Clin. exp. Immunol. (1992) 87, 99-104
Prevention of adjuvant arthritis in rats by a nonapeptide from the 65-kD mycobacterial heat shock protein: specificity and mechanism X.-D. YANG, J. GASSER & U. FEIGE Research Department, Pharmaceuticals Division, Ciba-Geigy, Basle, Switzerland
(Acceptedfor publication I August 1991)
SUMMARY In a previous study we have shown that Lewis rats were completely protected from adjuvant arthritis by pretreatment with a nonapeptide (residues 180-188) of the 65-kD mycobacterial heat shock protein. Here we address questions of specificity and mechanism(s) of protection. We demonstrate that complete protection against adjuvant arthritis can only be achieved by pre-immunization with the nonapeptide, while pretreatment with either the octapeptide (residues 181-188) of the 65-kD heat shock protein or unrelated immunogenic peptides failed to affect adjuvant arthritis. Interestingly, pretreatment with the nonapeptide of the 65-kD heat shock protein did not protect Lewis rats from type II collagen-induced arthritis. These results demonstrate that protection is both epitope and disease specific. Co-injection of the nonapeptide with mycobacterial antigen even at a weight ratio of 5:1 (nonapeptide: mycobacteria) failed to influence the disease, suggesting that the role of the nonapeptide is not as a 'blocking peptide'. T cells from rats immunized with nonapeptide respond to the nonapeptide as well as to mycobacteria in vitro, and adoptively transfer protection to naive recipients. The data indicate that the nonapeptide-induced protection may result from a T cellmediated specific suppression. Keywords T cell epitope autoimmune arthritis heat shock protein
INTRODUCTION Adjuvant arthritis (AA) can be induced in genetically susceptible strains of rats (e.g. Lewis rats) by a single injection of mycobacterial adjuvant . AA in Lewis rats has been extensively studied  as a model of human rheumatoid arthritis (RA), not only because of histopathological similarities of AA and RA , but also because several clinical symptoms observed in rats with AA have been observed in patients subjected to repeated treatment with mycobacterial adjuvant as part of immune therapy against cancer [4,5] and as sequelae after immunization against tuberculosis in childhood . AA can be transferred with T cells from diseased rats to naive recipients, and these T cells recognize mycobacterial components as well as self antigens, indicating that AA is a T cell-mediated autoimmune disease [7-9]. Nevertheless, some critical questions remain unanswered: how do mycobacterial antigens evoke an anti-self immune response leading to the autoimmune destruction of joints? What is common in the immunopathogenesis of AA in rats and RA in human beings? What can we learn about the etiology of RA from the AA model? To answer these questions, many investigators have recently studied the immune recognition of mycobacterial cell wall antigens, particularly the
65-kD heat shock protein (hsp65) of Mycobacterium tuberculosis and its epitopes in the pathogenesis of AA. hsp65 was found to be a dominant antigen of mycobacteria , and to be involved in AA [11,12]. An epitope consisting of nine amino acids (residues 180-188) of hsp65 appears to be critical in the pathogenesis of AA since this epitope is recognized both by arthritis-inducing (A2b) and arthritis-preventing (A2c) T cell clones [11,13]. Pretreatment of rats with mycobacterial hsp65 results in protection against AA [1 1,14], streptococcal cell wallinduced arthritis  as well as pristane-induced arthritis . Also, pretreatment with the peptide (180-188) has been shown to prevent AA [12,17]. hsp65 is produced endogenously in the synovial tissues of patients with RA and rats with AA [18,19], and significant immune responses to mycobacterial hsp65 were found both in the peripheral blood and synovial fluid of patients with RA and other chronic arthritides . hsp65, which is present ubiquitously both in prokaryotes and eukaryotes is highly conserved and immunogenic [21,22]. Importantly, immune responses to hsp were found in several autoimmune diseases such as systemic lupus erythematosus (SLE) in humans  or insulin dependent non-obese diabetes in NOD mice . These findings throw new light onto the study of autoimmune diseases in general and particularly provide possibilities for studying immunopathogenic mechanisms of autoimmune diseases at the molecular level by using recombinant hsp65 and its synthetic epitopes .
