Mesenchymal stem cells in rheumatoid synovium: enumeration and functional assessment in relation to synovial inflammation level.

OBJECTIVE: Achieving joint regeneration in rheumatoid arthritis (RA) represents a future challenge. Autologous synovial mesenchymal stem cells (MSCs) could be therapeutically exploited. However, the inflammatory milieu in the RA synovium could adversely affect endogenous MSC function. To test this hypothesis, the frequency and multipotency of RA synovial MSCs was evaluated in relation to existing synovial inflammation. METHODS: Synovial inflammation was measured using the arthroscopic visual analogue score (VAS) and further validated using immunohistochemistry and flow cytometry. Highly proliferative clonogenic in vivo MSCs were enumerated following fluorescence-activated cell sorting and expansion for 20 population doublings. MSC multipotency was quantified following standard in vitro culture expansion and trilineage differentiation assays. Real-time PCR, flow cytometry and ELISA were used to evaluate pro- and anti-chondrogenic molecules in standard polyclonal synovial MSCs. RESULTS: The arthroscopic VAS significantly correlated with synovial macrophage infiltration. In RA, synovial MSC chondrogenesis was inhibited in direct relation to VAS (r = -0.777, p<0.05) and reduced compared with control osteoarthritis (OA)-MSCs (p<0.05). In vivo, MSCs resided in the synovial fibroblastic/stromal fraction (CD45(-)CD31(-)) and were reduced in frequency in relation to VAS (r = -0.695, p<0.05). In RA-MSCs, CD44 levels correlated negatively with inflammation and positively with chondrogenesis (r = -0.830 and r = 0.865, respectively). Cytokine production and Sox9 expression was similar in RA-MSCs and OA-MSCs. CONCLUSIONS: There is a negative relationship between synovial MSC chondrogenic and clonogenic capacities and the magnitude of synovitis in RA. Effective suppression of joint inflammation is therefore necessary for the development of autologous MSC treatments aimed at cartilage regeneration in RA.

Abnormal T cell differentiation persists in patients with rheumatoid arthritis in clinical remission and predicts relapse.

OBJECTIVES: An abnormal CD4+ T cell subset related to inflammation exposure (inflammation-related cells, IRC) has been identified in rheumatoid arthritis (RA). Patients with inflammatory and non-inflammatory diseases were used to examine the relationship between inflammation and this T cell subset in vivo. METHODS: Blood was collected from healthy controls and patients with RA (active disease or in clinical remission), Crohn's disease and osteoarthritis. IRC and chemokine receptors were quantified by flow cytometry. Thymic activity and apoptotic factors were measured by real-time polymerase chain reaction. Circulating cytokines were measured by enzyme-linked immunosorbent assay. CXCR4 and SDF1 in synovial biopsies were measured using immunohistochemistry. RESULTS: IRC were identified in patients with RA (p<0.0001) and Crohn's disease (p = 0.005), but not in those with osteoarthritis. In RA in remission, IRC persisted (p<0.001). In remission, hyperproliferation of IRC was lost, chemokine receptor expression was significantly lowered (p<0.007), Bax expression dropped significantly (p<0.001) and was inversely correlated with IRC (rho = -0.755, p = 0.03). High IRC frequency in remission was associated with relapse within 18 months (OR = 6.4, p<0.001) and a regression model predicted 72% of relapse. CONCLUSIONS: These results suggest a model in which, despite the lack of systemic inflammation, IRC persist in remission, indicating that IRC are an acquired feature of RA. They have, however, lost their hyper-responsiveness, acquired a potential for survival, and no longer express chemokine receptors. IRC persistence in remission confirms their important role in chronic inflammation as circulating precursors of pathogenic cells. This was further demonstrated by much higher incidence of relapse in patients with high IRC frequency in remission.

Early rheumatoid arthritis is associated with a deficit in the CD4+CD25high regulatory T cell population in peripheral blood.

OBJECTIVE: Our aim was to test the hypothesis that there is a deficit in the CD4+CD25high regulatory T-cell population in early rheumatoid arthritis (RA), either in size or functional activity. METHODS: Peripheral blood mononuclear cells were examined from subjects with early active RA who had received no previous disease-modifying therapy (n = 43), from individuals with self-limiting reactive arthritis (n = 14), from subjects with stable, well-controlled RA (n = 82) and from healthy controls (n = 72). The frequencies of CD4+CD25high T-cells were quantified using flow cytometry, and function was assessed by the ability to suppress proliferation of CD4+CD25- T-cells. Paired blood and synovial fluid was analysed from a small number of RA and reactive arthritis patients. RESULTS: There was a smaller proportion of CD4+CD25high T-cells in the peripheral blood of early active RA patients (mean 4.25%) than in patients with reactive arthritis or in controls (mean 5.90 and 5.30%, respectively, P = 0.001 in each case). Frequencies in stable, well-controlled RA (mean 4.63%) were not significantly different from early active RA or controls. There were no differences in suppressor function between groups. Higher frequencies of CD4+CD25high T-cells were found in synovial fluid than blood in both RA and reactive arthritis. CONCLUSIONS: These data demonstrate a smaller CD4+CD25high regulatory T-cell population in peripheral blood of individuals with early active RA prior to disease-modifying treatment. This may be a contributory factor in the susceptibility to RA and suggests novel approaches to therapy.

