performed for the cytokines IL-10 and IFN-{gamma}. Values are mean and S.E.M. percentage of household gene (L32) expression (n=8 rats per group). *P<0.05 vs SOD/STI-treated controls.

 

Kinetics of antigen-induced cytokine mRNA expression
To examine whether the increased CD25-expression found on CD4+ T cells after treatment with HSP65/STI was accompanied by changes in the cytokine pattern, we examined antigen-induced cytokine mRNA expression levels in rat splenocytes. Splenocytes were harvested 48 h after one, two or three feedings with HSP65/STI or SOD/STI. The splenocytes were restimulated in vitro for 24 h with HSP65, Mt or Con A. We determined IL-10 and IFN-{gamma} mRNA expression, as representative of the TH2 and TH1 types of cytokine respectively, by RNase protection assay. No IL-4 mRNA expression was detectable using the RNase protection assay. As shown in Figure 2CGo and DGo, repetitive feeding of HSP65 resulted in a dynamic process of change in HSP65-induced cytokine mRNA expression in the spleen. One feeding of antigen resulted in an increase in HSP65-induced IFN-{gamma} and IL-10 mRNA expression in splenocytes from HSP65/STI-treated rats compared with SOD/STI-treated animals. After two feedings, the HSP65-induced expression of IFN-{gamma} mRNA declined significantly in splenocytes from HSP65-treated rats, whereas IL-10 mRNA expression stabilized. However, after the third oral administration of HSP65 the expression of IL-10 mRNA also decreased. At this time point, expression of both HSP65-induced IFN-{gamma} and IL-10 mRNA was significantly reduced in HSP65/ STI-treated rats compared with SOD/STI-treated animals.

As HSP65 is a major component of Mt, we also restimulated the splenocytes in vitro with Mt. As shown in Figure 2EGo and FGo, in vitro restimulation of splenocytes with Mt resulted in exactly the same pattern of antigen-specific IL-10 and IFN-{gamma} mRNA expression over time as was observed after in vitro restimulation with HSP65.

To investigate whether the observed changes in cytokine mRNA profiles was an antigen-specific phenomenon and not due to a generalized effect on T cells, we examined cytokine expression by splenocytes stimulated with the mitogen Con A. The results presented in Figure 2GGo and HGo clearly demonstrate that no significant differences in mitogen-induced cytokine expression were observed between the SOD/STI- and HSP65/STI-treated rats over time.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study we demonstrate that multiple oral administrations of mycobacterial HSP65 during the disease process of AA results in a dynamic change in antigen-specific cytokine production. From studies in which tolerance was induced before systemic immunization, it has become clear that two primary mechanisms mediate the induction of oral tolerance, depending on the dose of antigen: active suppression and clonal deletion or anergy [2, 4, 5]. In our experimental set-up, we administered a low dose of HSP65 (30 µg per rat), and therefore we hypothesize that active suppression via induction of changes in the cytokine pattern of antigen-specific T cells is the primary mechanism.

During the last few years, several regulatory subsets of CD4+ T cells have been described on the basis of expression of specific cell surface markers, such as IL-2R{alpha} (CD25). This subset of T cells has been shown to have suppressive properties both in vivo and in vitro, partly because they show higher expression levels of cytokines such as TGF-ß and IL-10 [9]. In an ovalbumin–T-cell receptor transgenic model, Zhang et al. [13] showed that oral administration of ovalbumin led to the emergence of CD25+CD4+ regulatory T cells. Furthermore, they showed that CD25+CD4+ T cells from ovalbumin-fed mice produce elevated levels of IL-10 and TGF-ß compared with unfed mice. Finally, it was shown that adoptive transfer of the CD25+CD4+ regulatory T cells into BALB/c mice resulted in suppression of in vivo delayed-type hypersensitivity responses. Also, Thorstenson and Khoruts [14] showed that transfer of T cells from RAG2–/ DO11.10 mice to BALB/c mice resulted in the generation of potentially immunoregulatory CD25+CD4+ cells in vivo after induction of peripheral tolerance with low-dose oral antigen. This suggests that oral antigen may be inducing CD25+CD4+ regulatory cells de novo.

Here we also show that CD25+CD4+ cells are up-regulated in splenocytes from AA rats fed with HSP65/STI compared with SOD/STI-treated animals. The high expression of CD25 suggests that these T cells are in an activated state and that this subset may mediate the suppression of activation and expansion of potentially pathogenic self-reactive T cells either directly via cell–cell contact or indirectly via production of anti-inflammatory cytokines. To further explore whether the induction of regulatory CD4+CD25+ T cells is accompanied by the induction of changes in the cytokine pattern, we analysed antigen-induced cytokine mRNA expression of splenocytes from tolerized and non-tolerized animals. We show here that increased levels of antigen-induced IFN-{gamma} mRNA are expressed after one oral feeding of HSP65/STI compared with SOD/STI-treated rats. The up-regulation of IFN-{gamma} is consistent with findings from other studies showing that IFN-{gamma} is up-regulated shortly after antigen feeding in both the spleen and the gut [15, 16]. A study by Lee et al. [17] suggests that the induction of IFN-{gamma} by orally administered antigen might affect the migration of effector T cells to the site of inflammation. In addition, results from studies on animal models of autoimmune diseases suggest that IFN-{gamma} plays a role in down-regulating tissue inflammation. In an experimental autoimmune encephalomyelitis model, treatment with anti-IFN-{gamma} soon after immunization resulted in aggravation of disease, whereas treatment with recombinant IFN-{gamma} resulted in amelioration of the clinical state [18, 19]. Therefore, the up-regulation of IFN-{gamma} after a single feeding of HSP65 might be an important first step in the optimal induction of tolerance. Moreover, a single feeding of HSP65/STI also results in increased levels of antigen-specific IL-10 expression in splenocytes compared with SOD/STI-treated rats. T cells expressing both IFN-{gamma} and IL-10 are characteristic of the Tr1 cells [11]. After a second oral feeding of HSP65/STI, the antigen-specific T cells in the spleen are characterized by high IL-10 mRNA expression and low IFN-{gamma}. Subsequent feeding of the third dose of antigen did not result in greater IL-10 expression in HSP65/STI-treated rats, but in a complete drop of IL-10 expression. The expression of IFN-{gamma} mRNA did not alter after the third feeding. The decline in IL-10 mRNA expression by HSP65-specific T cells was not due to deletion after three feedings of HSP65, as day-18 splenocytes from HSP65/STI-treated rats still proliferate upon in vitro stimulation with HSP65 [1]. Therefore, we suggest that after three feedings on day 17, HSP-specific cells producing IL-10 from the spleen are redistributed to other sites, e.g. the joints. This hypothesis is supported by the fact that the number of splenic CD4+CD25+ T cells also shows a sharp drop on day 17.

In conclusion, we showed that oral tolerance induction by HSP65 after induction of disease results in a dynamic kinetics of antigen-specific cytokine production. First, there is up-regulation of IFN-{gamma}, which may serve as a fast immunosuppressive signal. This is followed by the induction of HSP-specific T cells expressing IL-10.


    Acknowledgments
 
This work was supported by STER grant SW07 from the UMC Utrecht, The Netherlands.


    Notes
 
Correspondence to: C. J. Heijnen, Department of Immunology, Wilhelmina Children's Hospital of the University Medical Center Utrecht, Room KC3.68.0, Lundlaan 6, 3584 EA Utrecht, The Netherlands. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Submitted 23 August 2001; Accepted 17 February 2002