Immune responses to mycobacterial heat-shock protein 65 (Bhsp65) play a critical role in the immunopathogenesis of AA in the LEW rat 212224. The T-cell response to Bhsp65 determinant 180 to 188 has been implicated in disease induction in LEW rats 21. (Bhsp65 peptide 180 to 188 is cross-reactive with its longer version, Bhsp65 peptide 177–191 24.) With progression of AA, arthritic LEW rats raise an immunoregulatory T-cell response to Bhsp65 C-terminal determinants that apparently contributes to recovery from acute AA 24. Moreover, these rats spontaneously develop T-cell responses to (self) rat heat-shock protein 65 (Rhsp65) and the Rhsp65 C-terminal determinants, which are protective against AA 2627. The T cells against Bhsp65 C-terminal determinants are cross-reactive against Rhsp65 C-terminal determinants 27.

Generally, immune response to self-antigens is believed to initiate autoimmunity. Studies by other workers 28 and by us 2629, however, have shown that immune reactivity to self-hsp65 is immunoregulatory in the AA model 26 as well as in patients with juvenile idiopathic arthritis 28. Immunization of LEW rats with self-hsp65 affords protection against subsequent induction of AA by injection of Mtb in oil 26. In juvenile idiopathic arthritis, increased immune response to self-hsp65 correlates with a favorable outcome of the disease 28.

We have compared the arthritis-susceptible LEW rats with the arthritis-resistant WKY rats that share the same MHC haplotype (RT.1^l) 202430. Following Mtb challenge, both LEW rats and WKY rats raised comparable levels of recall T-cell proliferative response to Bhsp65 and its arthritogenic determinant Bhsp65 peptide 177 to 191/Bhsp65 peptide 180 to 188 30. Unlike LEW rats, however, WKY rats raised a T-cell response to Bhsp65 C-terminal determinants early after Mtb injection. Moreover, WKY rats gave a higher level of recall T-cell response to Rhsp65 compared with that of LEW rats before the appearance of any clinical signs of AA in the latter 20. These results suggest that the temporal kinetics of the appearance of a T-cell response against Rhsp65 and against Bhsp65 C-terminal determinants/Rhsp65 C-terminal determinants following Mtb injection is an important determinant of disease susceptibility in AA.

In a study on Wistar rats, a deficiency in T-cell response to Bhsp65 upon Mtb injection correlated with protection against AA 31. Wistar rats without arthritis gave a significantly reduced level of T-cell response to Bhsp65 compared with rats with arthritis, but both rat groups gave a comparable level of T-cell response to the immunogen Mtb.

Unlike LEW rats, F344 rats are relatively resistant to AA. Differences in the T-cell repertoire against Bhsp65 and its arthritogenic determinant Bhsp65 peptide 180 to 188 have been proposed as one of the reasons for the AA resistance of F344 rats 3233. Furthermore, F344 rats were neither defective in antigen/epitope processing and presentation nor displayed active suppression of arthritogenic T-cells in vitro 33.

New complexities regarding the role of cytokines in the immunopathogenesis of arthritis as well as other diseases are emerging as new information about cytokines comes forth 30343536. AA was shown to be adoptively transferred to naïve rats by a well-characterized T-cell clone, A2b, which is a CD4⁺T cell that secretes IFNγ 2137. Similarly, Mtb-primed T cells that can transfer disease are mostly T-helper type 1 (Th1) 37. Our studies in AA, however, have revealed a protective role of IFNγ and TNFα 3036.

Until recently, most of the available information regarding the temporal cytokine profiles in arthritic LEW rats pertained to antigen nonspecific ex vivo cytokine secretion or to cytokine mRNA levels 3839. In our recent studies in AA 3036, we observed higher Th1 cytokine levels against Bhsp65 during the recovery phase compared with those at the onset of or the peak phase of the disease. The cytokine response to Rhsp65 was found to be similar to that against Bhsp65 20. In general, the cytokine response of T cells against Bhsp65 and Rhsp65 as well as their defined antigenic determinants consisted predominantly of proinflammatory cytokines, IFNγ and TNFα . Furthermore, higher levels of proinflammatory cytokine secretion correlated with reduced severity of arthritis.

