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The link between metastasis-associated protein S100A4 and rheumatoid arthritis
Available online at www.sciencedirect.com
Joint Bone Spine 75 (2008) 242e249 http://france.elsevier.com/direct/BONSOI/
Abstracts 4th Meeting of Young Rheumatologists Across Europe (MYRACE) Vienna/Semmering, Austria, 17-19 January 2008 AB01 Difference between phenotypes of bone marrow and peripheral blood leukocytes indicate rheumatoid arthritis bone marrow as an important site for cell activation and proliferation
Acknowledgements: This work was partially supported by funds from the European Community’s FP6 Project 018661 Autocure.
A. Radzikowskaa, E. Warnawina, T. Burakowskib, E. Kontnyb, A. Gru¨tzkaua, A. Radbrucha, W. Maslinskia a Department of Pathophysiology and Immunology, Institute of b Rheumatology, Warsaw, Poland, Deutches, RheumaForschungzentrum, Berlin, Germany
AB02
Objectives: Although the pathogenesis of RA is still unknown, recent data suggest bone marrow compartment is an important site contributing to the initiation and perpetuation of chronic inflammation that may spread to the joint. The aim of the present study was to compare phenotypes of bone marrow and peripheral blood leukocytes isolated from rheumatoid arthritis (RA) and osteoarthritis (OA) patients that may indicate whether bone marrow is implicated in the activation and propagation of mature leukocytes. Methods: Mononuclear cells were isolated from peripheral blood and bone marrow of patients with RA and OA undergoing hip replacement. Sodium citrate was used as an anticoagulant. To assess cell phenotypes, specific monoclonal antibodies against: CD3, CD19, CD20, CD4, CD8, CD16, CD56, CD38, CD27, CD25, CD69, CD14, CD123, CD11c, HLA-DR, BDCA-1 and BDCA-2 were used for staining, followed by flow cytometric analysis. Results: Preliminary results indicate that RA bone marrow compartment contained different proportions of activated CD4+ and CD8+ T cell, B-cells, NK-cells and macrophages than paired peripheral blood. Unlike in peripheral blood from RA and OA, where relatively small differences in cell phenotypes were detected, composition of bone marrow mononuclear cells differed substantially between these two diseases. Conclusions: Phenotypic differences between leukocytes from bone marrow and peripheral blood indicate that in RA bone marrow microenvironment provides stronger signals leading to activation and maturation of leukocytes than in OA. Thus, these data support notion that bone marrow actively participates in the pathogenesis of RA.
The metastasis-associated protein S100A4 is a member of a large family of calcium-binding proteins that appears to play regulatory roles in cancer promoting activities. S100A4 protein regulates cell motility, proliferation, apoptosis, and stimulation of angiogenesis as well as remodeling of the extracellular matrix. It has been demonstrated that proliferating synovial fibroblasts in rheumatoid arthritis (RA) up-regulate expression of S100A4 mRNA. We have previously shown increased expression of S100A4 protein at the sites of joint destruction in RA synovial tissue. The aim of our current study was to examine the involvement of S100A4 in the pathogenesis of RA. Immunohistochemical and immunofluoresence studies were performed to analyze cell-specific distribution of S100A4 protein in RA and osteoarthritis (OA) synovial tissues. Western blotting was used to quantify the activation of the tumor suppressor protein p53 in synovial fibroblasts. Plasma concentrations of S100A4 from RA and OA patients were measured by sandwich ELISA. As we previously shown that S100A4 oligomer is involved in the regulation of several matrix degrading enzymes, in this study we could demonstrate that it can also modulate the transcriptional activation function of the tumor suppressor protein p53 in RA synovial fibroblasts. Moreover, we have identified increased expression of S100A4 in RA compared to OA synovial tissue and several S100A4 producing cells such as synovial fibroblasts, macrophages, mast cells, neutrophils, fraction of T-cells, endothelial cells and pericytes, but not B-cells, within the synovium. The local up-regulation of S100A4 was accompanied by high plasma and synovial fluid concentrations of the S100A4 protein in RA patients. Unlike other S100 proteins including S100A8/9 and S100A12, S100A4 plasma concentrations were not modified by TNF blocking therapy in active RA patients. Taken together, it can be speculated that increased S100A4 protein in circulation and locally
S1297-319X(08)00007-9
The link between metastasis-associated protein S100A4 and rheumatoid arthritis* L. Sˇenolt
Abstracts / Joint Bone Spine 75 (2008) 242e249
at sites of inflammation, particularly at sites of joint destruction, might be implicated in the process of aggressive fibroblast behavior contributing to the pathogenesis of rheumatoid arthritis. Keywords: S100A4; rheumatoid arthritis; apoptosis; matrix degrading enzymes; synovial fibroblasts
243
*
This work was supported by grant MRTN-CT-2004-005693 while Toma´sˇ Dallos, Monika Krivosˇ´ıkova´ and Elizabeth Za´nˇova´ were Marie-Curie fellows at the Department of Pathophysiology and Immunology of the Institute of Rheumatology, Warsaw, Poland.
