Experimental autoimmune arthritis in mice

CLINICAL

IMMUNOLOGY

AND

IMMIJNOPATHOLOGY

Experimental

48,

225-231 (1988)

Autoimmune

Arthritis

in Mice

II. Early Events in the Elicitation of the Autoimmune Phenomenon by Homologous Type II Collagen’

Induced

MARIE-CHRISTOPHEBOISSIER,*ARMELLE CARLIoz,'! AND CATHERINEFOURNIER* *INSERM U. 283 and fDepartment rue du Faubourg Saint-Jacques,

of Pathology, 75674 Paris

Hbpital Cochin, Cedex 14, France

27,

lntradermal injection of 100 pg of native homologous type II collagen (CII) into DBA/ l-susceptible mice induced a progressive and chronic polyarthritis. This experimental autoimmune arthritis (EAA) closely mimicked the clinical evolution of human rheumatoid arthritis (RA) except for the sex linkage. Males were highly susceptible to EAA induction even when the amount of autoantigen injected was reduced to 25 pg. Conversely, females remained resistant to the disease even when a booster injection of 50 pg was administered. With regard to age, no major difference in the incidence was observed, although younger males developed a more severe arthritis than older ones. Anti-C11 autoantibodies were detected in all immunized animals, regardless of the presence or absence of joint pathology. However, in arthritic mice, the onset of the disease was associated with a predominance of IgG2a autoantibodies. Kinetic studies revealed that females as well as males exhibited early histological lesions and detectable humoral responses toward mouse CII as of the second week postimmunization. Moreover, a specitic cellular autoreactivity to homologous CII occurred in different lymphoid organs with a higher intensity in females than in males. Taken together, these findings suggest that homologous CII injection induces an early subclinical arthritis that develops progressively in all immunized mice, but would be down-regulated several weeks after priming, exclusively in females. 0 1988 Academic Press, 1~.

INTRODUCTION For many years, immunization with native type II collagen (CII) of xenogeneic origin has been shown to induce a polyarthritis in both rats and mice (l-3). In the murine model of collagen-induced arthritis (CIA), Wooley ef al. (3) suggested that the susceptibility to the disease was linked to the I-A9 haplotype of the H-2 locus of the major histocompatibility complex. Recently, we, as well as others (4, 5), provided evidence that injection of homologous CII caused a progressive and chronic polyarthritis that shares many features with human rheumatoid arthritis (RA). In contrast with the bovine (B) CII arthritis which develops explosively 1 month after immunization and never relapses after remission, the experimental autoimmune arthritis (EAA) induced with a single injection of murine (M) CII was characterized by a progressive onset and an evolution fluctuating between remissions and recrudescences (4, 6). Surprisingly, the incidence of EAA was clearly preponderant in males, and furthermore, the few arthritic females developed a mild disease compared to the males. Despite their resistance to disease induction, ’ This work was supported by the French Medical Research Council, INSERM and the French Fund for Rheumatology Research. 225 0090-1229/88 $1.50 Copyright All rights

6 1988 by Academic Press. Inc. of reproduction in any form reserved.

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the females were able to secrete high levels of autoantibodies in response to homologous CII injection. Similarly, some inbred strains of rats and mice develop high titers of anti-collagen antibodies but are resistant to arthritis (3, 7, 8). These findings would argue against a humoral mechanism in the pathogenesis of EAA. On the other hand, a role for cellular immunity in initiating the disease is supported by the demonstration that nude rats cannot acquire CIA (9) and by the successful transfer of arthritis with CII reactive T-cell clones (IO). In our previous studies describing the EAA (4, 6), we showed that the clinical onset occurred late (about 6 weeks postimmunization) and thereafter the disease lasted for several months. In the present work, we further investigated this model of autoimmunity with particular emphasis on the earlier phases of the disease with regard to the histopathological findings and to the pattern of immunological responses to homologous CII in both male and female mice. We also report on the influence of the dose of antigen injected and the age of the animals at the time of immunization. MATERIALS AND METHODS mice were obtained from the Institut

