Exposure to dextran sulfate sodium (DSS) induces acute colitis, which is normally resolved after DSS removal. To study chronicity, mice are typically subjected to three to five cycles of weekly DSS exposures, each followed by a 1- to 2-wk rest period. Here, we describe a novel and convenient way of inducing chronic, progressive colitis by a single exposure to DSS. C57BL/6 mice exposed to DSS for 5 days developed acute colitis that progressed to severe chronic inflammation. The plasma haptoglobin levels remained high during the chronic phase, showing that the inflammation was active. Surprisingly, the mice regained their original weight along with the progression of colitis, and the only apparent symptom was loose feces. Histopathological changes 4 wk after DSS removal were dense infiltrates of mononuclear cells, irregular epithelial structure, and persistent deposits of collagen. A progressive production of the cytokines IL-1beta, IL-12 p70, and IL-17 correlated with the extensive cellular infiltration, whereas high IFN-gamma production was mainly found late in the chronic phase. Similar to C57BL/6 mice, BALB/c mice exposed to 5 days of DSS developed acute colitis as previously described. The acute colitis was accompanied by elevated plasma levels of haptoglobin and increased colonic levels of IL-1alpha/beta, IL-6, IL-18, and granulocyte colony-stimulating factor. However, soon after DSS removal, BALB/c mice recovered and were symptom free within 2 wk and completely recovered 4 wk after DSS removal in terms of histopathology, haptoglobin levels, and local cytokine production. In summary, these data stress the effect of genetic background on the outcome of DSS provocation. We believe that the present protocol to induce chronic colitis in C57BL/6 mice offers a robust model for validating future therapies for treatment of inflammatory bowel disease.

Immunization of mice with type II collagen (CII) leads to collagen-induced arthritis (CIA), a model for rheumatoid arthritis. T cell recognition of CII is believed to be a critical step in CIA development. We have analyzed the T cell determinants on CII and the TCR used for their recognition, using twenty-nine T cell hybridomas derived from C3H.Q and DBA/1 mice immunized with rat CII. All hybridomas were specific for the CII(256-270) segment. However, posttranslational modifications (hydroxylation and variable O-linked glycosylation) of the lysine at position 264 generated five T cell determinants that were specifically recognized by different T cell hybridoma subsets. TCR sequencing indicated that each of the five T cell epitopes selected its own TCR repertoire. The physiological relevance of this observation was shown by in vivo antibody-driven depletion of TCR Valpha2-positive T cells, which resulted in an inhibition of the T cell proliferative response in vitro towards the non-modified CII(256-270), but not towards the glycosylated epitope. Most hybridomas (20/29) specifically recognized CII(256-270) glycosylated with a monosaccharide (beta-D-galactopyranose). We conclude that this glycopeptide is immunodominant in CIA and that posttranslational modifications of CII create new T cell determinants that generate a diverse TCR repertoire.

The T cell recognition of type-II collagen (CII) in H-2q mice, susceptible to CII-induced arthritis, was analyzed. With the use of T cell hybridomas derived from rat CII-immunized mice, a peptide corresponding to amino acids 245-270 on chick CII was found to harbor a T cell epitope which is present on heterologous CII (chick, rat, human, and bovine CII) but not on autologous CII. It was shown that this epitope was located within amino acids 260-270, although flanking regions in either direction were necessary for proper recognition. A peptide corresponding to human CII (256-270) was used for further studies. A single amino acid difference at position 266 between mouse CII (aspartic acid) and heterologous CII (glutamic acid) strongly influenced recognition of this peptide. No response towards the mouse peptide was seen with any of the T cell hybridomas. Inhibition studies revealed that the mouse peptide did not bind as well to major histocompatibility complex as the corresponding heterologous peptide. Both peptides gave rise to a T cell response after immunization. However, immunization with the heterologous peptide resulted in a response strictly directed to rat CII and the immunogen while immunization with the autologous peptide elicited T cells which reacted in a heteroclitic fashion, with a stronger response to the heterologous peptide than to the autologous peptide, and did respond to rat CII but not to mouse CII. We suggest that aspartic acid in position 266 results in a cryptic determinant in mouse CII which is neither recognized after CII immunization nor capable of tolerance induction. A glutamic acid at position 266, however, gives rise to an immunodominant epitope which is recognized by a large proportion of the T cells activated after immunization with heterologous CII.

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SummaryA critical event in an immune response is the T cell recognition of peptides bound to major histocompatibility com~ex (MHC) molecules on the surface of an antigen presenting cell (APC). Although the majority of eukaryotic proteins are glycosylated, it has not yet been shown that T cell recognition of such proteins involves recognition of the bound carbohydrates. Type II collagen (CII), the major protein constituent of joint cartilage, is posttranslationally modified by hydroxylation and glycosylation of lysines. In this report we show that posttranslational modifications of the immunodominant peptide CII(256-270) generate a structural determinant that is distinct from the determinant represented by the corresponding synthetic peptide. Elimination of carbohydrates, present on CII, by two different biochemical methods revealed that the carbohydrates, O-linked to the hydroxylysines within the CII(256-270) determinant, were crucial for the reactivity towards the posttranslationally modified peptide. Furthermore, a T cell hybridoma specific for the glycosylated determinant was stimulated by tryptic CII-peptides presented by fixed APCs, thus showing that the carbohydrates are involved in the trimolecular complex T call receptor/peptide/MHC. Finally, the importance of the bound carbohydrates for the arthritogenicity of CII was investigated by comparing the development of arthritis after immunization with carbohydrate-depleted and glycosylated CII, respectively. Incidence, time of onset, and severity of the disease were significantly affected by the elimination of carbohydrates, whereas no significant difference in anti-CII antibody titers was seen.

Our data implicate MPO in atherosclerotic plaque instability and suggest that non-invasive imaging and pharmacological inhibition of plaque MPO activity hold promise for clinical translation in the management of high-risk coronary artery disease.

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The major histocompatibility complex (MHC) class II region is assumed to influence autoimmune diseases such as rheumatoid arthritis. In the mouse, the H-2q haplotype is associated with susceptibility to collagen-induced arthritis, while the H-2p haplotype is not. The class II A molecules of these haplotypes differ by only four amino acids in the first domain of the beta chain. To test if this difference accounts for the MHC influence on susceptibility to collagen-induced arthritis, H-2p mice were made transgenic with an Abp gene altered to resemble the Abq gene. The transgenic A beta chain hybridized with the A alpha p chain and was shown to be physiologically expressed by testing antigen-presentation capacity to Aq-restricted T cell hybridomas and with FACS analyses. These transgenic mice developed an autoimmune response to type II collagen and also collagen-induced arthritis. The data unequivocally suggest the Ab gene as a major genetic susceptibility locus for autoimmune collagen-induced arthritis.