hllIll O-

lmmunogenetics 34: 23-27, 1991

genetics

© Springer-Verlag 1991

The role of C5 and T-cell receptor Vb genes in susceptibility to collagen-induced arthritis Dominic G. Spinella 1'2'3, John R. Jeffers, Robert A. Reife 1'4, and John M. Stuart 1'2 i Veterans Administration Medical Center, Memphis, TN 38104, USA 2 Departments of Medicine, 3 Microbiology and Immunology, and 4 Anatomy, University of Tennessee, Memphis, TN 38163, USA Received December 4, 1990; revised version received January 22, 1991

Abstract. Collagen-induced arthritis (CIA) is a rodent arthritis model in which immunization with heterologous type II collagen induces an inflammatory polyarthritis. Susceptibility to the disease is mediated by major histocompatibility complex (MHC) genes as well as genes at other loci. Previous studies of the SWR/J mouse strain, which is resistant to CIA despite bearing the susceptible 1-1-2q haplotype, have suggested that this resistance is the result of a deletion of T-cell receptor (Tcr) Vb gene segments which is carried by this strain. Other studies have implicated a deficiency in complement component C5 as the cause for the resistance. In order to assess the relative importance of these two genes in susceptibility to CIA, and to provide an estimate of the number of independent genes involved in the disease, we analyzed 196 F 2 progeny of a (DBA/1 x SWR/J) cross for arthritis susceptibility, and expression of both C5 and Tcr genes. Thirty of the F 2 progeny developed arthritis. All of the arthritic mice had at least one copy of the wild-type C5 allele, while the Tcr-Vb haplotypes were distributed in Mendelian fashion. These results demonstrate that C5 sufficiency is an absolute requirement for CIA, but that TcrVb genes located within the SWR deletion have little influence. Genetic analysis of the incidence rate suggests that there is polygenic control of susceptibility to CIA and that in addition to H-2, 5-6 other independent loci (including C5) may be involved.

Introduction Collagen-induced arthritis (CIA) is a rodent model of arthritis in which susceptible strains of mice or rats develop an inflammatory polyarthritis after immunization with native type II collagen in Freund's adjuvant (for review Address correspondence and offprint requests to: D. G. Spinella, VA Medical Center (151), 1030 Jefferson Ave., Memphis, TN 38104, USA.

see Stuart et al. 1981). Both humoral and cellular immune mechanisms appear to play a role in the pathogenesis of this disease (Stuart et al. 1982; Stuart et al. 1983; Trentham et al. 1978; Holmdahl et al. 1986). Susceptibility to CIA is controlled by the major histocompatibility complex (MHC; Wooley et al. 1981; Griffiths et al. 1981) and in mice, strains bearing the A~ allele are particularly prone (Wooley et al. 1981, 1983). An interesting exception to this rule is the SWR mouse which bears the permissive H-2 q haplotype, but is completely resistant to CIA (Luthra et al. 1986). SWR mice have at least two potential immunologic deficits which could be responsible for this resistance. First, these animals are deficient in complement component C5 (Cinader et al. 1964; Erikson et al. 1964), an important mediator of inflammation. In addition, SWR is one of a group of five mouse strains that share the Vba (Klotz et al. 1989) haplotype which carries a deletion of approximately 50% of the known T-cell receptor (Tcr) Vb elements carried by wild-type (Vbb) strains (Behlke et al. 1986; Haqqi et al. 1989). Several studies have attempted to ascertain the relative importance of C5 or Tcrb-V genes to CIA susceptibility. Banerjee and co-workers (1988), in (SWRxB10)F 2 crosses, reported that susceptibility to CIA segregates with the Vbb (undeleted) haplotype in most cases suggesting that genes within the Tcr deletion are critical for the development of an arthritogenic autoimmune response. A later study by the same group (Banerjee et al. 1989), analyzed backcrosses between SWR and A/Sn (C5 deficient, Vbb) and C57L (C5 sufficient, Vba) and reported that only the SWR x A/Sn backcross (offspring of which have at least one undeleted Tcrb-V haplotype, but are still C5 deficient) restores full susceptibility to CIA again implicating Tcr genes located within the Vba deletion as important with C5 playing little or no role in disease susceptibility. In contrast to these studies, Reife and co-workers (1991) analyzed crosses between SWR and B10.D2/oSn and B10.D2/nSn which differ with