1. Mice. DBA/l Pasteur breeding center (Paris, France). 2. Anrigens. Native mouse cartilage collagen was prepared from the xiphoid appendix by pepsin digestion and subsequent purification as previously described (6). Native B-C11 was kindly supplied by D. Herbage (CNRS/UA 244, Lyon, France). 3. Immunization procedure. Lyophilized native M-C11 was dissolved at the appropriate concentration in 0.1 M acetic acid. The solution was then emulsified with an equal volume of complete Freund’s adjuvant (CFA, GIBCO Laboratories, NY) and injected intradermally into the right hind footpad of mice. In some experiments, female mice were boosted, 2 weeks after priming, with 50 kg of M-C11 in incomplete Freund’s adjuvant (IFA, GIBCO) at the base of the tail. 4. Arthritis evaluation. Animals were clinically observed daily from the third week postimmunization until sacrifice. The severity of the arthritis was determined for each mouse by separately scoring the two fore and the left hind paws on a graded scale as described previously (6). A maximum arthritic index was derived by combining the scores for all three non injected paws recorded for each mouse, regardless of when the measurement was made. It was used to compare the severity of arthritis obtained in mice immunized with different doses of autoantigen. The histopathological examination was performed at sacrifice. The three noninjected limbs of all animals (experimental and control mice) were dissected out for histological examination. Joints were slowly decalcified for 1 month and embedded in paraffin blocks. At least five sections were cut along a longitudinal axis, mounted, and stained with hematoxylin and eosin before blind evaluation. All stained sections from each mouse were examined and scored for two criteria: (a) score 1 represents the extent of the lesions and was evaluated by the proportion of affected joints among all the joints observed; (b) score 2 measures the intensity of the lesions based on the synovial proliferation (degree of multilayering and villi formation) and the presence of mononuclear cell infiltrates. Each joint

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was graded on a scale of 0 to 3. Additional scores were calculated for each mouse and then divided by the number of affected joints. 5. Measurement of anti-CZZ autoantibodies. Mice were bled on several occasions during the experiments as reported in the text. The individual sera were collected, aliquoted, and stored at -70°C until they were tested for antibodies directed against M-C11 using a standard enzyme-linked immunosorbent assay (ELISA) as already described in detail (6). The results were expressed as optical density (OD) x IO3 in experimental wells minus the background OD obtained in wells without the CII coating. 6. ZgG subclass derermination. A “sandwich” ELISA was used to evaluate the relative percentages of anti-C11 IgG subclasses. The serum samples (diluted 1: 100) were incubated overnight at 4°C on microtiter plates coated with 10 t.tg/ml of M-C11 as for the standard ELISA assay. Washed wells were then incubated (2 hr at room temperature) with appropriate dilutions of rabbit anti-mouse immunoglobulins (RAM, Nordic, Tilburg, The Netherlands) directed either against total IgG or different IgG subclasses (IgGl, IgG2a, IgG2b, IgG3). The microplates were again washed and absorbance values for total IgG or each IgG subclass were revealed with alkaline phosphatase-conjugated goat anti-rabbit IgG (GAR-AP, Miles, Yeda, Israel) and the appropriate substrate. Since in preliminary experiments, we have shown that the isotype specific antibodies bind to the relevant IgG subclasses with similar affinities, the observed absorbance values for each IgG autoantibody subclass in individual serum samples were added and relative percentages were calculated. 7. Proliferation assays. To measure proliferative responses of M-C11 immunized or control CFA-injected animals, different lymphoid organs (spleen, peripheral lymph nodes, and afferent popliteal lymph node) were dissected out under sterile conditions at varying intervals after priming as indicated in the text. Single cell suspensions were obtained after teasing of the lymphoid organs and two washes in Hanks’ balanced salt solution. The cells were finally suspended in complete culture medium which consisted of RPM1 1640 supplemented with glutamine, antibiotics (Flow Laboratories, Rockville, MD) and 10% heat-inactivated fetal calf serum. Cultures were conducted in triplicate in round-bottomed microplates (NUNC, Roskilde, Denmark) by mixing 2 x 10’ cells with several dilutions of M-C11 (25, 10, 5, and 1 t&ml). After a 4-day incubation at 37°C in a 5% CO, atmosphere, the cultures were pulsed with 1 &i of [3H]thymidine (CEA, Saclay, France) and further incubated for 18 hr before being harvested on glassfiber filters. The radioactivity incorporated was measured and the results were expressed as cpm calculated as follows: cpm of stimulated cultures minus cpm of unstimulated cultures. RESULTS I. Influence of the dose of M-CZZ injected on EAA induction. In a previous study (6), we showed that 100 u,g of M-C11 injected into DBA/l mice provoked a chronic polyarthritis in males, whereas most of the females were refractory to the induction of the disease. In order to test whether this sex linkage of EAA was related to the amount of autoantigen injected, we immunized DBA/l males with

