Eur. J. Immunol. 1991. 21: 1783-1785

CIA is not related to TcR VBgene polymorphism in Biozzi mice

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Short paper Laurent Vidard+, Thierry Rogerc, Jean Pierre BouvetD, Jacques Couderc+ and Michel Semanc

Resistance to collagen-induced arthritis in Biozzi mice is not associated with T cell receptor Vp gene polymorphism*

Institut Curie+, Unit6 d’Immunog6n6tique, CNRS URA 1413, Institut Jacques Monodo, Laboratoire d’Immunodifferenciation, CNRS-Universit6 Paris 7 and Institut Pasteurn, Unit6 d’Immunologie Microbienne, Paris

H-I and H-I1 high antibody-responder Biozzi mice, which express the H-24 permissive haplotype, were shown to be sensitive and refractory to collageninduced arthritis, respectively.To assess a possible role of T cell receptor (TcR) Vp gene deletion or polymorphism in the resistance to arthritis in H-I1 mice, the germinal structure of TcR Vg genes and their expression at the membrane level were compared in both lines. In contrast to H-2‘7 refractory SWR mice, which exhibit a deletion of about 50% of TcR Vp gene segments, H-I and H-I1lines have an identical and complete set of Vp genes and exhibit no difference in the average expression of Vg6, Vp8 and Vgll gene products on T cell surface. These results indicate that mechanisms other than Vp gene deletion or polymorphism can be involved in the resistance of H-24-positive mice to experimental arthritis.

1 Introduction

2 Materials and methods

Collagen-induced arthritis (CIA) is an autoimmune disease which occurs after injection of autologous [ l , 21 or heterologous [ l , 31 type I1 collagen (CII) in rodents and monkeys [4]. The mechanism of the joint lesions was, at first, attributed to T-dependent CII autoantibody production [5, 61. An additional requirement of effector T cells, involved in the perpetuation of passively transferred CIA, was also recently reported [7]. SWR mice were then shown to be refractory to CIA, although they exhibit the H-24 (Aq,E-)-permissive haplotype as DBA/1 susceptible mice [8] and develop high CII autoantibody responses [9]. Interestingly, SWR mice have deleted about 50% TcR Vp genes [lo]. It was, thus, suggested that specific Vg genes, absent in SWR mice, could be expressed in Tcells required for CIA development [8, 91.

2.1 Lines

In a recent study, we reported the induction of CIA in the high-responder H-I line of Biozzi mice, whereas no arthritis was observed in H-I1 mice although both lines share the H-24 haplotype and develop high CII autoantibody responses [ll]. These two lines were issued from independent selective breedings of outbred Swiss mice on the basis of a high antibody response to heterologous erythrocytes and were shown to have similar patterns of high responsiveness to a wide variety of unrelated antigens [12]. In the present study, TcR Vp gene polymorphism and expression was investigated in H-I and H-I1 mice to test the hypothesis of Vp gene deletion or polymorphism in H-I1 mice that would account for their resistance to CIA.

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This work was supported by Institut Curie, Contrat coopkratif 89-20-4.

Correspondence: Jacques Couderc, Unite d’Immunogknktique, Section de Biologie, URA 1413, Institut Curie, 26 Rue d’Ulm, F-75231, Paris Cedex 05, France Abbreviation: CIA: Collagen-induced arthritis 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991

H-I and H-I1Biozzi mice were bred in the animal unit of the Institut Curie. SWR mice were kindly provided by J. L. Guenet, Institut Pasteur, Paris. H-I, H-I1and SWR animals are H-24 (Kq, A4, E absent, Dq) [13]. BALB/c mice were purchased from IFFA-Credo (Les Oncins, France).

2.2 Southern blot analysis High-molecular weight genomic DNA was prepared from mice liver using standard procedures [14]. After complete digestion with 20 units of restriction endonucleases (EcoRI, BamHI, HindIII or MspI; Boehringer Mannheim, Mannheim, FRG), 10 pg of each DNA was separated on 0.8% agarose gels. After denaturation in 1.5 M NaCl, 0.5 M NaOH and neutralization with 3 M sodium acetate, gels were transferred to nylon membranes. Filters were prehybridized for more than 2 h in 5 ~Denhardt’s, 250 pg/ml salmon sperm DNA, 5 x SSC, 0.1% SDS, 50 mM phosphate buffer, 50% formamide, at 42°C and then hybridized to random primed 32P-labeledDNA probes at 42 “C overnight in prehybridization buffer completed with 10% dextran sulfate and 500 pg/ml salmon sperm DNA. Filters were then washed three times 20 min at 22°C in 2 x SSC, 0.1% SDS and three times 20 min at 55°C in 0.1 x SSC, 0.1% SDS before autoradiography. Subsequent hybridizations were performed after removal of the probes from nylon filters by two boiling bathes of 0.1 x SSC, 0.1% SDS. Probes were kindly given by Drs. D. Loh, St. Louis, MO (Vpl to Vg16) and J. Kappler, Denver, CO (Vgl7a).