24

D.G. Spinella et al. : Tcr and C5 genes in collagen-induced arthritis

respect to the presence of the wild type C5 allele. This study indicated that functional C5 sufficiency was critical to the susceptibility to CIA and supported earlier studies (Watson and Townes 1985; Watson et al. 1987) postulating an important role for C5 in the pathogenesis of CIA. In order to resolve this issue, and to provide an estimate of the number of genetic loci involved in controlling susceptibility to CIA, we analyzed 196 F 2 progeny from a (SWR× DBA/1) cross with respect to arthritis susceptibility and Tcr and C5 genotype. The choice of DBA/1 as the susceptible partner strain insured that all progeny were homozygous for the permissive H-2 q haplotype and avoided the potential problem of presentation of processed type II collagen by hybrid Ia molecules to an unusual T-cell population.

and there is a nucleotide sequence polymorphism in this gene that correlates with Vb haplotype of origin (Smith et al. 1990) and leads to the gain and loss of diagnostic restriction enzyme sites. DNA derived from DBA/1 mice has a cytosine residue at position 302 of the Vb 1 gene which is part of a Bam HI recognition site. In SWR DNA this cytosine is replaced with thymidine which destroys the Barn HI site but creates an Xba I site at the same location. Digestion of the amplified Vbl product with these enzymes provides a rapid means of genotyping F 2 animals for Tcr haplotype of origin. Details of the assay procedure are published separately (Spinella 1991).

Materials and methods Animals and immunizations. Parental mice of the DBA/1 lac and SWR/J strains were obtained from Jackson Laboratories (Bar Harbor, ME), and intercrossed in our animal facilities to produce F 1 and F 2 generations. Five DBA/1 and SWRJJ mice and ten I71 progeny were set aside for immunization. These animals, and a total of 196 F 2 mice were immunized at 8-10 weeks of age. Immunizations consisted of 100 gg native chick type II collagen prepared as described (Cremer 1988), emulsified in complete Freund's adjuvant, and injected at the base of the tail. Booster immunizations were given six weeks later with 100 gg of native type II collagen in incomplete Freund's adjuvant. Animals were observed weekly for development of arthritic signs and each joint was graded on a 0-4 scale as described (Cremer 1988), Maximum grades for each limb were added to provide an arthritic severity score for each afflicted animal. Statistical analyses (ANOVA, t-tests) were performed using software from BMDP Statistical Softare (Los Angeles, CA). C5 and Tcr genotyping. Serum levels of C5 among all the F 2 animals were assayed by radial immunodiffusion of whole serum using a polyclonal alloantiserum (B10.D2/oSn anti-B10.D2/oSn) specific for mouse C5. Comparison of precipitin ring diameters with control wells containing serum from DBA/1 and F 1 control mice allowed assignment of C5 genotype. DBA/1 female mice have lower levels of C5 than males and the F 1 animals have lower levels than the DBA/1 parents. By knowing the sex of the animals providing the serum, it was possible to unambiguously classify each F 2 animals as homozygous or heterozygous for the C5 gene. Tcr V/3 genotype of all arthritic mice was analyzed by restriction enzyme digestion of a portion of the Vbl gene amplified by polymerase chain reaction (PCR). The Vbl gene segment is located immediately 5' to the Vb a deletion (Chou et al. 1987)

Results

All of the immunized animals produced more or less equivalent levels of anti-type II collagen antibodies as assayed by enzyme-linked immunosorbent assay (ELISA) of serum obtained eight weeks post immunization (data not shown). This was not unexpected since both the susceptible DBA/1 and resistant SWR parental strains mount a good antibody response to this antigen. Nevertheless, the antibody assay was performed on all mice to insure that the animals had been appropriately immunized. Of 196 F 2 animals immunized with type II collagen, 30 exhibited signs of arthritis of varying levels of severity for an incidence of 15.3%. This contrasts with the F 1 animals in which 7/10 developed arthritis. Since the F 1 animals are genetically identical, one can derive a penetrance estimate for arthritis of 0.7. The arthritic F 2 animals also tended to develop less severe disease which took longer to develop compared to the F 1 control animals (Table 1). Among the 30 arthritic F 2 animals, the T-cell receptor locus was distributed in Mendelian fashion with eight animals homozygous for the Vba (deleted) haplotype, seven homozygous for the Vb b (wild type), and 15 heterozygotes (Table 1). These data therefore appear to exclude a critical role for Tcr genes located within the Vb a deletion in CIA susceptibility. In contrast to the Tcr haplotype, all of the afflicted animals had at least one copy of the wild-type C5 allele (Table 2). Moreover, homozygotes for this allele were over-represented and heterozygotes under-represented in the arthritic population (ten homozygotes expected, 17 observed; 20 heterozygotes expected, 13 observed; Chi-