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one-fourth the dose of M-CII (25 pg), while the females received greater amounts of antigen than in the initial experiments, since they were primed with 100 pg of M-C11 emulsified in CFA and they were boosted. 2 weeks later, with SO pg of M-C11 in IFA. These experiments were conducted in young and middle-aged animals and included control mice that were injected with CFA alone. At the time of sacrifice, i.e., 5 months postimmunization, almost all the males had developed clinical signs of arthritis, regardless of the dose of M-C11 injected or the age group considered (Table 1). Although the disease onset occurred earlier in young males injected with low doses of M-CII, the difference was not significant. Conversely, the arthritic score evaluated by a graded scale revealed that the disease was more severe in the younger group than in the older one, irrespective of the dose of antigen injected. However, the age-related decrease of disease severity was significant only in the group of males that were primed with high concentrations of homologous protein (Table 1). The clinical course of the arthritis did not differ from one group to another. It was characterized by fluctuating crises and perpetuation of the disease during the Smonth observation period. As shown on Table 2, few females developed clinical arthritis even if they received a booster injection. Moreover, the disease induced in females was usually mild. 2. HistologicaIfindings. At the time of sacrifice, males as well as females were examined for the presence of histological lesions. In the group of males, there was no major difference in the incidence of joint pathology whether mice were injected with high or low doses of M-CII. The number of joints affected or the severity of the lesions were highly variable from one mouse to another. These findings were thought to be consistent with the fluctuating clinical course of the disease that we observed (6). The most prominent histopathological changes consisted of mild to severe proliferation of the synovial lining cells and an increase in the villous projections of the synovial membrane into the joint cavity. Inflammatory infiltrates were found occasionally beneath the hyperplastic synovial cells. Interestingly, almost all the females presented mild synovitis at autopsy, suggesting that they developed subclinical arthritis. However, the lesions were minimal and infiltrates of mononuclear cells were rarely seen even when they received a booster injection. TABLE EAA Dose of mouse CII (I%) 0 25 loo

INDUCTION

1 IN MALE

Age (weeks)

incidence II (5%)

6-7 U-17 6-7 15-17 6-7 IS-17

O/6 (0) O/IO (0) 617 (85.7) 717 (100.0) 717 (100.0) I 1112 (91.6)

MICE

Disease onset (days)

34.6 40.1 47.2 38.3

+ 2 k 2

2.8 3.2 3.9 2.2

Note. Results of disease onset and arthritic score are expressed as means t- SEM. * P < 0.05 (Student’s t test).

Arthritic

X.1 4.4 9.4 6.1

L + 2 -L

score

2.3 0.5 1.4* 0.4*

AUTOIMMUNITY

TO HOMOLOGOUS TABLE 2 EAA INDUCTION IN FEMALE

Dose of mouse CII hz)

Age (weeks) 6-7 21 21 67 18

0

100 loo + 50

229

TYPE II COLLAGEN

Incidence n (%)

MICE

Disease onset (days)

o/5 (0) 015 (0) 3/8 (37.5) 217 (28.5) l/7 (14.2)

Arthritic

score

3.6 r 1.2 3.5 2 0.3 6.0

49.0 + 0.0 74.5 2 17.3 30.0

Note. Results of disease onset and arthritic score are expressed as means 2 SEM.

3. Humoral response toward M-CII in DBAIl males. Male mice injected with either 25 or 100 bg of M-C11 were bled at varying intervals postimmunization and their individual circulating anti-M-C11 antibody levels were determined using an ELISA. Since we did not observe age-linked differences. we pooled the results of young and old mice and compared the two groups, according to the dose of antigen administered. The serum IgG autoantibody levels were much higher when DBA/l males were immunized with low doses of M-CII. Moreover, the humoral response of mice injected with 25 pg of M-C11 peaked 7 to 9 weeks postimmunization and thereafter declined rapidly (Fig. 1). In contrast, the injection of 100 p,g of M-C11 appeared to be associated with a weaker autoantibody response that was scarcely perceptible 5 to 7 weeks after the priming and then decreased gradually to background levels defined by the amounts found in the sera of either the CFA-injected control group or naive mice (Fig. I). 4. Analysis of the IgG autoantibody subclass response. Our previous experiments (6), showing that nonarthritic females produced anti-C11 autoantibodies

I 100 i I d 50

0

5

9

12

16

2b

post-immunization

FIG. I. Influence of the dose of M-C11 injected on autoantibody responses in DBA/I males.

