3.1. Protection from EAT Induction by Elevating the Circulatory
Susceptible mice do not develop thyroiditis spontaneously, despite the continuous presence of low levels of circulatory Tg and the presence of autoreactive T cells. Indeed, thyroiditogenic, autoreactive T cells capable of responding to syn-geneic mTg administration and mediating thyroid pathology were demonstrated in susceptible, but not resistant, mice (ElRehewy et al., 1981). Clearly, suppressor mechanisms exist in susceptible mice, which can be overcome upon immunization with mTg. We subsequently observed that pretreatment of susceptible mice with mTg rendered the mice tolerant to immunization with mTg and adjuvant (Kong et al., 1982). Three parameters are used to measure this resistance: much reduced to absent infiltration of mononuclear cells into the thyroid, very low mTg antibody level, and minimal T cell proliferative response in vitro to mTg. Raising the circulatory mTg level 3- to 5-fold above baseline for 2-3 days is key to successful tolerance induction (Kong et al., 1982; Lewis et al., 1987). The increase can be achieved with three different protocols: (i) two intravenous doses of 100 |g deaggregated (d) mTg 7 days apart, or daily doses of 10 |g mTg for 10 days (Lewis et al., 1987), (ii) intravenous administration of 20 |g bacterial lipopolysaccharide (LPS) to reduce reticuloendothelial clearance 24 hr before two subtolerogenic, 20-|g doses of dmTg (Lewis et al., 1992), and (iii) TSH injections (Kong et al., 1982) or infusion with a peritoneal osmotic pump for 3 days to stimulate endogenous mTg release (Lewis et al., 1987). As circulatory Tg was a by-product of thyroid hormone release and had no apparent function, we hypothesized that there was a clonal balance between regulatory T cells and autoreactive T cells in the normal, susceptible host and that one major function of circulatory Tg was to activate low levels of suppressor T cells to keep autoreac-tive T cells in check (Kong et al., 1982). But this low level was unable to withstand insults from autoantigens and various polyclonal activators from infectious agents and other chronic inflammation. When these regulatory T cells were further expanded by mTg for 2-3 days, they altered the balance to favor protection against strong antigenic challenge.
3.2. CD4+ Regulatory T Cells as Mediators of Induced Resistance
Because mTg-activated regulatory T cells were few and no known markers existed at the time, unresponsiveness was relegated to the phenomenon of anergy by skeptics. However, anergy in autoreactive T cells induced by pretreatment with dmTg was insufficient to explain the finding that infusion of one spleen equivalent of normal T cells did not reconstitute the response of tolerant mice to challenge, indicating an active inhibition of autoreactive T cells (Kong et al., 1989). The hypothesis of active suppression was also tested by the transfer of cells from tolerant mice. This transfer of suppression was possible to demonstrate, but with some difficulty, because the use of adjuvant to induce autoimmune disease could overwhelm the regulatory T cells (Fuller et al., 1993). Since EAT tolerance can be established within 3 days after the two tolerogenic doses and lasts for at least 73 days (Fuller et al., 1993), this window was applied to examine suppression directly in the tolerized host. The availability of CD4 and CD8 mAbs for depletion studies in vivo enabled us to demonstrate that only depletion of CD4+ T cells abrogated tolerance, again supporting the hypothesis of active suppression. Thus, regulatory T cells in mice with established tolerance belonged to the CD4+ subset (Kong et al., 1989).
3.3. Effect of Cytokines on CD4+ Regulatory T Cell Induction and
To determine the mechanisms of CD4+ T cells regulating induced resistance to EAT induction, several cytokines with inhibitory or proinflammatory activity were examined. The possible role of Th2 cells in regulatory T cell function was suggested by the involvement of interleukins IL-4 and IL-10. Repeated doses of IL-10 into animals prevented the onset of diabetes (Pennline et al., 1994), experimental autoimmune encephalomyelitis (EAE) (Rott et al, 1994), and EAT (Mignon-Godefroy et al., 1995). Also, IL-4 was shown to protect animals from EAE (Racke et al., 1994) and diabetes (Rapoport et al., 1993). Accordingly, we examined the role of IL-4 and IL-10 in tolerance to EAT induction. Antibodies to IL-4 and IL-10 were initially given separately in conjunction with tolerance induction, and the animals were then challenged with mTg and LPS. No apparent effect of either cytokine was observed, and the mice became tolerant to EAT (Zhang and Kong, 1998). In the event that CD4+ regulatory T cells in tolerant mice released IL-10 to inhibit the response of autoreactive T cells during challenge, IL-10 mAb was injected in conjunction with immunization. No effect on the already established tolerance was seen and the animals remained unresponsive. We then combined IL-4 and IL-10 mAb administration at the time of tolerance induction, and, alternatively, administered IL-10 mAb to IL-4-knockout mice. Again, no interference with tolerance establishment was observed. The response of control IL-4-deficient mice to EAT induction was comparable to that of the wild type, indicating that the absence of IL-4 does not increase thyroiditis severity. These data demonstrate the noninvolvement of the two Th2 cytokines in EAT tolerance, but do not rule out any joint involvement with other cytokines, such as TGF-B.
3.3.2. Interference with Tolerance Induction by IL-1 and IL-12
EAT is a cell-mediated autoimmune disease involving Th1 cells and proin-flammatory cytokines, but the two major Th2 cytokines apparently are not involved in regulating EAT tolerance. We selected IL-1 and IL-12 as representatives of proinflammatory cytokines to test their capacity to interfere with tolerance induction. IL-1 has multiple biologic effects (Dinarello, 1989), one of which is its adjuvant effect for non-self-protein antigens (Staruch and Wood, 1983). Because of the short t1/2, IL-1B was found to be most effective when given 3 hr after, but not before, dmTg and less effective when given 24 hr after (Nabozny and Kong, 1992). IL-12 exerts its action primarily on Th1 cell differentiation and has been shown to accelerate the onset of and increase the incidence of cell-mediated autoimmune diseases, such as diabetes (Trembleau et al., 1995), collagen-induced arthritis (Germann et al., 1995), EAE (Leonard et al., 1997), and experimental autoimmune uveitis (Caspi, 1998). On interference with induction of EAT tolerance, IL-12, with a longer t1/2 than IL-1B, was effective when given twice, on the day of and the day after dmTg (Zhang et al., 2001). The treated animals were not protected against challenge, as tolerance had not been established. The interference of tolerance induction by IL-12 was not neutralized by the presence of anti-IFN-y. On the other hand, T cell costimulatory molecules may be involved through IL-12 activity. Either CD40L or CD28 mAb ameliorated the priming effect of dmTg and IL-12, compared to the IgG control group. Thus, both cytokines can convert a tolerogenic signal to a priming signal, thereby increasing thyroiditis severity after challenge. However, similar to the stronger adjuvants, complete Freund's adjuvant or LPS, neither cytokine can overcome established tolerance when given at the time of challenge. These data suggest that proinflammatory signals derived from ongoing immune responses or from cytokine immunotherapy could potentially interfere with the continuing induction of peripheral tolerance to self-antigens in susceptible individuals.
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