As AA is a Th1-mediated disease, this observation pointed to the role of Th1 cytokines in disease regulation. This inference indeed was supported by the cytokine profiles of Mtb-immunized, AA-resistant WKY rats. These rats produced high levels of Th1 cytokines early following Mtb injection, and the overall shape of the cytokine profiles was almost the opposite of that of LEW rats when followed against time post Mtb injection. Furthermore, treatment of LEW rats with TNFα or with Rhsp65 peptide 465 to 479, which primes a Th1 response, resulted in downmodulation of the clinical course of AA in LEW rats, albeit employing different mechanisms 3036. The levels as well as the timing of production of Th1 cytokines in response to Bhsp65/Rhsp65 therefore have a significant influence on the outcome of Mtb challenge of LEW/WKY rats. Other investigators have reported differences in the frequency/activity of T-helper type 2-cytokine secreting cells and CD8⁺ suppressor/regulatory T cells in AA-susceptible rats (LEW rats, Holtzman rats) versus AA-resistant rats (BN rats, Buffalo rats, and Wistar Albino Glaxo rats) 404142.

Two aspects of cytokine function are of significance when examining the susceptibility/resistance to AA. First, the pro-inflammatory cytokines (for example, TNFα and IFNγ) are not always pathogenic; instead, they can be immunoregulatory in nature 3036. Second, the T cells of arthritic LEW rats specific for the arthritogenic determinant of Bhsp65 (Bhsp65 peptide 180 to 188) produced IL-17 30. The contribution of IL-17 as well as the cytokines regulating IL-17 (for example, IFNγ, IL-4, and IL-27) in conferring susceptibility/resistance to AA, however, remains to be determined.

AA can be adoptively transferred to naïve syngeneic recipient rats via the T cells but not the serum antibodies of arthritic rats. Antibodies per se are therefore not considered arthritogenic in AA. Studies by other workers and us 2325, however, have revealed that antibodies against Bhsp65 and its mammalian homologue (Rhsp65) are protective in AA, and that the antibody profiles of AA-susceptible LEW rats are significantly different from that of AA-resistant BN rats and WKY rats.

In a study on Wistar rats immunized with Mtb, the nonarthritic subgroup of rats showed much higher antibody response compared with that of the arthritic subgroup of rats – suggesting a protective role of anti-Bhsp65 antibodies 31. In another study, it was shown that AA in LEW rats could be suppressed by a low dose of antibodies derived from the AA-resistant rat strains 43. The AA-protective antibodies were reactive against mycobacteria and the depletion of anti-mycobacterial antibodies abrogated the protective effect of the antibodies. The protective antibodies were found in AA-resistant rats (F344 rats and BN rats) but not in AA-susceptible rats (LEW rats and Wistar rats). Furthermore, the presence or absence of these antibodies in different rat strains was not much influenced by the housing environment (germ-free versus conventional) in which the rats were kept 43. In addition, serum transfer neither influenced the cell-mediated immune response to Mtb nor the protection against reinduction of AA in arthritis-recovered rats 43. In contrast to the above results, another study indicated that natural antibodies to Bhsp65 are present in LEW rats but there was no association between the level of natural antibodies and the severity of AA 44.

Testing of the anti-hsp65 antibody response in naïve LEW rats, naïve BN rats and arthritic LEW rats revealed important differences in AA-susceptible rat strains versus AA-resistant rat strains 23. Naïve LEW rats tested at different ages (6 weeks, 4 months and 9 months) showed spontaneous development of anti-Bhsp65 antibody response that gradually diversified in epitope reactivity. In contrast, naïve BN rats at an early age (6 weeks) showed reactivity to multiple epitopes of Bhsp65 that were also responded to by 9-month-old naïve LEW rats. Arthritic LEW rats showed reactivity to all these epitopes along with a couple of additional epitopes. The above three groups of rats also showed reactivity to two epitopes of self-hsp65, epitope 61 to 80 and epitope 436 to 455. Furthermore, pretreatment (tested separately) of naïve LEW rats with Bhsp65 peptide 31 to 46 and Bhsp65 peptide 37 to 52, Rhsp65 peptide 61 to 80, and antibodies to peptide 31 to 46 induced protection against subsequent AA 23.

In our study, both LEW rats and WKY rats showed antibody reactivity against multiple peptides of Bhsp65 and Rhsp65, but displayed opposite antibody profiles with time post Mtb injection 25. The antibody response in WKY rats was broad to begin with but contracted with time post Mtb injection, whereas that of LEW rats was initially restricted to a few epitopes but later spread to include other epitopes. Eventually, the antibody response of both rat strains was narrowed down to the epitopes 31 to 46, 211 to 226 and 349 to 364 of Bhsp65 and to epitope 61 to 80 of Rhsp65. These epitope regions 25 are included among the epitopes reacted by sera of LEW rats and BN rats 23. Moreover, adoptive transfer of sera of late phase arthritic LEW rats into naïve recipient LEW rats induced protection against AA 2325. There is therefore a common pattern and function of the humoral immune response to hsp65 shared by the arthritis-resistant WKY rats and BN rats, and this pattern in turn is different from that of arthritis-susceptible LEW rats.