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after immunization, strongly suggest that the magnitude of the total IgG autoantibody response was not associated with the susceptibility to arthritis. In order to examine the potential role for a particular anti-C11 IgG isotype in inducing the autoimmune arthritis, the IgG subclass response was analyzed in the sera of arthritic and nonarthritic mice collected at the initiation of the clinical joint pathology (5 to 7 weeks postimmunization). Table 3 reveals that the predominant IgG autoantibody response was confined to the IgG2a subclass in arthritic mice, averaging 43% of the total anti-C11 IgG secreted. In contrast, sera from nonarthritic mice exhibited a significantly lower (P < 0.02) percentage of IgG2a autoantibodies that was compensated by an increase in IgGl and IgG2b subclasses. IgG3 autoantibody responses of arthritic mice overlapped those of nonarthritic animals. These data suggest that IgG2a autoantibodies may play a pathogenic role in EAA. 5. Kinetics of EAA induction. With the aim of studying the early phases of EAA, we immunized DBA/l mice with 100 p,g of M-C11 in CFA and killed them 1, 2,4, or 6 weeks after priming. Each group included 6 immunized mice (3 males + 3 females) and 2 control CFA-injected mice (1 male + 1 female) that were examined for their histological lesions, and their humoral as well as cellular immunity in response to homologous CII. With regard to the histological incidence of arthritis, Table 4 shows that both males and females exhibited early abnormalities of the synovial tissue, without any macroscopic sign of clinical arthritis. Our kinetic studies clearly indicate that M-C11 injection caused a subclinical arthritis, that was already detectable in the noninjected limbs of most of the mice 1 week after immunization. Moreover, the extent of the lesions progressively increased until the fourth week postpriming, irrespective of the sex (Table 4, histological score 1). On the other hand, the severity of the lesions (evaluated by score 2) was identical in all the groups considered except for one male mouse that exhibited more severe pathologic changes in one joint 6 weeks after M-C11 injection. Interestingly, the clinical onset of arthritis in young males occurs between 6 and 7 weeks postpriming (Table 1). The earliest pathologic changes noted were the synovial cell proliferation and the formation of small synovial villi. Scarce inflammatory infiltrates were present even from the first week. No signs of cartilage destruction or bone erosion were observed.

COMPARISON

Mice Arthritic (n = 7) Nonarthritic (n = 9)

TABLE 3 OF IgG ALJTOANTIBODY SUBCLASS RESPONSES IN ARTHRITIC NONARTHRITIC MICE _--.~~~ .Percentages of anti-C11 IgG subclasses IgG 1

IgG2a

6.5 k 2.4 11.2 _' 2.8

Note. DBA/l mice were immunized collected 5 to 7 weeks postimmunization. using Student’s t test: *P < 0.02.

43.1* 2 5.3 26.51 +- 3.6

IgG2b

17.4 k 3.0 28.0 t 3.1

AND ~..~-

IgG3

33.0 5 3.6 34.3 ? 2.8

with 100 kg of M-C11 in CFA and their individual sera were Results are expressed as means t SEM. Statistical analysis

AUTOIMMUNITY

TO HOMOLOGOUS

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TYPE II COLLAGEN

TABLE 4 KINETICS OF EAA INDUCTION Histopathology Weeks postimmunization” 1 2 4 6 -.___-

a Groups of 6 2.4, or 6 weeks taneously tested b Histological and the intensity

Sex

Incidence (n)

M F M F M F M F

l/3 213 313 313 313 313 l/3 313

DBA0 mice (3 males + postimmunization. Two (data not shown). scores are expressed as of the lesions evaluated

