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Glucocorticoids have successfully been used in the treatment of rheumatoid arthritis. Data suggest that 7 alpha-hydroxy-dehydroepiandrosterone (7 alpha-OH-DHEA), an immunostimulating metabolite of dehydroepiandrosterone, can block glucocorticoid-induced immune suppression. Formation of 7 alpha-OH-DHEA is catalyzed by activity of cytochrome p450 enzyme 7b (Cyp7b). Recently, we reported that tumour necrosis factor (TNF)-alpha, IL-1 alpha, IL-1 beta and IL-17 enhance Cyp7b mRNA expression and induce a concomitant increase in the formation of 7 alpha-OH-DHEA by fibroblast-like synoviocytes (FLS) from rheumatoid arthritis patients. The aim of this study was to elucidate which signal transduction pathway is involved in the TNF-alpha-mediated induction of Cyp7b activity in FLS. We studied the effects of inhibitors of different signal transduction pathways on Cyp7b activity in FLS by measuring Cyp7b mRNA expression using reverse transcription PCR and by measuring the formation of 7 alpha-OH-DHEA. We applied SN50, an inhibitor of nuclear translocation of transcription factors (i.e. activator protein-1 [AP-1] and nuclear factor-kappa B [NF-kappa B]); PSI, a proteasome inhibitor that prevents I kappa B degradation and thereby NF-kappa B release; SP600125, a c-Jun N-terminal kinase (JNK) inhibitor; and the mitogen-activated protein kinase inhibitors PD98059 (extracellular signal-regulated kinase) and SB203580 (p38). Cyp7b is constitutively expressed in RA FLS and can be activated in response to TNF-alpha. SN50 and PSI prevented the TNF-alpha-induced increase in Cyp7b activity, whereas the mitogen-activated protein kinase inhibitors PD98059 and SB203580 had no effect. In addition, inhibition of Cyp7b mRNA expression and activity was observed with SN50, PSI and SP600125, suggesting that NF-kappa B and AP-1 induce Cyp7b transcription. These findings suggest that NF-kappa B and AP-1 are involved in the TNF-alpha-enhanced formation of the dehydroepiandrosterone metabolite 7 alpha-OH-DHEA. Our results are in accordance with presence of AP-1 and NF-kappa B binding sites in the Cyp7b promoter.

Considering the relation between synovial inflammation and global disease activity in rheumatoid arthritis (RA) and the distinct but heterogeneous histology of spondyloarthropathy (SpA) synovitis, the present study analyzed whether histopathological features of synovium reflect specific phenotypes and/or global disease activity in SpA. Synovial biopsies obtained from 99 SpA and 86 RA patients with active knee synovitis were analyzed for 15 histological and immunohistochemical markers. Correlations with swollen joint count, serum C-reactive protein concentrations, and erythrocyte sedimentation rate were analyzed using classical and multiparameter statistics. SpA synovitis was characterized by higher vascularity and infiltration with CD163(+) macrophages and polymorphonuclear leukocytes (PMNs) and by lower values for lining-layer hyperplasia, lymphoid aggregates, CD1a(+) cells, intracellular citrullinated proteins, and MHC-HC gp39 complexes than RA synovitis. Unsupervised clustering of the SpA samples based on synovial features identified two separate clusters that both contained different SpA subtypes but were significantly differentiated by concentration of C-reactive protein and erythrocyte sedimentation rate. Global disease activity in SpA correlated significantly with lining-layer hyperplasia as well as with inflammatory infiltration with macrophages, especially the CD163+ subset, and with PMNs. Accordingly, supervised clustering using these synovial parameters identified a cluster of 20 SpA patients with significantly higher disease activity, and this finding was confirmed in an independent SpA cohort. However, multiparameter models based on synovial histopathology were relatively poor predictors of disease activity in individual patients. In conclusion, these data indicate that inflammatory infiltration of the synovium with CD163+ macrophages and PMNs as well as lining-layer hyperplasia reflect global disease activity in SpA, independently of the SpA subtype. These data support a prominent role for innate immune cells in SpA synovitis and warrant further evaluation of synovial histopathology as a surrogate marker in early-phase therapeutic trials in SpA.

