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Fms-like tyrosine kinase 3 ligand (Flt3L) is known as the primary differentiation and survival factor for dendritic cells (DCs). Furthermore, Flt3L is involved in the homeostatic feedback loop between DCs and regulatory T cell (Treg). We have previously shown that Flt3L accumulates in the synovial fluid in rheumatoid arthritis (RA) and that local exposure to Flt3L aggravates arthritis in mice, suggesting a possible involvement in RA pathogenesis. In the present study we investigated the role of Flt3L on DC populations, Tregs as well as inflammatory responses in experimental antigen-induced arthritis. Arthritis was induced in mBSA-immunized mice by local knee injection of mBSA and Flt3L was provided by daily intraperitoneal injections. Flow cytometry analysis of spleen and lymph nodes revealed an increased formation of DCs and subsequently Tregs in mice treated with Flt3L. Flt3L-treatment was also associated with a reduced production of mBSA specific antibodies and reduced levels of the pro-inflammatory cytokines IL-6 and TNF-α. Morphological evaluation of mBSA injected joints revealed reduced joint destruction in Flt3L treated mice. The role of DCs in mBSA arthritis was further challenged in an adoptive transfer experiment. Transfer of DCs in combination with T-cells from mBSA immunized mice, predisposed naïve recipients for arthritis and production of mBSA specific antibodies. We provide experimental evidence that Flt3L has potent immunoregulatory properties. Flt3L facilitates formation of Treg cells and by this mechanism reduces severity of antigen-induced arthritis in mice. We suggest that high systemic levels of Flt3L have potential to modulate autoreactivity and autoimmunity.

RHO family proteins are important for the function of inflammatory cells. They are modified with a 20-carbon geranylgeranyl lipid in a process catalyzed by protein geranylgeranyltransferase type I (GGTase-I). Geranylgeranylation is viewed as essential for the membrane targeting and activity of RHO proteins. Consequently, inhibiting GGTase-I to interfere with RHO protein activity has been proposed as a strategy to treat inflammatory disorders. However, here we show that mice lacking GGTase-I in macrophages develop severe joint inflammation resembling erosive rheumatoid arthritis. The disease was initiated by the GGTase-I-deficient macrophages and was transplantable and reversible in bone marrow transplantation experiments. The cells accumulated high levels of active GTP-bound RAC1, CDC42, and RHOA, and RAC1 remained associated with the plasma membrane. Moreover, GGTase-I deficiency activated p38 and NF-κB and increased the production of proinflammatory cytokines. The results challenge the view that geranylgeranylation is essential for the activity and localization of RHO family proteins and suggest that reduced geranylgeranylation in macrophages can initiate erosive arthritis.

Proto-oncogene survivin has recently been identified as a prognostic marker distinguishing patients with destructive rheumatoid arthritis (RA). In the present material of 132 RA patients and 82 controls, the levels of survivin correlated to urokinase (uPA) (r= 0.46), a plasminogen activator over-expressed in inflamed joints and known to exhibit potent arthritogenic properties. Here we evaluate the functional relationship between these proteins using primary synovial fibroblasts and leucocytes of RA patients, human monocytic (THP-1) and fibroblast (MRC-5) cell lines. Using inhibitors of intracellular signalling, we show that uPA and survivin share common transduction pathways in synovial fibroblasts being dependent on the activity of tyrosine kinases, phosphatidylinositide 3 kinase and mitogen effector kinase. Moreover, uPA production is significantly reduced in fibroblasts if survivin synthesis has been silenced by siRNA. Importantly, silencing of survivin in fibroblasts prevented their invasive growth in knee joints of severe combined immune deficient mice. Interaction of uPA with receptor up-regulates survivin expression in leucocytes. In turn, survivin is required for the up-regulation of uPA receptor on the cell surface. These findings indicate that survivin is an essential mediator of arthritogenic properties of uPA regulating its synthesis in synovial fibroblasts and uPAR expression in leucocytes. Close correlation between survivin and uPA levels in patients with RA supports the importance of this connection for the pathogenesis of arthritis.

Dendritic cells (DC) express a functional NADPH oxidase and produce reactive oxygen species (ROS) upon interaction with microbes and T cells. Exposure to ROS leads to DC activation and maturation, as evidenced by phenotypic and functional changes. We have evaluated how endogenous ROS production affects the cytokine secretion pattern and T cell-activating capacity of bone marrow-derived murine DC. DC treated with ROS scavengers, as well as DC from mice that lack a functional NADPH oxidase (and thereby inherently deficient in ROS production) produced significantly increased levels of IL-1beta, IL-6, TNF-alpha and TGF-beta in response to microbial activation. DC deficient in ROS production induced high levels of IFN-gamma and IL-17 in responding T cells after Ag-specific or superantigen-induced activation. Finally, we show that ROS deficiency affected the induction of a T cell-dependent inflammatory condition, collagen-induced arthritis (CIA). C57BL/6 mice that lack a functional NADPH oxidase developed a severe and erosive CD4-dependent CIA, whereas the majority of the congenic wild-type animals remained healthy. These data suggest that ROS act as immunomodulators in DC-driven T cell activation and perhaps also in T cell-dependent immunopathology.

The prototype Staphylococcus aureus strain 8325-4 produces high levels of hemolysins and proteases. Recently it has been shown that this property depends on a deficiency of sigma factor B (SigB) activity controlling the activation of regulatory genes such as agr and sarA. SigB deficiency is in turn due to a mutation in the rsbU gene, which is required for posttranslational activation of SigB. The rsbU defect of strain 8325-4 has recently been repaired, and we used this strain (SH1000), along with its isogenic sigB-negative mutant, to investigate the contributions of RsbU and SigB in a murine model of septic arthritis. Intravenous inoculation with the rsbU-repaired isogenic strain SH1000 resulted in significantly more severe arthritis, weight decrease, and mortality compared to those of the parental strain 8325-4 (rsbU-negative) or the isogenic sigB-negative mutant (MJH502). SH1000 also persisted more in kidneys and joints of infected mice. Our data strongly suggest that RsbU and SigB regulate important virulence factors, thereby contributing significantly to the outcome of staphylococcal infection.