Histological scoresb 1 0.2 " k 2 -t t 0.2 0.3 2 0.4 0.4 0.5 0.6 0.6

2 1.0 1.9 _t 0.6 1.2 '- 0.1 1.3 i 0.2

0.1 0.1 0.1 0.2 0.2

1.3

0.1

3.0 1.5 k 0.2

1.8 2 0.3 2 0.1

Anti-M-C11 autoantibodies (OD x 10’) 5.0 23.6 646.3 199.0 539.0 850.0 913.3 572.3

2 " + 2 rf 2 -+ 2

3.6 19.3 249.9 143.3 147.8 320.8 51.2 137.8

3 females) were injected with 100 p,g of M-C11 and killed 1, control CFA-injected mice (1 male + I female) were simulmeans 2 SEM of the proportion by a graded scale (score 2).

of affected joints (score I)

The specific autoantibody response, as expected, was not observed before the second week postimmunization (Table 4). However, very disparate levels of autoantibodies were observed from one mouse to another, regardless of sex, a finding that we also observed in a previous work (6). As far as cellular autoreactivity was concerned, Fig. 2 represents the kinetics of proliferative responses to homologous CII. Given that the magnitude of the response differed according to the concentration of the antigen used for the in vitro stimulation and the origin of the responder cells, the results are reported for each experiment at the peak of the response. Taking into account the spontaneous activation of unstimulated cells which occurs with varying intensity in the different lymphoid organs (see Fig. 2 legend), all the data were expressed as the counts over the baseline levels (Acpm) thereby representing the specific stimulation. Several observations can be deduced from Fig. 2. First, in almost all the cases, the females exhibited higher specific proliferative responses than the males. Second, the maximal blastogenesis occurred in the popliteal lymph node afferent to the site of CII injection while peripheral lymph node cells or spleen cells proliferated at a lower rate. Third, the kinetics of specific reactivity to M-CII revealed that the maximal stimulation of spleen cells was noted 2 weeks after immunization and then declined progressively. In contrast, in the draining lymph node, the number of cells specific for CII increased until the fourth week postimmunization. In the peripheral lymph nodes, the situation was not as clear, since males exhibited a substantial proliferative response 4 weeks after priming, while that of the females was already decreasing. No CII-specific cells could be detected 1 week after immunization, except in the popliteal lymph node of the 3 females and in the peripheral Iymph nodes of 1 out of 3 females. Finally, Fig. 2 also shows that CFA-injected males and females did not develop specific cellular immunity toward M-CII in the popliteal or peripheral lymph nodes. Unexpectedly, spleen

‘37 -_ -

ROISSIER,

1

CARLIOZ,

AND

2 W‘...kc.

FOURNIER

1, ,,‘>=.l

(1

~1l11rT1~,lIl,.lflrll

FIG. 2. Kinetics of specific cellular autoreactivity of M-Cl1 in different lymphoid organs. The males (0). 3 proliferation assays were expressed as mean Acpm x IO- ’ 2 SEM of 3 M-U-injected M-CII-injected females (O), and 2 CFA-injected mice (A). Average background counts (cpm X 10. ‘) given by unstimulated cells were 2.9 -t 0.5 for spleen cells. I .9 -+ 0.7 for peripheral lymph nodes, and 3.7 2 1.O for afferent popliteal lymph nodes.

cells from control mice displayed a proliferative response to M-C11 and to a lesser extent to B-CII, that was similar to that of the immunized males. As shown in Fig. 3, lymphoid cells from mice primed with homologous CII responded to both M-C11 and B-CII. However, a substantial part of the cellular reactivity was directed against species-restricted epitopes of the autoantigen since in almost all the cases M-C11 induced higher DNA synthesis than B-C11 when tested on the same cell suspensions. Using the paired t test, the differences were highly significant for the whole group in the spleen (P < 0.001) or in the afferent popliteal lymph nodes (P < 0.01) but were more modest and not significant in the peripheral lymph nodes. DISCUSSION The present study was undertaken in order to further investigate the arthritic pathology that arises in DBA/l mice when they are immunized with homologous CII (4-6). This experimental model is unique among the CIA described for many

AUTOlMMUNITY

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,

5

TYPE I1 COLLAGEN

FF

MAL

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I-c1

FIG. 3. Specificity of the cellular reactivity against CII in different lymphoid organs. Male and female mice were injected with M-CII and their lymphoid cells tested for their proliferative responses to M-C11 and B-CII. Each line represents the comparative Acpm given by T cells from a single mouse in response to M-C11 and B-CII.

