Genetic control of leucocyte--endothelial cell interaction in collagen-induced arthritis.

OBJECTIVE: Despite considerable work on defining disease pathways, several aspects of collagen-induced arthritis (CIA) remain poorly defined, in particular those contributing to the initiation phase of the disease. It is thought that in CIA the activation of circulating leucocytes, their interaction with the endothelial lining followed by subsequent transendothelial migration and infiltration into tissue represents the first and determining step in a complex sequence of processes mediating tissue injury. In this study we attempted to define the genetic basis of this stage of disease using genetic linkage studies, in-vivo imaging and expression profiling. METHODS: A genome scan with 132 informative markers was performed on 155 (DBA/1JxFVB/N) F2 mice. Linkage analysis was performed by combining genotyping data from the genome scan and the phenotypic data of leucocyte adherence, leucocyte rolling fraction, functional capillary density, centre line red blood cell velocity and capillary width as well as the expression level of the selected genes Cd44, Il13ralpha1, Ccr3, Defb3, Sele, Sell, Selp, Xcl1, Il1beta, Tnfalpha and Ifngamma as traits. RESULTS: Multiple classic quantitative trail loci (QTL) controlling leucocyte-endothelial cell interactions were identified on chromosomes 8 and 17 as well as expression QTL controlling the expression of several differentially expressed adhesion molecules and cytokines on chromosomes 1, 2, 5, 6, 7, 8, 12, 15, 16 and 17. CONCLUSION: The study describes for the first time QTL controlling the CIA initiating leucocyte-endothelial cell interaction.

Impact of short-term liquid storage on human CD133(+) stem cells.

Stem cell transplantation is a viable strategy for regenerative medicine. However, it is inevitable to have cells undergo storage for several hours or days due to processing and transportation. Therefore, it is crucial to have rigidly controlled conditions ensuring the therapeutic benefit of isolated stem cells. In the present study, we investigated the impact of short-term storage on human CD133(+) cells. CD133(+) cells were isolated from human bone marrow and kept at standardized nonfreezing storage conditions for up to 72 h. Cell viability (apoptosis/necrosis) and expression of CD133 and CXCR4 were analyzed by flow cytometry. Metabolic activity was determined using an MTT assay; colony-forming ability, as well as endothelial-like differentiation, was further evaluated. A qRT-PCR array was employed to investigate the expression of stemness genes. CD133 and CXCR4 expressions were preserved at all time points. After 30 h, cell number and metabolic activity decreased, although no significant changes were detected in cell viability and proliferation as well as endothelial-like differentiation. Cell viability and proliferation decreased significantly only after 72 h of storage. Our results indicate that storage of isolated human CD133(+) bone marrow stem cells in liquid allows for high viability and functionality. However, storage time should be limited in order to avoid cell loss.

Perforin deficiency attenuates collagen-induced arthritis.

Collagen-induced arthritis (CIA), an approved animal model for rheumatoid arthritis, is thought to be a T cell-dependent disease. There is evidence that CD8+ T cells are a major subset controlling the pathogenesis of CIA. They probably contribute to certain features of disease, namely tissue destruction and synovial hyperplasia. In this study we examined the role of perforin (pfp), a key molecule of the cytotoxic death pathway that is expressed mainly in CD8+ T cells, for the pathogenesis of CIA. We generated DBA/1J mice suffering from mutations of the pfp molecule, DBA/1J-pfp-/-, and studied their susceptibility to arthritis. As a result, pfp-deficient mice showed a reduced incidence (DBA/1J-pfp+/+, 64%; DBA/1J-pfp-/-, 54%), a slightly delayed onset (onset of disease: DBA/1J-pfp+/+, 53 +/- 3.6; DBA/1J-pfp-/-, 59 +/- 4.9 (mean +/- SEM), and milder form of the disease (maximum disease score: DBA/1J-pfp+/+, 7.3 +/- 1.1; DBA/1J-pfp-/-, 3.4 +/- 1.4 (mean +/- SEM); P < 0.05). Concomitantly, peripheral T cell proliferation in response to the specific antigen bovine collagen II was increased in pfp-/- mice compared with pfp+/+ mice, arguing for an impaired killing of autoreactive T cells caused by pfp deficiency. Thus, pfp-mediated cytotoxicity is involved in the initiation of tissue damage in arthritis, but pfp-independent cytotoxic death pathways might also contribute to CIA.

Fas ligation on macrophages enhances IL-1R1-Toll-like receptor 4 signaling and promotes chronic inflammation.

The nonapoptotic functions of Fas ligation are incompletely characterized. In contrast to expectations, we show here that Fas-deficient mice developed less-severe collagen-induced arthritis than did control mice. Despite having milder arthritis, Fas-deficient mice had more of the critical pro-inflammatory mediator interleukin-1 beta (IL-1 beta) in their joints, suggesting inefficient activation through IL-1 receptor 1 (IL-1R1) when Fas signaling is deficient. In primary human macrophages and macrophages from Fas- or Fas ligand (FasL)-deficient mice, interruption of Fas-FasL signaling suppressed nuclear factor-kappa B activation and cytokine expression induced by IL-1 beta and lipopolysaccharide. This cross-talk was mediated by the Fas-associated death domain through interaction with myeloid differentiation factor 88. These observations document a unique mechanism whereby Fas-FasL interactions enhance activation through the IL-1R1 or Toll-like receptor 4 pathway, which may contribute to the pathogenesis of chronic arthritis.

A proinflammatory role for Fas in joints of mice with collagen-induced arthritis.

Collagen-induced arthritis (CIA) is a chronic inflammatory disease bearing all the hallmarks of rheumatoid arthritis, e.g. polyarthritis, synovitis, and subsequent cartilage/bone erosions. One feature of the disease contributing to joint damage is synovial hyperplasia. The factors responsible for the hyperplasia are unknown; however, an imbalance between rates of cell proliferation and cell death (apoptosis) has been suggested. To evaluate the role of a major pathway of cell death - Fas (CD95)/FasL - in the pathogenesis of CIA, DBA/1J mice with a mutation of the Fas gene (lpr) were generated. The susceptibility of the mutant DBA-lpr/lpr mice to arthritis induced by collagen type II was evaluated. Contrary to expectations, the DBA-lpr/lpr mice developed significantly milder disease than the control littermates. The incidence of disease was also significantly lower in the lpr/lpr mice than in the controls (40% versus 81%; P < 0.05). However DBA-lpr/lpr mice mounted a robust immune response to collagen, and the expression of local proinflammatory cytokines such as, e.g., tumor necrosis factor alpha (TNF-alpha) and IL-6 were increased at the onset of disease. Since the contribution of synovial fibroblasts to inflammation and joint destruction is crucial, the potential activating effect of Fas on mouse fibroblast cell line NIH3T3 was investigated. On treatment with anti-Fas in vitro, the cell death of NIH3T3 fibroblasts was reduced and the expression of proinflammatory cytokines TNF-alpha and IL-6 was increased. These findings suggest that impairment of immune tolerance by increased T-cell reactivity does not lead to enhanced susceptibility to CIA and point to a role of Fas in joint destruction.