Table 1. Arthritis severity score and onset as a function of T-cell receptor genotype. Animals

Tcr genotype

(DBA/1 × SWR/J)FI

~bb/~b

(DBA/1 × SWR/J) F 2

* - SEM.

No. of animals

Severity*

Date of onset* (weeks)

7

7.1 + 1.2

7.7 + 0.3

Wb/l~b

8

4.1 --+0.8

10.0 -- 0.7

Wb/~

15

5.2+0.4

9.3+0.7

V~b/~

7

6.1 + 1.2

9.5 + 0 . 9

25

D.G. Spinella et al.: Tcr and C5 genes in collagen-induced arthritis Table 2. Arthritis severity score and onset as a function of C5 genotype. Animals

C5

genotype (DBA/1 x SWR/J)F1 (DBA/1 x SWR/J)F2

C5+/C5C5+/C5+ C5+/C5C5-/C5-

No. of animals

Severity*

Date of onset* (weeks)

7 17 13

7.1 + 1.2 5.7 + 0.1 5.2 + 0.1

7.7 +0.3 9.1 +-0.1 9.3 + 0.7

0

* ___SEM. square=7.4, P < 0 . 0 1 ) . Thus, functional C5 sufficiency appears to be a requirement for CIA susceptibility, at least among strains of mice homozygous for the n - 2 q haplotype. In order to assess whether Tcr and C5 genotype influence severity or time of onset of arthritis in addition to susceptibility, all arthritic animals were graded for disease severity and post-immunization latency. As shown in Tables 1 and 2, there is a trend toward increasing disease severity and reduced latency with increased gene dosage of both the wild-type C5 and Tcr haplotypes. A two factor analysis of variance was performed on the data to test the significance of this trend. The results indicate that neither trend is statistically significant given this sample size. However, there is a significant interaction component between C5 and Tcr genotype in that animals homozygous for the wild-type C5 allele develop significantly more severe disease as the number of undeleted (Vb b) chromosomes rises from 0 to 2 (P_< .03) as shown in Fig. 1. Thus, the Tcr genotype, while not a critical determinant of CIA susceptibility per se, may influence disease severity.

gesting that these mice carried recombinant Tcr haplotypes. However, despite these attempts to postulate a role for specific Vb genes in CIA, it should be pointed out that ratio of homozygotes to heterozyotes among the 11 F2 mice analyzed by Banerjee and co-workers (1988; 3:6:2) is not significantly different from the 1:2:1 expected Mendelian ratio. Thus, even these data could be interpreted as excluding a role for deleted Tcrb-V elements in CIA susceptibility. Note that this does not necessarily imply that specific Tcrb or Tcra genes are not relevant to CIA, but merely that the resistance of SWR mice to CIA is not related to the absence of T c r b - V g e n e s located in the region of the Tcr locus deleted in Vb a strains. While the Tcr genotype of the F 2 animals does not appear to influence direct disease susceptibility, it does 12

10

8

Discussion The reason for the discrepancy between these results and the work of Banerjee and co-workers (1988, 1989) is not clear. The earlier study analyzed a total of 11 arthritic ( S W R x B 1 0 ) F 2 animals and found two Vb" (deleted) homozygotes, three Vb b (wild-type) homozygotes, and six heterozygotes. The Vb" homozygotes were found to be heterozygous at the M H C (i. e., H-2q/H-2b). Thus, one possible interpretation put forth by the authors is that hybrid Ia molecules in these animals presented an arthritogenic epitope of the type II collagen molecule to Tcell bearing receptors encoded outside the deletion. The Vb analysis in this study was performed serologically using the F23.1 monoclonal antibody (mAb; Staerz et al. 1985) that detects expressed products of the Vb8 family (Behlke et al. 1986) which is among those deleted in Vb a strains. Subsequent Southern blot analysis of the two anomalous mice appeared to show that one of the Vb elements (Vb6) was derived from the Vbb haplotype sug-

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