Correspondence: Dr X.-D. Yang, Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA
X.-D. Yang, J. Gasser & U. Feige Table 1. Alignment of the amino acid residues of synthetic peptides
Amino acid sequence
SNP SOP pAE
T-F-G-L-Q-L-E-L-T Ac-F-G-L-Q-L-E-L-T A-A-A-A-A-E-E-E-E
Source of the peptide
Synthetic nonapeptide of hsp65 (residues 180-188) Synthetic octapeptide of hsp65 (residues 181-188) Synthetic nonapeptide with polyalanine and glutamic acid Synthetic decapeptide with random amino acids and sequence
Our recent finding that the synthetic nonapeptide (residues 180-188, SNP) of hsp65 is not arthritogenic but could prevent and suppress AA [12,17] and furthermore that the protective activity of SNP could be improved by applying SNP in incomplete Freund's adjuvant emulsion  prompted us to study the epitope and disease specificity of SNP pretreatment as well as the underlying mechanism leading to resistance against AA. MATERIALS AND METHODS
Animals Inbred male Lewis rats weighing 100- 150 g at the start of the experiment were used.
Induction of arthritis AA was induced by injection of 0-1 ml complete Freund's adjuvant (CFA) containing 5 mg/ml heat-killed M. tuberculosis (Difco, Detroit, MI) intradermally at the base of the tail. Bovine collagen type II was prepared and collagen arthritis was induced as previously described . Briefly, collagen was dissolved overnight at a concentration of 2 mg/ml in 0-1 N acetic acid. An emulsion was made with incomplete Freund's adjuvant (IFA) (Difco) at a ratio of 1:1. Rats were immunized by i.d. injection of 0-1 ml of emulsion (0- 1 mg collagen per rat) at the base of the tail. An identical booster injection was given 7 days later. The solutions and emulsions were prepared at 4CC or in an ice bath to prevent denaturation of the collagen before immunization. The day of CFA injection or of the first collagen injection was designated as day 0 . Evaluation of arthritis Rats were examined daily for clinical signs of arthritis (erythema, swelling and deformity) and disease severity was examined by an arthritis score assigned: 0, no evidence of disease; 1, inflammation of small digital joints; 2, inflammation of ankle and joints; 3, inflammation of footpad; 4, inflammation of entire foot. Each paw was given a score between 0 and 4, the highest possible score being 16 per rat. Arthritis was also confirmed and evaluated radiographically. Rats were anaesthetized and radiographs were taken of the fore- and hindlimbs with a Mammomat instrument (Siemens, Zurich, Switzerland), on Kodak X-omat MA film exposed at 30 kV for 160 ms. Separate scores of 0 to 4 were assigned to each joint for periosteal reaction, bony erosions, joint space narrowing and joint space destruction. A total of 20 joints of each limb were evaluated resulting in a maximum possible score of 320 per rat .
Synthetic peptides The nonapeptide (residues 180-188) and the N-acetylated octapeptide (residues 181-188) of mycobacterial hsp65 (Table 1) were synthesized by the solid-phase method on a polystyrene resin with the p-benzyloxy-benzylalcohol linker as described previously . Peptides pAE and plO (Table 1) were purchased from Bachem, Feinchemikalien (Bubendorf, Switzerland). Treatment regimens Peptides dissolved in phosphate-buffered saline (PBS) were emulsified in an equal volume of IFA. Rats were injected intraperitoneally with 0-1 ml IFA emulsion containing either PBS alone or 0-1 mg of peptide at days -35, -20 and -5 before induction of arthritis.
Lymphocyte proliferation assays Spleen cells were obtained at day 0 from rats after three immunizations with peptides at days -35, -20 and -5. Mononuclear cells (MNC) were separated with Ficoll-Paque (Pharmacia, Uppsala, Sweden) and incubated at 5 x 105 cells/ well in flat-bottomed microtitre plates (Falcon, Oxnard, CA) in the presence of antigen or mitogen for 72 h. Each test was done in triplicate. Six hours before harvesting 0-5 ,uCi of tritiated thymidine ([3H]-TdR) (Amersham, Amersham, UK) was added to each well. The cells were harvested onto glass fibre filters using a cell harvester, and the incorporation of [3H]-TdR was measured by liquid scintillation counting and expressed as counts per minute (ct/min). Culture medium was RPMI 1640 (GIBco, Grand Island, NY) supplemented with 5% heatinactivated fetal calf serum (FCS), 2-mercaptoethanol (5 x 10-5 M), 10-9 M L-glutamine, vitamins and amino acids (GIBCO).
