A recurrent valpha17/vbeta10 TCR-expressing T cell clone is involved in the pathogenicity of collagen-induced arthritis in DBA/1 mice.

Collagen-induced arthritis (CIA) is an experimental model that mimics clinical and histological features of rheumatoid arthritis. In this disease, a crucial role in initiating the pathological changes has been assigned to T lymphocytes expressing the Th1 phenotype. Aiming at identifying type II collagen (CII)-specific T cells involved in CIA, T cell clones were generated in vitro from the lymph nodes (LN) of CII-immunized DBA / 1 mice. In three independent experiments, we repeatedly isolated CD4(+) Th1 clones recognizing the immunodominant epitope in the CB11 fragment of bovine CII and expressing a unique alpha betaTCR produced by the rearrangement of Valpha17/Jalpha20 and Vbeta10/Dbeta1.1/Jbeta2.5 gene segments. By reverse transcriptase-PCR, we demonstrated the presence of mRNA transcripts specific for the beta complementary-determining region 3 of this clonotype in the LN of the majority (73%) of mice with CIA whereas it was never detected in control animals. When transferred to CII-immunized DBA/1 mice, this recurrent Th1 clone augmented the incidence, aggravated significantly the clinical signs of CIA and greatly enhanced the anti-CII antibody response. Altogether, these results provide evidence that a CD4(+) Th1 clone belonging to the public arm of the response toward the immunodominant epitope of CII is involved in the cascade of events leading to CIA.

CD4+ Th2 cells specific for mycobacterial 65-kilodalton heat shock protein protect against pristane-induced arthritis.

Previous studies showed that mice with pristane-induced arthritis (PIA) and those protected from the disease by preimmunization with mycobacterial 65-kDa heat shock protein (hsp65), possess raised immune responses to hsp65. Thus, a paradox exists whereby T cells from both arthritic and hsp65-protected animals proliferate vigorously in response to the same Ag. Here we demonstrate that T cells from mice with PIA and hsp65-protected mice produce different cytokines in vitro in response to hsp65. The use of a sensitive CelELISA to measure Ag-driven lymphokine production revealed that spleen cells from hsp65-protected mice, but not those from pristane-injected or normal mice, produced the Th2-associated cytokines IL-4, IL-5, and IL-10 in response to stimulation with hsp65. By contrast, the Th1-associated cytokines IL-2 and IFN-gamma were produced by spleen cells from mice of all groups in response to hsp65. Furthermore, there was a dramatic increase in the IgG1 to IgG2a ratio of anti-hsp65 Abs from arthritic to protected mice. Thus, it appears that a Th2 response is protective against PIA. To examine this theory, a regimen of IL-12 administration which polarizes the hsp65-specific (Th2) immune response toward Th1 was identified. This regime abolished hsp65-mediated protection against PIA. Other experiments revealed that the specificity of the response to hsp65 was important, as other bacterial proteins known not to protect against PIA induced similar Th2-associated cytokines in vitro. It is considered that the protection afforded by hsp65 preimmunization is mediated by Th2-associated cytokines produced by hsp65-specific CD4+ T cells.

Conversion in vivo from an early dominant Th0/Th1 response to a Th2 phenotype during the development of collagen-induced arthritis.

Over the past decade, the central role of T cells in the process of collagen-induced arthritis (CIA) has been extensively documented. The inflammatory features of CIA and its successful modulation after treatment in vivo with Th2 lymphokines, known to down-regulate proinflammatory cytokines, classify CIA as a Th1-mediated disease. However, no direct evidence for the presence of the different T helper subsets has been obtained. To identify the collagen-specific CD4+ T cell subset(s) developing during the course of CIA, lymph nodes from susceptible DBA/1 mice (H-2q) were harvested at different times after injection of bovine type II collagen in Freund's complete adjuvant and checked by enzyme-linked immunospot assay for the production of interferon (IFN)-gamma and interleukin (IL)-4. The results clearly showed that type II collagen-specific T cells secreting either IFN-gamma, IL-4, or both, develop early in vivo, before the onset of arthritis: the number of IFN-gamma-secreting cells was already maximal 15 days after immunization, whereas more IL-4-secreting cells were found at day 30, just before the onset of clinical arthritis. Another strategy was to establish collagen-specific CD4+ T cell lines and sublines in vitro and to analyze their lymphokine secretion pattern. Lines generated 8 days after immunization displayed a mixed lymphokine secretion pattern characteristic of Th0 cells or of a mixture of Th1 and Th2 cells. After limiting dilution of a day 8 line, 60% of the growing sublines were Th0-like (secreting IFN-gamma, IL-4, and IL-5), and 25% were Th1 (secreting IFN-gamma). By day 25 post-immunization, 33% of the generated sublines were Th0-like, 11% Th1, and 56% Th2 (secreting IL-4 and IL-5). Moreover, all the sublines raised from the lymph nodes of arthritic mice harvested at day 55 secreted high amounts of Th2 lymphokines, and only 3 out of 14 also produced some IFN-gamma. This study demonstrates that during the course of CIA the collagen-specific CD4+ T cell response shifts in vivo from a dominant Th0/Th1 response to a clear Th2 phenotype. These results contribute to our understanding of the collagen-specific CD4+ T helper subsets which develop during the induction and clinical phases of CIA.

