Clin. exp. Immunol. (1990) 81, 189-194
Prevention of adjuvant arthritis in rats by a nonapeptide from the 65-kD mycobacterial heat-shock protein X.-D. YANG, J. GASSER & U. FEIGE Research Department, Pharmaceuticals Ditision, Ciba-Geigy,
(A ccepted for publication 8 May 1990)
SUMMARY model of T cell-mediated
autoimmune arthritis resembling Adjuvant arthritis in Lewis rats is a human rheumatoid arthritis. A nonapeptide from the 65-kD heat-shock protein of Mycobacterium boais BCG, amino acid sequence 180-188, has been described to carry the dominant immunogenic epitope(s) for both arthritis-protective and arthritogenic T cell clones. Here we demonstrate that immunizations with the synthetic nonapeptide completely protected rats against adjuvant arthritis induced by M. tuberculosis. Interestingly, deletion of the N-teminal threonine of the nonapeptide resulted in loss of the protective activity. Pretreatments with the nonapeptide resulted in an immune response to the nonapeptide and to M. tuberculosis. After immunizations with the synthetic nonapeptide, only low titres of nonapeptide-specific antibodies were produced, whereas a significant cellular immune response to the nonapeptide was observed. In addition, the protection was transferable to naive rats by spleen T cells. These findings document the requirement of a T cellspecific immune response to the dominant epitope of the 65-kD mycobacterial heat-shock protein for the protection against adjuvant arthritis and suggest the feasibility of immune intervention in autoimmune arthritis through the use of synthetic peptides.
Keywords heat shock protein autoimmunity inflammatory arthritis
synthetic nonapeptide (SNP) would either activate an A2b-like T cell subset to induce arthritis or it would stimulate an A2c-like T cell subset resulting in a protective response against AA (van Eden et al., 1988; Yang et al., 1990). In a previous study, we have shown that administration of SNP suspended in mineral oil did not induce AA but was able to suppress AA partially in Lewis rats (Yang et al., 1989, 1990). In the present experiments, Lewis rats were immunized with SNP emulsified with Freund's incomplete adjuvant (FIA) prior to induction of AA. In separate experiments, 11 out of 16 rats were completely protected against subsequently induced AA. Our results demonstrate that the induction of an immune response to the critical epitope might be involved in the prevention of autoimmune arthritis.
INTRODUCTION Recent studies have demonstrated that the dominant immunogenic epitopes of some T cell-mediated experimental autoimmune diseases can be precisely defined by using disease inducing T cell clones as tools (Zamvil et al., 1986; van Eden et al., 1988). In adjuvant arthritis (AA), a model of autoimmune arthritis inducible in certain strains of rats such as in Lewis rats, by heatkilled Mycobacterium tuberculosis (Pearson, 1956; 1964; Taurog, Argentieri & McReynolds, 1988), a 65-kD mycobacterial heat-shock protein (HSP) has been proposed to be involved in the pathogenesis of AA (van Eden et al., 1988). Administration of the 65-kD HSP led to protection against AA and streptococcal cell-wall-induced arthritis in Lewis rats (van Eden et al., 1988; van den Broek et al., 1989; Billingham et al., 1990). A nonapeptide (Thr-Phe-Gly-Leu-Gln-Leu-Glu-Leu-Thr), representing amino acid sequence 180-188 of the 65-kD mycobacterial HSP, has been suggested to carry the critical epitope(s) for AA because of its ability to stimulate the arthritis-inducing T cell clone A2b as well as the arthritis-protective T cell clone A2c in vitro (Holoshitz, Matitiau & Cohen, 1984; Cohen et al., 1985; van Eden et al., 1988; van der Zee et al., 1989). Based on these observations, it was suspected that immunization with the
MATERIALS AND METHODS Animals Inbred male Lewis rats were obtained from Versuchstierzucht Ciba-Geigy, Werk Stein. The rats were fed a standard diet and tap water. Rats weighed 100-150 g at the start of the experiments.
