Comparative analysis of collagen type II-specific immune responses during development of collagen-induced arthritis in two B10 mouse strains

Abstract

Introduction: Immune responses against collagen type II (CII) are crucial for the development of collagen-induced arthritis (CIA). The aim of the present study was to evaluate and compare the CII-directed T cell and antibody specificity at different time points in the course of CIA using two mouse strains on the B10 genetic background - B10.Q, expressing Aq MHC class II molecules, and B10.DR4.Ncf1*/*, expressing human rheumatoid arthritis-associated MHC II DR4 molecules (DRA*0101/DRB*0401).

Methods: B10.Q and B10.DR4.Ncf1*/* mice were immunized with CII emulsified in adjuvant and development of CIA was assessed. T cells from draining lymph nodes were restimulated in vitro with CII peptides and interferon-gamma (IFN-γ) levels in culture supernatants were evaluated by ELISA. CII-specific antibody levels in serum samples were measured by ELISA.

Results: At four different CIA time points we analyzed T cell specificity to the immunodominant CII epitope 259-273 (CII259-273) and several posttranslationally modified forms of CII259-273 as well as antibody responses to three B cell immunodominant epitopes on CII (C1, U1, J1). Our data show that CII-specific T and B cell responses increase dramatically after disease onset in both strains and are sustained during the disease course. Concerning anti-CII antibody fine specificity, during all investigated stages of CIA the B10.Q mice responded predominantly to the C1 epitope, whereas the B10.DR4.Ncf1*/* mice also recognized the U1 epitope. In the established disease phase, T cell reactivity toward the galactosylated CII259-273 peptide was similar between the DR4- and the Aq-expressing strains whereas the response to the non-modified CII peptide was dramatically enhanced in the DR4 mice compared with the B10.Q. In addition, we show that the difference in the transgenic DR4-restricted T cell specificity to CII259-273 is not dependent on the degree of glycosylation of the collagen used for immunization.

Conclusions: The present study provides important evaluation of CII-specific immune responses at different phases during CIA development as well as a comparative analysis between two CIA mouse models. We indicate significant differences in CII T cell and antibody specificities between the two strains and highlight a need for improved humanized B10.DR4 mouse model for rheumatoid arthritis.

Figure 1

CIA disease course in B10.Q and B10.DR4.Ncf1^*/* mice*. Experimental animals were immunized with 100 μ g rCII in CFA (day 0) and three weeks later received a booster injection of 50 μ g rCII in IFA (day 21 postimmunization). CIA severity and incidence were evaluated two to three times weekly until the end of experiment (day 65 postimmunization). (A) Mean arthritis score in B10.Q (n = 10) and B10.DR4.Ncf1^*/* mice (n = 7). Data are presented as mean ± SE and include both arthritic and healthy animals. (B) CIA incidence in B10.Q and B10.DR4.Ncf1^*/* mice. * Data represent CIA assessment only in mice examined until the end of experiment. CIA data from mice killed for in vitro T cell assays are included in Table 1. CIA, collagen-induced arthritis; IFA, incomplete Freund's adjuvant; rCII, rat collagen type II.

Figure 2

T cell specificity to the CII259-273 epitope and its posttranslationally modified forms. B10.Q and B10.DR4.Ncf1^*/* mice were immunized with 100 μ g CII in CFA (day 0) and boosted 21 days later with 50 μ g CII in IFA. Five to ten mice per group were sacrificed at day 15, 35, 50 and 65 postimmunization. Single cell suspensions from collected inguinal lymph nodes were incubated in vitro with collagen peptides or (conA) for 96 h. Culture supernatant was then used for measurement of INF-γ content. (A) T cell recall response in B10.DR4.Ncf1^*/* strain. The symbol '*' marks responses that are significantly higher than the GalOK264 response. *, P <.05; **, P <.01; ***, P <.001. Mean IFN-γ values for the conA stimulated positive control: d. 15 - 10.276 ± 0.899 ng/ml; d. 35 - 4.386 ± 0.269 ng/ml; d. 50 - 15.915 ± 1.349 ng/ml; d. 65 - 7.996 ± 1 ng/ml. (B) CII-specific T cell response in B10.Q mouse strain. The symbol '†' marks significantly lower responses compared to GalOK264 response. †, P <.05; ††, P <.01. Mean IFN-γ values of the positive control: d. 15 - 1.927 ± 0.321 ng/ml; d. 35 - 7.004 ± 0.231 ng/ml; d. 50 - 4.569 ± 0.631 ng/ml; d. 65 - 2.4 ± 0.3 ng/ml. Background values (IFN-γ levels secreted by cells cultured in standard medium without CII peptides or conA) are subtracted from the presented data. All data represent mean ± SE of triplicates. CFA, complete Freund's adjuvant; con A, concanavalin A; CII, collagen type II; IFA, incomplete Freund's adjuvant; IFN, interferon.