Transfer of protection MNC were obtained as described above. Non-adherent cells were prepared by incubating MNC on petri dishes (Falcon) for 1 h followed by an additional 1-h incubation with nylon wool to enrich for T cells . These nylon wool-enriched T cells were injected intravenously into syngeneic naive rats (5 x 10710 x 107 cells/rat). In parallel, spleen MNC cells obtained from SNP-immunized rats were re-stimulated in vitro by culturing the MNC with 20 yg/ml SNP. After 72 h of incubation, cells were harvested and washed. The T cells were prepared and transferred as described above. AA was induced in recipients immediately after cell transfers. RESULTS SNP has been identified as a critical epitope in AA, recognized by two functionally distinct T cell clones, A2b and A2c . We
65-kD heat shock protein and adjuvant arthritis Table 2. Effect of SNP on collagen-induced arthritis* 0
Incidence of arthritis
Onset of arthritis
Arthritic scores (mean + s.d.)
10 15 20 25 30 Days after induction of arthritis
Fig. 1. Epitope-specific prevention of adjuvant arthritis (AA) by SNP. Lewis rats were pretreated intraperitoneally with SNP, SOP, pAE or p lO (01 mg) emulsified with incomplete Freund's adjuvant (IFA) or IFA alone at days -35, -20, and -5 (10-20 rats per group). AA was induced by challenging rats with complete Freund's adjuvant (CFA) containing M. tuberculosis (0 5 mg) at day 0. Rats were monitored daily for clinical symptoms (left). Radiographs were taken at day 35 and scored for disease severity (right). Left panel: arthritis incidence (%). Right panel: mean radiographic scores + s.d. *, SNP; & and M, SOP; v and M, pAE; * and plO; 0 and IFA only. U,
have previously shown that while SNP did not induce AA, it could prophylactically prevent as well as therapeutically partially suppress AA [12,17]. However, it remained unclear whether the protection is epitope-specific since the failure of the octapeptide (residues 181-188) of hsp65 to prevent AA may be simply because of its non-immunogenicity. Thus in addition to SOP, two unrelated synthetic peptides, pAE and plO, which have a molecular size similar to that of SNP (for sequences see Table 1), were selected as controls. Neither pAE nor plO crossreact with M. tuberculosis or SNP (data not shown). Following three pretreatments with the synthetic peptides emulsified in IFA at days -35, -20 and -5, AA was induced at day 0. As shown in Fig. 1, only 20% (four out of 20) of SNPpretreated rats showed signs of arthritis while the majority of the IFA control (90%, nine out of 10), the SOP- (950%, 19 out of 20), and the pAE- and plO- (90%, nine out of 10) immunized rats developed AA. The radiographic scores (Fig. 1, right panel), reflecting the disease severity, confirmed the clinical observations. Based on these results it can be concluded that the protection induced by SNP is antigen (epitope)-specific. Lewis rats are susceptible to AA as well as to collageninduced arthritis (CIA), an autoimmune arthritis induced by immunization with type II collagen. CIA has distinct immunogenic and pathogenic features in comparison with AA . It can be seen from Table 2 that pretreatment of Lewis rats with SNP was without effect on the incidence, day of onset, or severity of CIA, indicating that prevention of AA with SNP is disease-specific. We had previously shown that treatment with SNP at the time of the arthritogenic challenge with M. tuberculosis resulted in a slight suppression of AA . This effect could have been due to a so-called 'blocking peptide' mechanism . It can be seen from Table 3 that SNP or SOP did not affect the disease process when SNP or SOP were injected simultaneously with M. tuberculosis into rats even at a weight ratio of 5: 1 or 10: I (SNP or SOP: M. tuberculosis) and thus are not 'blocking peptides'.
* Lewis rats were pretreated with 0.1 ml incomplete Freund's adjuvant (IFA) emulsion containing 0 1 mg SNP at days -35, -20 and -5. Collagen arthritis was then induced at day 0 as described in Materials and Methods. The rats were checked daily for induced arthritis.
Table 3. Effect of SNP or SOP on the induction of adjuvant arthritis (AA) by M. tuberculosis
Peptide/ M. tuberculosis
Incidence Arthritic scores of AA (mean + s.d.)