2.3 mAb mAb from rat hybridoma cell lines 44-22-1 (anti-Vp6), KJ16 (anti-Vg8.1 and -Vp8.2) and K T l l (anti-Vgll) were gifts of Drs. H. Hengartner, J. Kappler and K. Tomorani, respectively. Anti-CD3 145-2C11 hamster mAb was a gift of Dr. 0014-2980/91/0707-1783$3.50+ .25/0

Eur. J. Immunol. 1991. 21: 1783-1785

L.Vidard, T. Roger, J. P. Bouvet et al.

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Bluestone. Anti-CD3 mAb was used as FITC-conjugate in immunofluorescence. Anti-Vp mAb were biotinylated with Act-Biotin (Becton Dickinson, Mountain View, CA). 2.4

F C M analysis

Vp gene expression was evaluated by two-color analysis in FCM. Aliquots of spleen and LN cells were stained with biotinylated anti-Vp6, Vp8 or Vpll antibodies and FITCconjugated anti-CD3. After washing, cells were then labeled with streptavidin-PE (Becton Dickinson). Fluorescence was recorded on a FACStar cell sorter (Becton Dickinson).

3 Results and discussion In an attempt to explain the difference in CIA susceptibility between H-I and H-I1 high antibody-responder Biozzi mice, which are both H-2q,TcR Vp gene polymorphism was investigated at genomic DNA level in Southern blot using Vp-specific probes. As shown in Figs. 1 and 2, identical RFLP were observed between H-I and H-II mice, with any of the probes on either BamHI, Eco RI or HindIII digests as suggested from previous studies [15]. In particular, the Vp5,8,9,11,12,13 deletion, which characterizes the Vp haplotype of the CIA' (resistant) SWR mouse strain, was not found in the CIA' H-I1 line. This ruled out the hypothesis that a large deletion of Vp genes is responsible

for CIA resistance in H-I1 mice in contrast with the mechanism proposed by Banerjee et al. [8, 161 to explain the CIA resistance in SWR mice. These authors postulated that such a large deletion would impair theTcell recognition of arthritic CII epitopes, a mechanism in agreement with the hypothesis of autoimmune T cells as the major pathogenic agents during CIA [7, 8, 11, 161. Genes of the Vp8 family have themselves already been involved in the susceptibility to EAE [17], another autoimmune disease. However, this gene family appears unlikely to be involved in the differences in CIA susceptibility between the lines of Biozzi mice, since both H-I and H-11animals have the same Vp8 RFLP patterns and exhibit no difference inVp8.1 and Vg8.2 expression in spleen and LN. As shown in Fig. 3, about 15% of CD3+ LN Tcells are stained with KJ-16 in both lines. Similarly, no significant difference was detected between H-I and H-I1 mice inTcell usage of theVpl1 gene segment. Segregation studies in (SWR x B1O)FZ progeny suggested that Vp6 polymorphism could also be involved in CIA resistance based on the observation that the two parental strains exhibit different RFLP patterns in Southern blot analysis upon Msp I digestion with aVp6-specificprobe [9]. Results in Fig. 2 indicate that H-I and H-I1 mice have the sameVp6 RFLP pattern, similar to that of BALB/c or B10 mice, in MspJ digests. Moreover, no major difference is observed between the two lines in Vp6 expression among spleen (not shown) and LN Tcells (Fig. 3). This suggests that Vg6 cannot impart for CIA resistance. Consistent with

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Figure 2. Summary of TcR Vp gene genomic configuration in SWR, H-I and H-I1 mice as determined by Southern blot analysis.The first line shows the restriction endonucleases used to study the different TcR Vg gene regions (B = BamHI, E = Eco RI,H = HindIII and M = MspI). (P) Means polymorphic, (-) deleted and (+) nonpolymorphic configuration as compared with BALB/c.

Eur. J. Immunol. 1991. 21: 1783-1785

CIA is not related to TcR Vp gene polymorphism in Biozzi mice

Altogether, our results strongly suggest that, in addition to MHC genes, genes located outside MHC and Vg regions are also likely to be involved in the genetic control of CIA susceptibility. Such gene(s) might have a role in collagen processing or might encode a cross-reacting self antigen involved in a negative selection of the T cell repertoire.

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Received January 23, 1991.