In one study, the expression within the joints of two mammalian hsps (hsp47 and hsp72) of arthritis-susceptible DA rats was compared with that of arthritis-resistant AO rats 45. Also examined was the relative T-cell proliferation and antibody response to hsp47 and mycobacterial hsp71. Important differences were revealed in these parameters in DA rats and AO rats following an arthritogenic challenge with Mtb.

Immunization with Mtb led to a significant increase in the expression of hsp47 in the joints of DA rats but not AO rats 45. The expression of hsp47 presumably correlated with its role in procollagen production and processing. In contrast, no change in hsp72 expression in the joints was found in either rat strain, suggesting that the disease induction was not related to the local expression of hsp72. Both hsp47 and hsp71 caused an inhibition of lymph node cell (LNC) proliferation in AO rats but not in DA rats following in vitro restimulation of LNC with these hsps 45. For the antibody response, an opposite pattern was observed for anti-hsp47 and anti-hsp71 antibodies 45. The former were increased in DA rats, implying their association with a pathogenic response, while the latter were increased in AO rats, suggesting their link with protection against AA.

Another factor implicated in the AA resistance of AO rats is the increased production of ROS 10. The ROS production was enhanced by hsp47 but not by hsp71, suggesting the role of hsp47 in controlling the disease process as well 10. The protective effect of hsp71 in AA has also been reported in studies using the LEW rat 46. hsp47 induces certain effector responses in the synovial tissue that facilitate disease induction/propagation, whereas the same protein may also contribute to downmodulation of the disease via increasing ROS 104546. The precise conditions (the level and timing of expression of hsp47) that lead to these two different outcomes need to be defined.

Studies in experimental arthritis models have shown that LEW rats, which are susceptible to AA as well as to streptococcal cell wall-induced arthritis, have reduced plasma levels of adrenocorticotropic hormone and corticosterone in response to streptococcal cell wall fragments as well as in response to IL-1α when compared with that of arthritis-resistant F344 rats 51. The development and severity of arthritis in these two rat strains could be modulated in the opposite direction by administration of dexamethasone to LEW rats (which caused a reduction in arthritis severity) and by injection of a glucocorticoid receptor antagonist to F344 rats (which caused increased arthritis severity) 51.

A comparative study using electric tail shock to induce acute stress in five inbred rat strains, including LEW rats, F344 rats, and WKY rats – which were also subjected to altered (increased/attenuated) glucocorticoid negative feedback – showed that LEW rats displayed reduced response to stress (as assessed by measurement of adrenocorticotropic hormone and corticosterone) compared with F344 rats and WKY rats 52. Furthermore, glucocorticoids can cause immune cells to undergo a Th1 to T-helper type 2 shift and increase the secretion of anti-inflammatory cytokines IL-10 and IL-4, while decreasing the secretion of proinflammatory cytokines TNFα and IL-1 53. This change in cytokine milieu, coupled with alteration in chemokines and chemokine receptor expression, can have a significant influence on the development and progression of arthritis, including AA, which is a Th1-mediated disease.

The involvement of the HPA axis in regulating peripheral inflammation was further validated by experiments showing that intracerebroventricular transplantation of fetal hypothalamic tissue from F344 rats to LEW rats led to a significant reduction in carrageenan-induced inflammation in recipient rats 54. This transplant resulted in the expression of hypothalamic factors (for example, corticotropin-releasing hormone and corticosterone) in response to inflammatory stimuli.

With regard to behavior of rat strains and arthritis susceptibility, experiments in different rat strains (DA rats, LEW rats, AO rats, PVG rats, outbred Wistar rats) indicated that the development of arthritis and severity of the disease upon Mtb injection correlated well with specific behavioral patterns of the rat strains 55. A study comparing apomorphine-susceptible versus apomorphine-unsusceptible Wistar rats showed that the former were relatively resistant to AA, whereas the latter developed arthritis of moderate severity 56. In another study, Wistar rats were separated into two groups based on their learned helplessness (which also forms the basis for an animal model of depression), and were then immunized with Mtb 57. Learned helplessness-negative rats developed AA earlier and of higher severity compared with learned helplessness-positive rats, although the corticosterone response to acute stress was much higher in learned helplessness-negative rats than that of learned helplessness-positive rats 57.

An interesting correlation has been observed between susceptibility/resistance of inbred rat strains to arthritis and drug addiction 58. Rat strains that are highly susceptible to AA (for example, LEW rats and DA rats) are also prone to addiction to drugs like cocaine. Similarly, strains such as BN and F344 that are resistant to arthritis are also relatively resistant to addiction. As these two diseases/behavioral phenotypes also correlate with the level of HPA axis activity, common mechanisms underlying these three physiopathological parameters have been suggested 58.