Considering the relation between synovial inflammation and global disease activity in rheumatoid arthritis (RA) and the distinct but heterogeneous histology of spondyloarthropathy (SpA) synovitis, the present study analyzed whether histopathological features of synovium reflect specific phenotypes and/or global disease activity in SpA. Synovial biopsies obtained from 99 SpA and 86 RA patients with active knee synovitis were analyzed for 15 histological and immunohistochemical markers. Correlations with swollen joint count, serum C-reactive protein concentrations, and erythrocyte sedimentation rate were analyzed using classical and multiparameter statistics. SpA synovitis was characterized by higher vascularity and infiltration with CD163(+) macrophages and polymorphonuclear leukocytes (PMNs) and by lower values for lining-layer hyperplasia, lymphoid aggregates, CD1a(+) cells, intracellular citrullinated proteins, and MHC-HC gp39 complexes than RA synovitis. Unsupervised clustering of the SpA samples based on synovial features identified two separate clusters that both contained different SpA subtypes but were significantly differentiated by concentration of C-reactive protein and erythrocyte sedimentation rate. Global disease activity in SpA correlated significantly with lining-layer hyperplasia as well as with inflammatory infiltration with macrophages, especially the CD163+ subset, and with PMNs. Accordingly, supervised clustering using these synovial parameters identified a cluster of 20 SpA patients with significantly higher disease activity, and this finding was confirmed in an independent SpA cohort. However, multiparameter models based on synovial histopathology were relatively poor predictors of disease activity in individual patients. In conclusion, these data indicate that inflammatory infiltration of the synovium with CD163+ macrophages and PMNs as well as lining-layer hyperplasia reflect global disease activity in SpA, independently of the SpA subtype. These data support a prominent role for innate immune cells in SpA synovitis and warrant further evaluation of synovial histopathology as a surrogate marker in early-phase therapeutic trials in SpA.

In inflammatory processes, the p38 mitogen-activated protein kinase ( MAPK) signal transduction route regulates production and expression of cytokines and other inflammatory mediators. Tumor necrosis factor alpha (TNF-alpha) is a pivotal cytokine in rheumatoid arthritis and its production in macrophages is under control of the p38 MAPK route. Inhibition of the p38 MAPK route may inhibit production not only of TNF-alpha, but also of other inflammatory mediators produced by macrophages, and indirectly of inflammatory mediators by other cells induced by TNF-alpha stimulation. Here we investigate the effects of RWJ 67657, a p38 MAPK inhibitor, on mRNA expression and protein production of TNF-alpha and other inflammatory mediators, in monocyte-derived macrophages. A strong inhibition of TNF-alpha was seen at pharmacologically relevant concentrations of RWJ 67657, but also inhibition of mRNA expression of IL-1beta, IL-8, and cyclooxygenase-2 was shown. Furthermore, it was shown that monocyte-derived macrophages have a high constitutive production of matrix metalloproteinase 9, which is not affected by p38 MAPK inhibition. The results presented here may have important implications for the treatment of rheumatoid arthritis.

In inflammatory processes, the p38 mitogen-activated protein kinase ( MAPK) signal transduction route regulates production and expression of cytokines and other inflammatory mediators. Tumor necrosis factor alpha (TNF-alpha) is a pivotal cytokine in rheumatoid arthritis and its production in macrophages is under control of the p38 MAPK route. Inhibition of the p38 MAPK route may inhibit production not only of TNF-alpha, but also of other inflammatory mediators produced by macrophages, and indirectly of inflammatory mediators by other cells induced by TNF-alpha stimulation. Here we investigate the effects of RWJ 67657, a p38 MAPK inhibitor, on mRNA expression and protein production of TNF-alpha and other inflammatory mediators, in monocyte-derived macrophages. A strong inhibition of TNF-alpha was seen at pharmacologically relevant concentrations of RWJ 67657, but also inhibition of mRNA expression of IL-1beta, IL-8, and cyclooxygenase-2 was shown. Furthermore, it was shown that monocyte-derived macrophages have a high constitutive production of matrix metalloproteinase 9, which is not affected by p38 MAPK inhibition. The results presented here may have important implications for the treatment of rheumatoid arthritis.

