CD4+CD25+ immunoregulatory T cells may not be involved in controlling autoimmune arthritis.

Accumulating evidence suggests that regulatory T cells play a crucial role in preventing autoimmunity. Recently, a naturally occurring CD4+CD25+ T-cell subset that is anergic and also suppressive has been shown to suppress autoimmunity in several animal models. We used proteoglycan-induced arthritis (PGIA) as a study model to investigate the role of the CD4+CD25+ regulatory T cells in autoimmune arthritis. There was no significant change in the percentage of CD4+CD25+ T cells during the immunization period when proteoglycan- or ovalbumin-immunized BALB/c and C57BL/6 mice were compared. An adoptive transfer study showed that the CD4+CD25+ T cells did not protect severe combined immunodeficient mice from arthritis when they were cotransferred with splenocytes from arthritic animals. Similarly, depletion of the CD4+CD25+ T cells did not enhance the onset of the disease or disease severity in severe combined immunodeficient mice. Moreover, CD28-deficient mice, which have very few CD4+CD25+ T cells, were highly resistant to PGIA. These findings indicate that the CD4+CD25+ regulatory T cells may not play a critical role in controlling PGIA.

Development of inflammation in proteoglycan-induced arthritis is dependent on Fc gamma R regulation of the cytokine/chemokine environment.

FcgammaRs are specialized cell surface receptors that coordinately regulate immune responses. Although FcgammaR expression is a prerequisite for the development of several immune complex-mediated diseases, the mechanism responsible for FcgammaR-dependent regulation in autoimmunity remains unclear. Therefore, we assessed FcgammaR-dependent regulation of inflammation in proteoglycan-induced arthritis (PGIA) using FcgammaR(-/-) mice. FcgammaRIIb(-/-) mice developed arthritis at an earlier time point and with a greater severity than wild-type (WT) mice. In gamma-chain(-/-) (FcgammaRI(-/-) and FcgammaRIII(-/-)) mice, no clinical or histological evidence of inflammation was observed. Exacerbation of arthritis in FcgammaRIIb(-/-) mice correlated with enhanced PG-specific Ab production, but did not significantly affect PG-specific T cell priming. In gamma-chain(-/-) mice, the absence of arthritis did not correlate with serum Ab responses, as PG-specific Ab production was normal. Although PG-specific T cell proliferation was diminished, spleen cells from gamma-chain(-/-) mice successfully adoptively transferred arthritis into SCID mice. Our studies indicated that the mechanism responsible for FcgammaR regulation of PGIA development was at the level of inflammatory cytokine and beta-chemokine expression within the joint. FcgammaRIIb regulated the development of PGIA by controlling the initiation of cytokine and chemokine expression within the joint before the onset of arthritis, whereas the expression of FcgammaRI and or FcgammaRIII controlled cytokine and chemokine expression late in the development of PGIA during the onset of disease. These results suggest that FcgammaRs are critical for the development of inflammation during PGIA, possibly by maintaining or enhancing inflammatory cytokine and beta-chemokine production.

IL-4 and IL-12 regulate proteoglycan-induced arthritis through Stat-dependent mechanisms.

IL-4, a well-recognized modulator of macrophage activation, is perceived as an anti-inflammatory cytokine; however, under certain circumstances IL-4 may function as a proinflammatory cytokine. We have previously demonstrated that IL-4 treatment of mice with proteoglycan-induced arthritis (PGIA) inhibited the development of disease. To determine whether the capacity of IL-4 to inhibit disease is dependent on IL-4-mediated regulation of IL-12, we assessed the requirement for IL-4 in modulating development of PGIA. Immunization of mice, lacking IL-4 and Stat6, with proteoglycan results in a significant increase in arthritis severity in comparison to wild-type controls, suggesting that arthritis severity is regulated by IL-4 through a Stat6-dependent mechanism. Concomitant with exacerbated disease in IL-4(-/-) mice, there is a significant increase in the systemic production of proinflammatory cytokines IL-12, TNF-alpha, and IFN-gamma and in levels of mRNA transcripts for proinflammatory cytokines and chemokines in joints. Disease is suppressed in Stat4(-/-) mice indicating that elevated levels of IL-12 contribute to exacerbation of arthritis and that suppression is accompanied by reduced levels of IFN-gamma production. In support of this, IFN-gamma(-/-) mice are protected from PGIA and the degree of inflammation is similar to Stat4(-/-) mice. The decrease in disease severity in IFN-gamma(-/-) and Stat4(-/-) mice correlates with diminished TNF-alpha levels but there is no switch to a Th2-type response. Taken together, these results suggest that IL-4 regulates the severity of disease in PGIA by controlling IL-12 production, which in turn regulates the magnitude of IFN-gamma expression through a Stat4-dependent pathway.

Genetic rescue of chondrodysplasia and the perinatal lethal effect of cartilage link protein deficiency.

The targeted disruption of cartilage link protein gene (Crtl1) in homozygous mice resulted in a severe chondrodysplasia and perinatal lethality. This raised the question of whether the abnormalities seen in Crtl1 null mice are all caused by the absence of link protein in cartilage or whether the deficiency of the protein in other tissues and organs contributed to the phenotype. To address this question we have generated transgenic mice overexpressing cartilage link protein under the control of a cartilage-specific promoter, and then these transgenic mice were used for a genetic rescue of abnormalities in Crtl1 null mice. While the overexpression of cartilage link protein resulted in no abnormal phenotype, the cartilage-specific transgene expression of link protein could completely prevent the perinatal mortality of link protein-deficient mice and, depending on the level of the link protein expression, rescue skeletal abnormalities. Although link protein was originally isolated from cartilage, we found and determined Crtl1 transcripts and corresponding proteins in every organ tested from mouse embryos to aging animals. We also identified three additional members of the link protein family, all co-localized with hyaluronic acid-binding proteoglycans in the mouse genome. The ubiquitous presence of link protein suggests a general and systemic function of link protein in the organization of extracellular matrix in a number of tissues, possibly interacting with other proteoglycans, such as versican, brevican, and neurocan.