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Figure 3. Vp6, Vp8 (8.1, 8.2) and Vpll expression in LN T cells from SWR, H-I and H-I1 mice determined by two-color FCM analysis. Numbers indicate the percentage of Vp-positive cells among CD3+ T lymphocytes.

our results, Holmdahl et al. [18] have observed that, in contrast to EAE, CIA is not prevented by in vivo administration of anti-Vg6 or -Vg8 mAb in DBA/1 mice, although these treatments lead to the depletion of Tcells expressing the corresponding Vp gene segments. Our present results suggest that, in contrast with the SWRlBlO or SWRIDBAIl models, difference in CIA susceptibility between H-I and H-I1 Biozzi mice cannot be accounted for by major Vg gene deletion. In addition, the analysis of Vp6,Vg8 and Vp15 genes polymorphism does not seem to explain H-I1 resistance either. Thymic selection of the T cell repertoire can lead to the elimination of T cell subsets expressing particular Vg gene segments such asVp8.1 and Vp6 in Mls-la mice [19,20] or Vgl7a and Vgll in I-E-positive animals [21,22]. Fluorescence analysis of Vp6,Vp8 and Vgll gene expression does not provide evidence for such negative selection in H-I or H-II mice. This also tends to eliminate the hypothesis that one of these Vg genes would participate in the control of CIA susceptibility in these two lines. However, this analysis should be extended to other Vg gene products and further investigation is needed to define Vp gene usage among arthritis-inducing T cells in H-I mice. Experiments are in progress to analyze the CII-specificTcell repertoire in H-I and-H-11.

4 References 1 Trentham, D. E.,Townes, A. S. and Kang, A. H., J. Exp. Med. 1977. 146: 857. 2 Holmdahl, R., Jansson, L., Gulberg, D., Rubin, K.,Forsberg, F! 0. and Klareskog, L., Clin. Exp. Immunol. 1985. 62: 639. 3 Courtenay,J. S., Dallman, M. J., Dayan, A. D., Martin, A. and Mosedale, B., Nature 1980. 283: 666. 4 Cathcart, E. S., Hayes, K. C. and Kang, A. H., Lab. Invest. 1986. 54: 26. 5 Klareskog, L., Holmdahl, R., Larsson, E. and Wigzell, H., Clin. Exp. Immunol. 1983. 51: 117. 6 Stuart, J. M., Cremer, M. A. ,Townes, A. S. and Kang, A. H., J. Exp. Med. 1982. 155: 1. 7 Seki, N., Sudo, Y., Yoshioka, T., Sugihara, S., Fujitsu, T., Sakuma, S., Ogawa,T., Hamaoka,T., Senoh, H. and Fujiwara, H., J. Immunol. 1988. 140: 1477. 8 Banejee, S., Haqqi, T. M., Luthra, H. S., Stuart, J. M. and David, C. S., J. Exp. Med. 1988. 167: 832. 9 Haqqi,T. M., Banejee, S., Behlke, M. A., Dungeon, G., Loh, D.Y., Stuart, J., Luthra, H. S. and David, C. S., FASEB J. 1988. 2: A661. 10 Behlke, M. A., Chou, H. S., Huppi, K. and Loh, D.Y., Proc. Natl. Acad. Sci. USA 1986. 83: 767. 11 Bouvet, J. P., Couderc, J., Bouthillier,Y., Franc, B., Decreusefond, C. and Mouton, D., J. Immunol. 1989. 143: 1537. 12 Biozzi, G., Mouton, D., Sant'Anna, 0. A., Passos, H. C., Gennari, M., Reis, M. H., Ferreira,V C. A., Heuman, A. M., Bouthillier,Y.,Ibanez, 0.M., Stiffel, C. and Siqueira, M., Curr. Top. Microbiol. Immunol. 1979. 85: 31. 13 Frangoulis, B., Mouton, D., Sant'Anna, 0. A.,Vidard, L. and Pla, M., Immunogenetics 1990. 31: 389. 14 Davis, L. G., Dibner, M. D. and Battey, J. F., Basic Methods in Molecular Biology, Elsevier, New York 1986, p. 388. 15 Vidard, L., Roger, T., Pham, G., Couderc, J., Bouthillier,Y., Mevel, J. C., Mouton, D. and Seman, M., Immunogenetics 1990. 32: 27. 16 Banejee, S. G., Anderson, D., Luthra, H. S. and David, C. S., J. Immunol. 1989. 142: 2237. 17 Acha-Orbea, H., Mitchell, D. J., Timmerman, L., Wraith, D. C., Tausch, G. S.,Waldor, M. K., Zanviln, S. S., McDevitt, H. 0. and Steinman, L., Cell 1989. 54: 263. 18 Goldsmith,T. L., Janson, L. and Holmdahl, R., Immunology 1990. 69: 508. 19 Pullen, A. M., Marrack, P. and Kappler, J.W., Nature 1988.335: 796. 20 Kappler, J. W., Staerz, U., White, J. and Marrack, F!, Nature 1988. 332: 35. 21 Kappler, J. W., Roehm, N. and Marrack, P., Cell 1987. 49: 273. 22 Bill, J., Kanagawa, 0.,Woodland,D. L. and Palmer, E., J. Exp. Med. 1989. 169: 1405.

Resistance to collagen-induced arthritis in Biozzi mice is not associated with T cell receptor V beta gene polymorphism.

H-I and H-II high antibody-responder Biozzi mice, which express the H-2q permissive haplotype, were shown to be sensitive and refractory to collagen-i...
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