Figure 3

CII-specific antibody response at different time points after CIA induction. Blood samples were taken from B10.Q and B10.DR4.Ncf1^*/* mice at four time points after rCII immunization (day 15, 35, 50 and 65). Extracted sera were used to assess the levels of anti-CII antibodies, anti-C1, anti-U1 and anti-J1 epitope-specific antibodies. (A) Total anti-CII antibody concentration in sera of B10.DR4.Ncf1^*/* mice (number of animals per group: day 15 - n = 22, day 35 - n = 16, day 50 - n = 10, day 65 - n = 5). (B) Total anti-CII antibodies concentration in B10.Q mice (n = 20 per each time point). (C) Anti-C1, anti-U1 and anti-J1 antibody levels, measured in B10.DR4.Ncf1^*/* mouse strain (number of animals per group: day 15 - n = 22, day 35 - n = 16, day 50 - n = 10, day 65 - n = 5). (D) Levels of anti-CII epitopes (C1, U1, J1) antibodies in B10.Q mice (n = 20 per each time point). Results indicate mean ± SE. CIA, collagen-induced arthritis; CII, collagen type II.

Figure 4

Comparison of CII-specific T cell and antibody responses in arthritic vs. nonarthritic animals (day 50 postimmunization). (A) IFN-γ recall response to CII259-273 peptides, measured in sick and healthy B10.DR4.Ncf1^*/* mice. Mean positive control values: arthritic mice - 17.294 ± 1.274 ng/ml; nonarthritic animals - 14.696 ± 2.266 ng/ml. (B) CII-specific T cells response in arthritic vs. nonarthritic B10.Q mice. Mean IFN-γ values for the positive control: arthritic animals - 5.199 ± 0.819 ng/ml; nonarthritic mice - 3.624 ± 0.903 ng/ml. Background INF-γ levels were subtracted from all T cell responses data. (C) and (D) Anti-CII antibody concentrations in arthritic vs. nonarthritic B10.DR4.Ncf1^*/* and B10.Q mice, respectively. (E), (F) Levels of antibodies, specific to C1, U1 and J1 CII epitopes in B10.DR4.Ncf1^*/* and B10.Q sick and healthy mice, respectively. All data display mean ± SE. *, P <.05; **, P <.01; ***, P <.001. CII, collagen type II; IFN, interferon.

Figure 5

T cell specificity to the CII259-273 epitope and its posttransationally modified forms in B10.DR4.Ncf1^*/* mice following immunization with huCII. Experimental animals were immunized with 100 μ g huCII emulsified in CFA at day 0. Twenty-one days later the mice received a booster immunization of 50 μ g huCII in IFA. At four particular time points after immunization (day 15, 35, 50, 65) five mice per group were sacrificed and inguinal lymph nodes were harvested for further analysis. (A) T cell recall responses in huCII-immunized B10.DR4.Ncf1^*/* mice following in vitro restimulation with CII259-273 peptides. Mean IFN-γ values for the conA positive control at each time point: d. 15 - 2.884 ± 0.313 ng/ml; d. 35 - 2.137 ± 0.485 ng/ml; d. 50 - 4.732 ± 0.53 ng/ml; d. 65 - 4.632 ± 0.291 ng/ml. Responses that are significantly higher than the GalOK264 response were marked with '*'. Background IFN-γ values are subtracted from the presented data. (B) Total CII-specific antibody levels in B10.DR4.Ncf1^*/* mice immunized with huCII. (C) CII epitope-specific antibody levels B10.DR4.Ncf1^*/* mice immunized with huCII. Data designate mean ± SE. *, P <.05; **, P <.01; ***, P <.001. CFA, complete Freund's adjuvant; con A, concanavalin A; (hu)CII, (human) collagen type II; IFA, incomplete Freund's adjuvant; IFN, interferon.