Prevention of autoimmune disease due to lymphocyte modulation by the B-subunit of Escherichia coli heat-labile enterotoxin.

We demonstrate that the receptor binding moiety of Escherichia coli heat-labile enterotoxin (EtxB) can completely prevent autoimmune disease in a murine model of arthritis. Injection of male DBA/1 mice at the base of the tail with type II collagen in the presence of complete Freund's adjuvant normally leads to arthritis, as evidenced by inflammatory infiltration and swelling of the joints. A separate injection of EtxB at the same time as collagen challenge prevented leukocyte infiltration, synovial hyperplasia, and degeneration of the articular cartilage and reduced clinical symptoms of disease by 82%. The principle biological property of EtxB is its ability to bind to the ubiquitous cell surface receptor GM1 ganglioside, and to other galactose-containing glycolipids and galactoproteins. The importance of receptor interaction in mediating protection from arthritis was demonstrated by the failure of a non-receptor-binding mutant of EtxB to elicit any protective effect. Analysis of T cell responses to collagen, in cultures of draining lymph node cells, revealed that protection was associated with a marked increase in interleukin 4 production concomitant with a reduction in interferon gamma levels. Furthermore, in protected mice there was a significant reduction in anti-collagen antibody levels as well as an increase in the IgG1/IgG2a ratio. These observations show that protection is associated with a shift in the Th1/Th2 balance as well as a general reduction in the extent of the anti-type II collagen immune response. This suggests that EtxB-receptor-mediated modulation of lymphocyte responses provides a means of preventing autoimmune disease.

Pristane-induced arthritis is CD4+ T-cell dependent.

The development of arthritis induced in mice by intraperitoneal injection of the non-antigenic mineral oil pristane (2,6,10,14-tetramethylpentadecane) was shown to depend on the presence of CD4+ T cells. Initial experiments assessed the influx of lymphoid cells into the peritoneal cavity of CBA/Igb mice after pristane injection. Both CD4+ and CD8+ cell numbers were maximal around 50 days. Other experiments confirmed our original observation that irradiated pristane-treated mice failed to develop arthritis unless they were reconstituted with spleen cells from normal donors. This finding has been extended by showing that the population of transferred splenic lymphoid cells must contain CD4+ T cells, while CD8+ T cells and B cells were not required for reconstitution. Conventionally housed and hsp 65-immunized animals are known to harbour T cells reactive with hsp 65. In addition, hsp 65-immunized mice are resistant to the development of pristane-induced arthritis (PIA). Thus, additional experiments assessed the population of splenic T cells activated and proliferating against mycobacterial 65,000 MW heat shock protein (hsp 65). In cultures of purified splenic T cells derived from both conventional and hsp 65-immunized mice, removal of CD4+ T cells significantly reduced the proliferative response to hsp 65, while removal of CD8+ T cells often enhanced the response. These proliferative responses were also shown to be major histocompatibility complex (MHC) class II restricted. The present findings demonstrate that PIA is CD4+ T-cell mediated, and immunodominant environmental antigens such as hsp 65 activate this population of lymphocytes. The CD4+ hsp 65-reactive population may be pathogenic or protective in PIA, depending upon the route of sensitization.

Collagen-induced arthritis in DBA/1 mice: cytokine gene activation following immunization with type II collagen.

Immunization of DBA/1 mice with bovine type II collagen (CII) in complete Freund's adjuvant (CFA) leads to collagen-induced arthritis as evidenced by joint inflammation. In this study, reverse transcription-polymerase chain reaction (RT-PCR) was used to demonstrate the activation of genes encoding for IL-2, IFN-gamma, and IL-10 in the lymph nodes from both CII-immunized and control CFA-immunized DBA/1 mice, at Days 10, 40, and 70 after immunization, in the absence of any IL-5 or IL-13 transcription. By quantitative RT-PCR, the levels of IFN-gamma mRNA in response to CII could not be quantitatively differentiated from the IFN-gamma transcribed in response to CFA alone. In the joints of CII-immunized mice, IL-1beta and IL-10 mRNA were found in the absence of IL-5 or IFN-gamma. Synovial IL-1beta and IL-10 were expressed most strongly at the time of clinical symptoms, 40 days after immunization. Together, these findings suggest that immunization with CII in CFA induces a type 1 response against the adjuvant, giving a cytokine environment which influences the T cells responding to CII to become type 1 T cells. This is manifested here by the appearance of gene activation in synovial tissue of collagen-immunized mice, but not in adjuvant-immunized control animals.