Introduction: High Mobility Group Box 1 (HMGB1) is a nuclear non-histone protein. HMGB1, which is secreted by inflammatory cells and passively released from apoptotic and necrotic cells, may act as a pro-inflammatory mediator. As apoptotic cells accumulate in systemic lupus erythematosus (SLE), HMGB1 levels might be increased in SLE. HMGB1 may also serve as an autoantigen, leading to the production of anti-HMGB1 antibodies. In this study we determined levels of HMGB1 and anti-HMGB1 in SLE patients in comparison to healthy controls (HC) and analysed their relation with disease activity.Methods: The study population consisted of 70 SLE patients and 35 age-and sex-matched HC. Thirty-three SLE patients had quiescent disease, the other 37 patients were selected for having active disease. Nineteen of these had lupus nephritis. HMGB1 levels were measured with both Western blot and ELISA. Anti-HMGB1 levels were measured by ELISA. Clinical and serological parameters were assessed according to routine procedures.Results: HMGB1 levels in SLE patients could be measured reliably by Western blotting only, and were significantly increased compared to HC. During active disease HMGB1 levels increased, in particular in patients with renal involvement. Serum HMGB1 levels correlated with SLEDAI, proteinuria, and anti-dsDNA levels, and showed a negative correlation with complement C3. Anti-HMGB1 levels were significantly increased in SLE patients compared to HC, and positively correlated with HMGB1 levels.Conclusions: Levels of HMGB1 in the sera of SLE patients, in particular in those with active renal disease, are increased. Serum HMGB1 levels are related to SLEDAI scores and proteinuria, as well as to levels of anti-HMGB1 antibodies. These findings suggest that besides HMGB1, HMGB1-anti-HMGB1 immune complexes play a role in the pathogenesis of SLE, in particular in patients with renal involvement.

Introduction: There is growing evidence that interleukin 17 (IL-17) producing T cells are involved in the pathogenesis of systemic lupus erythematosus (SLE). Previous studies showed that increased percentages of T-cell subsets expressing the costimulatory molecules CD80 and CD134 are associated with disease activity and renal involvement in SLE. The aim of this study was to investigate the distribution and phenotypical characteristics of IL-17 producing T-cells in SLE, in particular in patients with lupus nephritis, with emphasis on the expression of CD80 and CD134.Methods: Thirty-four patients (3 male, 31 female, mean age 41 +/- 15 years) fulfilling at least four of the American College of Rheumatology (ACR) revised criteria for the diagnosis of SLE and 24 healthy controls were enrolled. T-cells from the peripheral blood were analysed by fluorescence activated cell sorting (FACS) for their expression levels of CD80, CD134 and CCR6. In vitro stimulated CD3(+)IL17(+) cells were also investigated for the expression of these costimulatory markers. Finally, renal biopsies from SLE patients were evaluated for the presence of CD134 expressing T-cells.Results: Percentages of IL-17 expressing T-cells were significantly increased in patients with active disease as compared to healthy controls (1.46 +/- 0.58% versus 0.93 +/- 0.30%, P = 0.007). The percentage of IL-17 producing T-cells was correlated with disease activity as assessed by systemic lupus erythematosus disease activity index (SLEDAI) (r = 0.53, P = 0.003). In patients, most of the IL-17 producing T-cells were confined to the CCR6(+) T-cell subset (80 +/- 13%). Expression of CD80 and CD134 on the IL-17 producing T-cell subset was higher in SLE than in healthy controls (HC) (CD134: 71.78 +/- 14.51% versus 51.45 +/- 16.58%, P = 0.002; CD80: 25.5 +/- 14.99% versus 14.99 +/- 5.74%, P = 0.02). Also, patients with lupus nephritis expressed higher levels of CD134(+) on CD3(+)IL-17(+) cells as compared to HC (72.69 +/- 11.54% versus 51.45 +/- 16.58%, P = 0.006). Furthermore, renal biopsies of lupus nephritis patients showed infiltration of CD134(+) T cells.Conclusions: Percentages of IL-17 expressing T-cells correlate with disease activity. Further, these cells show increased expression of costimulatory markers such as CD134 and CD80. The presence of CD134(+) T-cells in renal biopsies of lupus nephritis patients suggest that these cells migrate to the kidney and might contribute to inflammatory processes through IL-17 secretion.

The authors thank Carmen Pena-Pena for providing outstanding technical assistance. MS-G is the recipient of a FPU predoctoral bursary of the Spanish Ministry of Education. The present work was supported by Fondo de Investigación Sanitaria of the Instituto de Salud Carlos III (Spain), grants 04/1651 and 06/0620 that are partially financed by the Fondo Europeo de Desarrollo Regional program of the European Union, by grants from the Xunta de Galicia, and by BMBF KN Rheuma grant C2.12 (to TW)., Suarez-Gestal, M., Calaza, M., Endreffy, E., Pullmann Jr., R., Ordi-Ros, J., Domenico Sebastiani, G., Ruzickova, S., Jose Santos, M., Papasteriades, C., Marchini, M., Skopouli, F.N., Suarez, A., Blanco, F.J., D'Alfonso, S., Bijl, M., Carreira, P., Witte, T., Migliaresi, S., Gomez-Reino, J.J., Gonzalez, A., Kovacs, A., Balada, E., Dostal, C., Vinagre, F., Kappou-Rigatou, I., Scorza, R., Mavromati, M., Gutierrez, C., Rego, I., Barizzone, N., Kallenberg, C.G., Schmidt, R.E.