Induction and evaluation of arthritis AA was induced by injection of 01 ml of Freund's complete adjuvant (FCA) containing 5 mg/ml heat-killed Mycobacterium
Correspondence: Dr X.-D. Yang, R-1056. 1.18, Ciba-Geigy, CH4002 Basle, Switzerland.
X.-D. Yang, J. Gasser & U. Feige
tuberculosis H37Ra (Difco) intradermally at the base of the tail. The day of arthritis induction was designated as day 0. Rats were examined daily for clinical signs of arthritis (erythema, swelling and deformity) and an arthritis index assigned as described (Taurog et al., 1988): 0, no evidence of disease; 1, inflammation of small digital joints; 2, inflammation of ankle and joints; 3 inflammation of foot-pad; and 4, inflammation of entire foot. Each paw was given a score between 0 and 4, the highest score being 16 per rat. Arthritis was also confirmed and evaluated by examination of radiographs (Taurog et al., 1988; Yang et al., 1990). Rats were anaesthetized with Evipan-sodium (100 mg/kg, Bayer) and radiographs were taken for fore and hind limbs of rats with a Mammomat (Siemens), on Kodak Xomat MA film exposed at 30 kV for 160 msec. Scores of 1-4 were assigned to each joint for peri-osteal reaction, bony erosions, joint space narrowing and joint space destruction. A total of 20 joints ofeach limb were evaluated, resulting in a maximum score of 320 per rat.
Synthetic peptides Two peptides (SNP, Thr-Phe-Gly-Leu-Gln-Leu-Glu-LeuThr, representing sequence 180-188; and SOP, CH3CO-PheGly-Leu-Gln-Leu-Glu-Leu-Thr, representing sequence 181-188 of the 65-kD HSP of M. bovis BCG; van Eden et al., 1988; van der Zee et al., 1989) were synthesized for this study. Peptides were prepared by solid-phase method on a polystyrene resin with the p-benzyloxy-benzylalcohol linker. Coupling reactions were performed with Fmoc amino acid trichlorophenylesters, and the side-chains of threonine and glutamic acid were protected by tert.butyl ether and tert.butyl ester, respectively. After completion, the free peptide was obtained by cleavage with 95% counter-current distribution in the solvent system n-butanol/methanol/I N aqueous ammonia (5/1/5) and obtained in chromatographically pure form. Treatment regimen Peptides dissolved in phosphate-buffered saline (PBS) were emulsified with an equal volume of FIA. Rats were injected intraperitoneally with 0 1 ml FIA emulsion containing 0-1 mg of the synthetic peptides at days -35, -20 and -5 before induction of AA. Control rats received 01 ml PBS/FIA emulsion.
Lymphocyte proliferation assays Spleen cells were obtained from rats before or 35 days after induction of AA and mononuclear cells (MNC) were prepared by Ficoll-Paque (Pharmacia) separation. MNC (5 x 105 cells per well) were incubated in flat-bottomed microtitre plates (Falcon No. 3072) with the following stimuli: SNP and SOP (25 pg/ml), M. tuberculosis (10 pg/ml) and concanavalin A (Con A) (2.5 pg/ ml) for 72 h. Culture medium was RPMI 1640 (GIBCO) supplemented with 5% heat-inactivated fetal calf serum (FCS), 2-mercaptoethanol (5 x 10-5 M), 10-9 M glutamine, vitamins and amino acids (GIBCO). Each test was performed in triplicate. Six hours before harvesting the cells, 0 5 pCi of 3H-TdR (Amersham) was added to each well. The cells were harvested onto glass fibre filters using a cell harvester (LKB Wallac 1295-001) and the incorporation of 3H-TdR was measured by liquid scintillation counting (LKB Wallac 1205 Betaplate) and expressed as ct/min.
