B cells are important as antigen presenting cells for induction of MHC-restricted arthritis in transgenic mice

Abstract

Rheumatoid arthritis and its animal model, collagen-induced arthritis, are known as a T and B cell dependent disease. To analyze the role of B cells in arthritis, we generated B cell deficient (microMT) mice carrying HLA-DQ8 as transgene, Abetao.DQ8.micromt mice. HLA-DQ8 transgenic mice (Abetao.DQ8) are susceptible to collagen induced arthritis, an animal model for inflammatory arthritis. Deletion of IgM gene led to the absence of B cells while T cells were comparable to Abetao.DQ8 mice. Arthritis and autoantibodies was completely abrogated in B cell deficient DQ8 mice. T cell response and proinflammatory cytokine production in response to type II collagen and its derived peptides in vitro was significantly decreased despite an increased number of Mac-1 positive cells in DQ8.micromt mice compared to DQ8 mice suggesting B cells could be important for antigen presentation as well. In vitro substitution of B cells from wild type mice restored the response in DQ8.micromt mice. B cells could also present CII-derived peptides to antigen-specific DQ8-restricted hybridomas reinforcing the role of B cells in presentation of antigens to T cells. The data suggest that B cells can be involved in pathogenesis of arthritis by producing autoantibodies and antigen presentation.

Figure 1

Peptide ELISA using sera from DQ8 mice showed that a differential B cell reactivity with known DQ8-restricted T cell epitopes. A) Sera from primed were tested on plates coated with known DQ8-restricted T cell epitopes of human type II collagen-derived peptides. Peptides sequences are described in methods. B) Sera from primed mice were tested for anti bovine type II antibodies.

Figure 2

DQ8.μmt mice are resistant to develop collagen-induced arthritis. A) DQ8 and DQ8.μmt mice were immunized with CII and followed for onset of arthritis. B) Anti-CII antibodies in primed DQ8 and DQ8.umt mice, showing no detectable levels of antibodies in the latter.

Figure 3

Antigen specific response in DQ8.μmt mice is lower when compared to DQ8 mice. A) In vitro T cell response to CII and CII derived peptide 284−303 in B cell sufficient and deficient DQ8 mice. DQ8.umt mice produce low amounts of cytokines B) IFNγ and IL-4.

Figure 4

B cells from parent strain can restore in vitro antigen specific response. A) T cells from CII-derived peptide 284−303 primed DQ8.μmt mice co-cultured in vitro with B cells isolated from primed DQ8 mice in 2 different concentrations. The response was restored to the level of B cell sufficient mice as shown in Figure 3. B) Cytokine, IFN-γ and IL-4, levels were also restored to that of DQ8 mice as shown in Figure 3. Cytokines were measured by ELISA using supernatants from cultures in 4A.

Figure 5

Presentation of a CII-derived peptide, HII-44 (554−573), and its minimal epitope by B and dendritic cells to antigen-specific and DQ8-restricted T cell hybridoma shows B cells can present the peptide. A) T cell proliferation in vitro with peptide 554−573 and its minimal epitope presented by APCs. B) B cells and dendritic cells isolated from naïve mice and mice primed with HII-44 and minimal peptide 44S were cultured in vitro with T cells hybridoma specific for HII-44 in the presence or absence of the peptides. IL-2 levels were measured by ELISA from the in vitro culture supernatants after 24 hrs. Also, B and dendritic cells were pre-incubated with the peptides HII-44 and HII-44S for 24 hrs, washed and then used as antigen presenting cells to specific hybridoma without the presence of peptide. *p<0.05.

Figure 6

Cell profile of A) naïve and B) immunized mice show that DQ8.μmt mice have higher frequency of CD3+ and Mac1+ cells. After priming, a significant difference was observed in frequency of cells with costimulatory molecules. *p<0.05, ** p<0.01.