M. tuberculosis SNP+ M. tuberculosis SNP + M. tuberculosis SNP+ M. tuberculosis SNP + M. tuberculosis SNP
0/5 (0/0 5) 5/1 (2 5/0 5) 3/1 (1 5/0 5) 1/1 (0 5/0 5) 1/5 (0-1/0-5) 5/0(0 5/0)
10/10 10/10 10/10 10/10 10/10 0/7
tuberculosis tuberculosis tuberculosis tuberculosis
10/1 (5-0/0-5) 3/1 (1-5/0-5) 1/1 (0-5/0-5) 1/5 (0 1/0-5) 5/0 (0-5/0
5/5 5/5 5/5 5/5 0/5
SOP+M. SOP+M. SOP+ M. SOP+ M. SOP
7-2+0-8 6-3+2-9 7-8 + 2-8
5-2+1-5 7-3 + 3-1 6-6+ 1-3 7-6+2 1 6 2 +±04 6-6+ 1 5
* Peptides were mixed with M. tuberculosis at different ratios and suspended in incomplete Freund's adjuvant (IFA). Lewis rats were injected with 0-1 ml IFA containing M. tuberculosis plus peptides, M. tuberculosis alone or peptides alone intradermally at the base of the tail. The rats were monitored and scored for arthritis.
Splenic T cells from rats protected against AA by SNPpretreatment responded to SNP in vitro and could transfer protection to naive recipients, whereas splenic T cells from rats with AA could not . However, it was difficult to draw a conclusion from this observation since the immune responses were examined after immunization with the peptides and the arthritogenic challenge with M. tuberculosis , in other words the T cells used for transfer protection were exposed to SNP as well as M. tuberculosis. Therefore, spleen MNC were taken at day 0 from peptide-pretreated rats before the arthritogenic challenge with M. tuberculosis. As shown in Fig. 2, responses to SNP, M. tuberculosis and PPD were observed only in SNPpretreated rats. MNC from SOP-pretreated rats responded slightly to SOP but not to SNP, M. tuberculosis or PPD, indicating that deletion of the N-terminal threonine of SNP resulted in a loss not only of protective activity but also of immunogenicity. The responses to concanavalin A (Con A) are similar in all SNP- and SOP-treated, and in IFA control rats,
X.-D. Yang, J. Gasser & U. Feige 25
f ~~~~100 _ /
25 C dd)
60 _W Q 0 10 100 0. 4 tuberculosis M. (Jtg/ml)
10 _ 5 041
Con A (ug/mi)
Fig. 2. Proliferation assay ([3H]-TdR incorporation) of spleen mononuclear cells (MNC) from SNP plus incomplete Freund's adjuvant (IFA) (-), SOP plus IFA, (-), or IFA (0) immunized rats in the presence of SNP (a), M. tuberculosis (b), Con A (c), SOP (d), PPD (e) or ovalbumin (OVA) (f).
demonstrating that the resistance to AA is not due to nonspecific suppression (Fig. 2c). It is intriguing to note that MNC from SNP-immunized rats respond to SNP as well as to M. tuberculosis and PPD, indicating that SNP is a dominant epitope of the whole M. tuberculosis (Fig. 2a, b, e). The higher proliferative response of MNC from SNP-treated rats to whole M. tuberculosis or its soluble components, PPD, might reflect that free SNP and the processed SNP epitope of M. tuberculosis are presented with different efficiencies. The fact that the rats after SNP pretreatment respond to SNP indicates that the 14
12 uo Co
prevention of AA is not due to induction of unresponsiveness or tolerance to hsp65. Next we asked whether the T cell population responding to SNP (described above) could transfer protection against AA in naive recipients. Spleen T cells obtained from SNP-pretreated rats were transferred into naive rats either directly or after 3 days of in vitro re-stimulation with SNP. After transfer of T cells from SNP- or SOP-pretreated rats, AA was induced in recipients. Fig. 3 shows that only rats which received T cells from SNPtreated donors were protected from the subsequently induced AA. The transfer of T cells from SOP-pretreated rats did not affect AA. The protection transferable with T cells seems to be independent of in vitro re-stimulation of the T cells with SNP since no significant difference between these two transfer groups was observed (Fig. 3). These results suggest that the protection against AA observed in SNP-pretreated rats is mediated by SNP-specific T cells and these T cells possess the potential for protection once they are specifically activated by SNP in vivo. Interestingly, the T cell population which mediates the protection seems not to be activated in rats during AA since T cells from arthritic rats transferred 35 days after induction of AA did not prevent AA in recipient rats at all .