Delayed-type hypersensitivity (DTH) reaction In order to test the in vivo cellular immune response, DTH reactions to peptides and M. tuberculosis were checked as described (Powell & Greene, 1988). The rats were injected subcutaneously with 50 Sl PBS containing 1 mg/ml of synthetic peptides or M. tuberculosis into an ear before or 33 days after induction of AA. The ear swelling was measured after 24 h and 48 h using a Vernier caliper and expressed as A (increase) in ear
swelling. ELISA for anti-peptide or anti-mycobacteria antibodies Rats were bled 35 days after induction of AA and serum samples were collected and pooled for determination of specific antibody responses by ELISA. Flat-bottomed microtitre plates (Dynatech) were coated with peptides or M. tuberculosis (100 pl/well, I mg/ml) overnight. After washing, dilutions of serum samples were added. Following incubation for 4 h at room temperature, the plates were washed and incubated with biotinylated sheep anti-rat IgG (Sera-Lab). Four hours later, the plates were washed and streptavidin-biotinylated horseradish peroxidase complex (Sera-Lab) was added. After 2-h incubation, the plates were washed and developed by adding o-phenylenediamine (0 4 mg/ml) and H202 (0.02%). The reciprocal of serum dilution resulting in 50% of maximum absorbance at 490 nm is given as titre.
Transfer of spleen T cells to naive rats Spleens were removed from donor rats aseptically and single cell suspensions were prepared. Erythrocytes were eliminated by Ficoll-Paque gradients. Adherent cells were removed by 1-h incubation on Petri dishes (Falcon 3003) and by a further 1-h incubation with nylon wool (van den Broek et al., 1989). After extensive washing, 5-10 x 107 nylon wool-enriched T cells were transferred intravenously to naive rats. AA was induced in recipients immediately after cell transfer. RESULTS SNP is a nonapeptide representing the amino acid sequence 180-188 of the 65-kD HSP of M. bovis BCG (van Eden et al., 1988). The nonapeptide carries the epitope recognized by both arthritogenic and arthritis-protective rat T cell clones (van der Zee et al., 1989). We have previously shown that prophylactic (at day -20 or at days -2, - 1, 0) or therapeutic (at days 7, 8, 9, 10) i.p. treatment with SNP in oil did result in a partial suppression of AA in Lewis rats (Yang et al., 1989, 1990). These results raised the question whether the potential of SNP to prevent AA could be improved. In the present study, rats were treated with SNP emulsified in FIA intraperitoneally at days -35, -20, -5 (Table 1). During the 35-day pretreatment period none of the rats showed any clinical symptoms of AA, indicating that SNP is not arthritogenic (Billingham et al., 1990; Yang et al., 1990). In two separate experiments, we found after induction of AA that 11 out of 16 rats were completely protected (Table 1); furthermore on radiographs no indication of arthritis was found in these rats (Table 2). In addition, in SNP-pretreated groups, rats that did develop AA were found to have lower clinical as well as radiographical scores compared with rats of the FIA-pretreated control groups (Tables I and 2). Interestingly, SOP, an N-acetylated octapeptide lacking the N-terminal threonine of
Protection against adjuvant arthritis by a synthetic epitope of HSP
Table 1. Prevention of AA by SNP: clinical observations* Experiment I Immunizations witht
SNP+FIA SOP+FIA FIA
Incidence of AA
Maximal arthritis index of individual rats
Incidence of AA
Maximal arthritis index of individual rats
0, 0, 0, 0, 9, 9, 10 ND 12, 12, 13, 14, 14, 14, 14
2/9 9/9 9/9
0, 0, 0, 0, 0, 0, 0, 4, 9 10,11,11,12,14,14,14,16,16 9, 10, 10, 12, 12, 13, 14, 14, 16
* Rats were checked daily for signs of arthritis and the severity of the disease was scored as described in Materials and Methods. t Rats were immunized with synthetic peptides according to the treatment regimen described in Materials and Methods. AA, adjuvant arthritis; SNP, synthetic nonapeptide; SOP, synthetic octopeptide; FIA, Freund's incomplete adjuvant; ND, not done.