years in its ability to induce a progressive and chronic polyarthritis that mimics human RA. However, the male mice were much more susceptible to the disease. Therefore, experiments were designed to challenge the resistance of the females by increasing the amount of autoantigen injected. Such treatment did not produce any major change in either the clinical incidence of arthritis or the severity of the disease, although mild histological lesions were frequently noted. In contrast, the disease could be induced in males even when the dose of antigen injected was reduced from 100 to 25 p,g per mouse. A similar dose-independent disease incidence was found by others using chick CII as the antigen, although these investigators did not specify the sex of the immunized mice (11). With regard to the age of the mice, no major difference in the incidence was observed in our study between mice injected with low or high doses of M-CU. However, the younger males developed a more severe arthritis than the older ones. Our data point out discrepancies between arthritis induced with homologous and heterologous CIT

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since previous reports showed that susceptibility to arthritis in DBA/l mice was increased with age after injection of rat CII (12) whereas it was decreased when Bl0.Q mice (II) or rats (13) were immunized with chick CII. Whether these differences reside in the species source of CII injected and/or in the species or strain of the recipients is a possibility that is supported by previous reports on the immunogenetic regulation of the immune response to CII (11, 14). Regardless, we provided evidence that the sex linkage of the resistance to arthritis in the homologous CII model is not related to the amount of autoantigen injected. It may be suspected that female hormones exert a suppressive action on the development of arthritis as recently demonstrated for arthritis induced with heterologous CII (15). Autoantibody formation against CII occurred in all groups of mice irrespective of the dose of antigen injected. Confirming our previous findings (6) and the conclusions reached by others (5) in the same model of EAA, the levels of anti-C11 antibodies failed to correlate with the arthritic status of the mice. Surprisingly, a low dose of homologous CII elicited a delayed and much higher humoral response in males than the usual dose of 100 pg. This finding may reflect a suppressive mechanism conferred by suppressor T cells that are activated in the presence of high CII concentrations. Alternatively, high autoantibody titers may be generated during the 2 or 3 weeks following priming with 100 pg of CII and then the formation of immune complexes would deplete the circulating autoantibodies. By whatever mechanism involved, an important feature of the humoral response to homologous CII was the observation that the clinical initiation of the arthritis was associated with a predominance of IgG2a autoantibodies. Similar conclusions were reached by others after injection of heterologous CII into either rats (16, 17) or mice (18, 19). Given that the injection of affinity purified anti-C11 antibodies from arthritic rats or mice was successful in passively transferring the disease to naive syngeneic recipients (20, 21), a pathogenic role for the IgG2a subclass of anti-C11 antibodies may be suspected in EAA induction. Interestingly, the antiCII antibodies from nonarthritic rats failed to transfer the disease (20). Moreover, the resistance to induction of CIA observed after intragastric administration of CII was associated with a decrease in the magnitude of the IgG2a response or a switch in isotype predominance (19). Aside from the link between susceptibility to arthritis and IgG2a subclass, a pathogenic role for the fine specificity of the anti-C11 antibodies must be taken into account. The evidence of arthritogenic epitopes on the CII molecule is supported by the demonstration that sera from different CIIimmunized mouse strains display varying capacities in transfer experiments (22). Moreover, the injection of two different monoclonal anti-C11 antibodies bearing the IgG2a isotype into DBA/l mice caused a mild histological synovitis without macroscopically observable lesions, despite a strong in vitro reactivity with M-C11 (23). In addition, as reported in our previous study (6), the consistently higher humoral response against the immunizing M-C11 than against B-C11 supports the existence of one or several species-specific epitopes on the collagen molecule that trigger autoimmune reactivity. The few female mice that developed arthritis after injection of homologous CII presented a macroscopically mild disease with very low arthritic scores. At the time of sacrifice (4 to 5 months after priming), the histological sections showed