Fluvastatin induces apoptosis of vascular endothelial cells: blockade by glucocorticoids.

Statins block de novo synthesis of cholesterol by inhibiting the enzyme, HMG CoA reductase. The product of this reaction, mevalonic acid, is also a precursor of isoprenoids, molecules required for the activation of signaling G-proteins, such as Ras. Signal transduction pathways involving Ras are important for cell survival and this may be why statins induce apoptotic death of several cell types. Given that statins are used to treat vascular disease, surprisingly no studies have been conducted on vascular endothelial cells. Here we show that fluvastatin (FS), at concentrations from 1-2 microM, blocks growth and induces apoptosis of the endothelial cell line, EA.hy 926. Considerable redundancy is known to exist in cell signaling and in vivo toxicity of FS might be prevented by other signaling pathways, like those activated by adrenal or sex steroids. RT-PCR analysis revealed the expression of the androgen and glucocorticoid receptor in EA.hy 926 cells. Although the androgen, dihydrotestesterone (DHT) had no effect, the glucocorticoid, dexamethasone (Dex), blocked FS-induced apoptosis. Cell cycle analysis revealed that 24 h exposure to FS prevented cells from leaving G(1) and 24-48 h later a marked sub-G(1) peak was observed. Dex was able to reduce the sub-G(1) peak, but it failed to block accumulation of cells in G(1), indicating that it's effect was specific for blockade of apoptosis, and not specific to an effect on FS alone. This study strongly suggests that glucocorticoids have a role to play in preventing vascular injury and they may provide the reason why statins are not inherently toxic to vascular endothelial cells, in vivo.

Statin-induced apoptosis of vascular endothelial cells is blocked by dexamethasone.

Statins block de novo synthesis of cholesterol by inhibiting the enzyme, HMG CoA reductase. The product of this reaction, mevalonic acid, is also a precursor of isoprenoids, molecules required for the activation of signalling G-proteins, such as Ras. Signal transduction pathways involving Ras are important for cell survival and this may be why statins induce apoptotic death of several cell types. Given that statins are used to treat vascular disease, it is surprising that no studies have been conducted on vascular endothelial cells. For this reason, we have tested the effect of fluvastatin (FS) on the endothelial cell line EA.hy 926. Here we show that FS, at concentrations from 1 to 2 microM, blocks growth and induces apoptosis of the endothelial cell line, EA.hy 926. As considerable redundancy exists in cell signalling pathways for cell survival, toxicity of FS under more physiological conditions might be prevented by pathways that do not require Ras, such as those activated by adrenal or sex steroids. To test this hypothesis, first RT-PCR analysis was performed for nuclear receptor mRNA expression. This revealed the presence of mRNA for the androgen receptor (AR) and glucocorticoid receptor (GR). The effect of the AR agonist, dihydrotestosterone (DHT), and the GR agonist, dexamethasone (Dex), was then tested. Whilst DHT (100 nM) had no effect on FS-induced cell death, Dex (1 microM) blocked FS-induced apoptosis. Cell cycle analysis revealed that 24 h exposure to FS prevented cells from leaving G(1) and 24-48 h later a marked sub-G(1) peak was observed. Dex was able to reduce the sub-G(1) peak, but it failed to reduce accumulation of cells in G(1). Further studies revealed that, in addition to blocking FS-induced apoptosis, Dex was able to block apoptosis of EA.hy 926 cells induced by serum deprivation, tumour necrosis factor-alpha, oxidants, DNA damage and mitochondrial disruption. This study strongly suggests that glucocorticoids have a role to play in preventing vascular injury and they may provide a reason why statins are apparently not toxic to vascular endothelial cells in vivo.

Functional inactivation of the oestrogen receptor by the antioestrogen, ZM 182780, sensitises tumour cells to reactive oxygen species.

Reactive oxygen species (ROS) play a fundamental role in both apoptotic and necrotic cell death. Their importance is highlighted by studies showing that they mediate cell death in response to radiotherapy and to some forms of chemotherapy. Here we provide the first evidence for a role of ROS in response to an antiendocrine agent currently undergoing clinical trials. Using the oestrogen receptor (ER) containing rat pituitary GH3 cell line, we show that cell death is induced by the pure steroidal antioestrogen, ZM 182780, and that this is blocked by the antioxidant, N-acetyl cysteine (NAC). By flow cytometry, we show that, prior to the onset of DNA breakdown measured by ELISA, ZM 182780 exposure has no significant effect on intracellular oxidant concentrations. In contrast, ZM 182780 exposure greatly increases sensitivity to oxidants generated by blocking cellular antioxidant pathways and from exogenous administration of hydrogen peroxide (H2O2). As both necrosis and apoptosis are controlled by mitochondrial function, further experiments conducted to determine mitochondrial membrane potential (Delta|gWm) have indicated that the ZM 182780-induced loss of ER function increases the ease with which oxidants collapse mitochondrial activity and, as a consequence, cell death.