Days after induction of arthritis Fig. 3. Transfer of protection against adjuvant arthritis (AA) by spleen T cells from SNP-immunized rats. After immunization with SNP or SOP, spleen T cells were prepared as described in Materials and Methods. The T cells from SNP or SOP immunized rats were transferred intravenously into naive rats. Also T cells from SNP-immunized rats were transferred after restimulation by SNP in vitro. Each group contains 7- 10 rats. AA was induced in recipients after cell transfer. Rats were monitored and scored for clinical arthritis (left). Radiographs were taken at day 35 and scored (right). Left panel: mean arthritic scores + s.d. Right panel: mean radiographic scores + s.d. * and transfer of T cells from SNP immunized rats; and from SOP immunized rats; * and 0, no cell transfer; and *, after in vitro restimulation with SNP. U,
DISCUSSION We had shown previously that the route of pretreatment with SNP is important for the induction of protection against AA in rats, and only i.p. treatment is effective . Splenic T cells from rats protected against AA by SNP-pretreatment could transfer the protection to naive recipients . These results left the question open whether exposure of rats to mycobacteria (the arthritogenic challenge) was necessary for the in vivo activation of the protective T cell population. Therefore we have transferred T cells from donors after SNP pretreatment (before the arthritogenic challenge with M. tuberculosis) to rats and induced
65-kD heat shock protein and adjuvant arthritis AA in recipients (Fig. 3). We found that SNP-pretreatment alone was sufficient for the induction of the splenic T cell population mediating resistance against AA, and interestingly that this T cell population had not to be activated or restimulated in vitro before transfer to be active. Also we have found that pretreatment intraperitoneally with SNP induces a splenic T cell population responding in vitro to SNP but not to SOP. This pattern of response is similar to that of T cell clones (arthritogenic) A2b and (protective) A2c , and might be taken as an indication that SNP treatment activates T cells similar to the A2b and A2c clones. However, we have never observed arthritis after immunization with SNP , suggesting that i.p. pretreatment with SNP would favour the induction of T cells functionally similar to the clone A2c. AA in rats is, so far, a unique case in which apparently a single epitope is recognized by two functionally distinct T cell clones which were reported to have identical T cell receptors (TCR) [11,29]. If this were also true for other autoimmune diseases, one might anticipate that autoimmune responses could be manipulated using the relevant T cell epitopes. This is supported by recent findings in autoimmune diabetic NOD mice where hsp65 plus PBS (non-immunogenic form) rather produces resistance, while hsp65 plus IFA (immunogenic form) induces an episode of disease, suggesting that the same antigen can produce or protect against autoimmune disease depending on the form (immunogenic or non-immunogenic) in which the antigen is administered . Recently it has been shown that vaccination with the peptide (p277) of human hsp65, which is recognized by diabetogenic T cell clones, leads to resistance against spontaneous diabetes in NOD mice . The present study indicates that it is possible to prevent autoimmune disease through an antigen-specific modulation of the immune reponse to the critical antigen or its epitopes in a model of AA, and that epitope-specific regulatory mechanisms appear to play an important role in the development of AA. Considering the fact that the 65-kD mycobacterial heat shock protein seems to be involved in several autoimmune diseases [19,20,24,25,30], multiple autoreactive T cells with specificities to different or identical epitopes on the (auto-)antigens, like hsp65, could conceivably be involved in RA as well as in other autoimmune diseases. Thus, it is extremely important to identify which epitopes are critical in the disease process. To extrapolate from the results presented here to RA in man appears difficult at present. T lymphocytes of healthy individuals can recognize shared epitopes on human and bacterial hsp65, but whether these epitopes are involved in the process of autoimmune disease is an open question [31,32]. On the other hand no T cell response has been found against SNP in rats with AA or in patients with RA . However, the apparent absence of a T cell response to an epitope does not exclude the possibility that this epitope is involved in the disease process . The successful strategy to investigate AA in rats, based on the arthritogenic clone A2b and the protective clone A2c to define SNP as an epitope involved in disease, obviously cannot be applied to RA in man directly. However, with T cell clones derived from RA patients, and based on the results of T cell vaccination obtained with those T cell clones in patients, it may be possible to develop a peptide vaccination for RA similar to that presented here for AA. We have shown here that epitopespecific and disease-specific intervention in (auto)immune disease appears feasible.
ACKNOWLEDGMENTS We would like to thank Dr P. Lipsky, and Drs J. R. Green, Y. Fang and T. Lopez for stimulating discussions and critically reading the manuscript. We are also grateful to Dr M. Glatt for continuous support and to Dr B. Riniker for the synthetic peptides.
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