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moderate lesions of synovitis, even in animals that were not considered as clinically arthritic. Although they were performed on a limited number of mice of each sex, the kinetic studies that we have conducted herein revealed the existence of very early histological abnormalities. Moreover, the incidence and severity of synovitis were similar for both sexes. On the other hand, we were able to directIy demonstrate the existence of a cell-mediated immunity directed toward the homologous protein that was always detected with more intensity in the females than in the males. One week after immunization in the footpad with M-CII, the popliteal lymph node afferent to the site of injection contained CII-specific T cells and their number increased during the following weeks, probably as a result of autoantigen retention in the footpad and the continuous elicitation of the immune response (24). In the spleen and peripheral lymph nodes, M-CII-specific cells were detectable as of the second week postimmunization but their proportion was much lower than in the draining lymph node. It must be noted that spleen cells from the two control mice, that were killed 2 weeks after injection of CFA, exhibited a weak but specific proliferative response to CII. These observations are reminiscent of findings under similar circumstances in the rat (25) and may result from the nonspecific triggering of T-cell clones which exhibit autoreactivity to CII. Using chick or bovine CII as the immunogen, Stuart et al. showed that both rats (13, 26) and mice (27) displayed in vitro proliferative responses to the immunizing protein. Recently, evidence for in vivo cellular sensitization to CII was provided by induction of delayed-type hypersensitivity (DTH) using a radiometric ear assay (28, 29). In the homologous CII model described herein as well as in the heterologous CIA (27, 29). the peak of specific cell-mediated immunity occurred prior to the onset of clinical arthritis. Interestingly, lymphoid cells from mice immunized with M-C11 proliferated in vitro in response to B-CII, indicating that the T cells recognize determinants common to both species. However, we consistently found a higher magnitude of proliferation against M-C11 than against B-CII. This result is reminiscent of our findings on the humoral response to M-C11 (6) and is in agreement with the conclusions reached in the study cited above (29) which measured the DTH reactivity to M-C11 and B-W. On the other hand, a lack of species specificity in the cellular responses to two CII of xenogeneic origin was reported by others in DBA/l mice injected with chick CII (27). Our data showing that M-CII-specific T cells can be generated in response to homologous CII further support a role for the importance of cellular immunity in inducing the disease. In fact, convincing arguments for the participation of T cells in heteroIogous CIA were provided from experiments showing that thymusdeficient rats do not develop arthritis (9), that CIA is prevented after in vivo treatment with anti-T-cell autoantibodies (30), or that the disease can be transferred with CII-reactive T-cell lines (10). On the basis of our kinetic experiments, we may postulate that injection of M-C11 into the footpads of susceptible strains of mice provokes an immune reaction in the draining lymph node. This would result, within 1 week, in the generation and then in the amplification of T-cell populations specific for different M-C11 epitopes. Progressively, the sensitization of T cells occurs in the central and peripheral lymphoid organs. Concomitant to the development of the immune reaction to M-CII, early onset of synovitis is

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apparent in the distal joints. Whether the histopathological lesions are initiated by the migration to the synovial tissue of T cells specific for arthritogenic determinants of M-C11 or through the production of lymphokines by such activated T ceils (3 1) remains to be determined. Moreover, the binding of anti-C11 autoantibodies to cartilage may also contribute to the perpetuation of the disease (32). The fact that homologous CII induces a chronic disease, whereas heterologous CII causes an explosive arthritis without relapses, favors the hypothesis that the arthritogenie epitopes of the CII molecule responsible for EAA are restricted to M-CII. Alternatively, it is possible that EAA induction involves the cooperation of different T-cell clones, one (or several) of which recognize(s) mouse-specific determinants. From our experiments, it is evident that females as well as males exhibited immunological reactivity to M-C11 and exhibited early histopathological lesions. Therefore, it appears that most of the females develop an early subclinical arthritis which lasts several months without any detectable sign of joint pathology whereas males progressively show joint swelling. These observations seem to imply that the suppressive mechanism(s) occurring m females are initiated several weeks after immunization and are able to revert the ongoing autoimmune process. Additional work is required to analyze the role and the regulation of arthritogenic T lymphocytes in EAA. ACKNOWLEDGMENTS The authors thank D. Herbage for generously providing the bovine type II collagen. They are indebted to Mr. J. L. Olivier for preparing mouse type II collagen and to Mr. J. C. Mussard and Mrs. S. Mistou for their excellent technical assistance. They acknowledge Ms. J. Jacobson for her editorial help and Mrs. J. Decaix for typing the manuscript.

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