Systemic lupus erythematosus (SLE) is associated with premature and accelerated atherosclerosis. Circulating progenitor cells (CPCs) are circulating bone-marrow derived cells that play an important role in the repair of vascular damage that underlies the development of atherosclerosis. The objective of this study was to determine the number and functionality of CPCs in patients with SLE. The study included 44 female SLE patients in an inactive stage of disease and 35 age-matched female controls. CPC numbers in the circulation were determined by FACS with monoclonals against CD14, CD34 and CD133. Peripheral blood-derived mononuclear cell (PBMNC) fractions were cultured in angiogenic medium. The endothelial-like phenotype was confirmed and the colony forming unit (CFU) capacity, migratory capacity and the potential to form clusters on Matrigel were determined. Expression of apoptosis inhibiting caspase 8L was analyzed in PBMNCs and CPCs by gene transcript and protein expression assays. The number of CD34-CD133 double-positive cells (P

In order to selectively block nuclear factor kappa B (NF-kappa B)-dependent signal transduction in angiogenic endothelial cells, we constructed an alpha v beta 3 integrin specific adenovirus encoding dominant negative I kappa B (dnI kappa B) as a therapeutic gene. By virtue of RGD modification of the PEGylated virus, the specificity of the cell entry pathway of adenovirus shifted from coxsackiadenovirus receptor dependent to alpha v beta 3 integrin dependent entry. The therapeutic outcome of delivery of the transgene into endothelial cells was determined by analysis of cellular responsiveness to tumor necrosis factor (TNF)-alpha. Using real time reverse transcription PCR, mRNA levels of the cell adhesion molecules E-selectin, vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1, the cytokines/growth factors IL-6, IL-8 and vascular endothelial growth factor (VEGF)-A, and the receptor tyrosine kinase Tie-2 were assessed. Furthermore, levels of ICAM-1 protein were determined by flow cytometric analysis. RGD-targeted adenovirus delivered the dnl kappa B via alpha v beta 3 to become functionally expressed, leading to complete abolishment of TNF-alpha-induced up-regulation of E-selectin, ICAM-1, VCAM-1, IL-6, IL-8, VEGFA and Tie-2. The approach of targeted delivery of dnl kappa B into endothelial cells presented here can be employed for diseases such as rheumatoid arthritis and inflammatory bowel disease where activation of NF-kappa B activity should be locally restored to basal levels in the endothelium.

In order to selectively block nuclear factor kappa B (NF-kappa B)-dependent signal transduction in angiogenic endothelial cells, we constructed an alpha v beta 3 integrin specific adenovirus encoding dominant negative I kappa B (dnI kappa B) as a therapeutic gene. By virtue of RGD modification of the PEGylated virus, the specificity of the cell entry pathway of adenovirus shifted from coxsackiadenovirus receptor dependent to alpha v beta 3 integrin dependent entry. The therapeutic outcome of delivery of the transgene into endothelial cells was determined by analysis of cellular responsiveness to tumor necrosis factor (TNF)-alpha. Using real time reverse transcription PCR, mRNA levels of the cell adhesion molecules E-selectin, vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1, the cytokines/growth factors IL-6, IL-8 and vascular endothelial growth factor (VEGF)-A, and the receptor tyrosine kinase Tie-2 were assessed. Furthermore, levels of ICAM-1 protein were determined by flow cytometric analysis. RGD-targeted adenovirus delivered the dnl kappa B via alpha v beta 3 to become functionally expressed, leading to complete abolishment of TNF-alpha-induced up-regulation of E-selectin, ICAM-1, VCAM-1, IL-6, IL-8, VEGFA and Tie-2. The approach of targeted delivery of dnl kappa B into endothelial cells presented here can be employed for diseases such as rheumatoid arthritis and inflammatory bowel disease where activation of NF-kappa B activity should be locally restored to basal levels in the endothelium.