Table 2. Prevention of AA by SNP: radiographic examination*
Radiographic scores of individual rats
Incidence of AA
Radiographic scores of individual rats Day 35
Incidence of AA
0, 0, 0, 0, 27, 33, 41
62, 64, 72, 74, 76, 79, 82
0, 0, 0, 0, 0, 0,0,8,9 9, 11, 14, 17, 18, 19, 19, 21, 23 19, 23, 25, 26, 27, 29, 30, 30, 37
0, 0, 0, 0, 0 0,0,24,28 17,21,27,34,38, 42,49,51,57 30, 30, 33, 35, 36, 38, 40, 42, 42
0, 0, 0, 0, 0 0,0,32,34 42,50,60,66,66, 68,82,83,93 59, 63, 69, 72, 78, 80, 81, 83, 90
* Radiographs were taken 15,25, and 35 days after induction of AA and the radiographical scores were evaluated as described in Materials and Methods. t Immunization protocol as in Table 1. Abbreviations as in Table 1.
SNP, did not have any protective capacity (experiment 2, Tables I and 2). AA incidence and severity in SOP-pretreated rats were not different from FIA-pretreated controls (Tables 1 and 2). This points to the specificity of the SNP effect in AA. It has been postulated that suppression of autoimmune disease in animals is usually caused by the induction of tolerance to the autoimmune disease-inducing antigen (Higgins & Weiner, 1988; Thomson et al., 1988; Clayton et al., 1989), i.e. the protection against mycobacteria-induced arthritis after pretreatment with whole mycobacteria or the 65-kD HSP, is due to induction of tolerance (Grey & Waksman, 1967; Cozine et al., 1972; van den Broek et al., 1989). This led us to investigate the cellular immune response of the rats to SNP, SOP and M. tuberculosis in vivo and in vitro after 35 days of pretreatment but before induction of AA and also 35 days after induction of AA. Before induction of AA, SNP-pretreated rats showed a DTH to SNP, but not to M. tuberculosis (Table 3). Normal rats and FIA-pretreated rats did not show a DTH to SNP. An in vitro proliferative response of splenic MNC to both SNP and M. tuberculosis is found in SNP-pretreated rats, but not in normal or FIA-pretreated rats (Table 3). Interestingly, no crossreactivities between SOP and SNP or M. tuberculosis were
observed (Table 3). This is consistent with the fact that T cell clones A2b and A2c do not recognize the epitope without the N-terminal threonine (van der Zee et al., 1989). Therefore it can be concluded that the threonine at position of 180 of the 65-kD mycobacterial HSP is important for immunogenicity of the nonapeptide. After induction of AA (day 35) DTH to M. tuberculosis was identical in rats from all groups (Table 3). However, rats with AA did not show a DTH to SNP. In contrast, SNP-pretreated and AA-protected rats showed a DTH to SNP. Interestingly, SNP-pretreated rats which developed AA did show a DTH to SNP in experiment I but not in experiment 2. The proliferative responses of splenic MNC to M. tuberculosis, like the DTH, were comparable in all rats (Table 3). Splenic MNC from SNPpretreated rats, but not from rats of the control groups, proliferated in response to SNP. Antibody titres specific to M. tuberculosis were found to be high in rats from all groups (Table 4). In SNP-pretreated, but not in rats from the other groups, a low titre antibody response to SNP was measured. From the results above it was concluded that pretreatments with SNP do not result in an induction of tolerance to SNP or
X.-D. Yang, J. Gasser & U. Feige
Table 3. Cellular immune response*
DTH response (A mm x 10-2 mean + s.d.)
Induction of AA
none SNP+IFA FIA
8 10 10
no no no