AB04 Synovial glycosidases in joint diseases
*
This work was supported by Czech Ministry of Health - NR/9082-4 and Pfizer EU ARTICULUM Fellowship.
AB03 Bone marrow derived mesenchymal stromal cells provide survival signals to B-cells in vitro e no major role for BAFF* T. Dallosa,c, M. Krivosˇ´ıkova´b,c, M. Chora˛zy-Massalskac, E. Warnawinc, E. Za´nˇova´c,d, W. Rudnickac, A. Radzikowskac, W. Maslin´skic a 2nd Department of Paediatrics, Comenius University, Bratislava, Slovak Republic, b Institute of Immunology, Medical Faculty, Comenius University, Bratislava, Slovak Republic, c Department of Pathophysiology and Immunology, Institute of Rheumatology, Warsaw, Poland, d National Institute of Rheumatic Diseases, Piesˇˇtany, Slovak Republic Background: Mesenchymal stromal cells (MSCs) are a unique cell type that has strong anti-proliferative effects on co-cultured activated T and B-cells in vitro. Based on our observation of significant differences between rheumatoid arthritis (RA) and osteoarthritis (OA) bone marrow B-cell compartments, we hypothesized that RA bone marrow MSCs may contribute to the pathogenesis of RA by enhancing B-cell survival. Objectives: To compare the effect of RA and OA bone marrow derived MSCs (RA-MSCs, OA-MSCs) on the survival of healthy donor purified B-cells. Methods: RA-MSCs (n¼7) and OA-MSCs (n¼5) were isolated from patients undergoing hip replacement surgery, and cultured in vitro for 2-5 passages. Washed cells were co-cultured with CD20+ B-cells for 60 hours in 17 different co-culture experiments. Cell survival was analyzed using 7-amino-actinomycin D (7AAD) labelling and flow-cytometric analysis and compared to the survival of B-cells cultured without MSCs (n¼8). Expression of B-cell activating factor (BAFF) mRNA and protein was determined by RT-PCR and flow-cytomery after labelling with BAFF-specific antibodies. Results: We observed that the presence of both RA-MSCs and OAMSCs in the cultures significantly enhanced B-cell survival (70,117,28% and 54,6511,83% viable cells, respectively) as compared to controls (35,1313,83%, p < 0,001, Kruskal-Wallis ANOVA), the effect being more prominent in RA-MSCs (p < 0,05, Tukey-Kramer test). Both RA-MSCs and OA-MSCc displayed expression of BAFF mRNA and protein. We did not observe a convincing enhancement of BAFF mRNA expression by TNF-a. Blocking BAFF signalling by specific BAFF and BAFF-R antibodies, reduced the survival of B-cells by 20%, but did not abrogate the positive effect of MSCs on B-cell survival. Conclusions: MSC interaction with B-cells may provide additional stimuli for lymphocyte survival via an as yet unidentified factor and therefore contribute to the pathogenesis of RA. BAFF, though produced by MSCs, is of minor importance in this setting. Further studies to identify the molecular basis of our observation are warranted.
M. Pa´szto´ia, G. Nagyc, P. Ge´herc, T. Lakatosc, K. To´thd, P. Po´czaa, M. Mercedesza, A. Falusa,b, E.I. Buzasa a Department of Genetics, Cell and Immunobiology, Semmelweis University, Medical School, Budapest, Hungary, b Inflammation Biology and Immunogenomics Research Group, Hungarian Academy of Sciences-Semmelweis University, c Department of Rheumatology, Semmelweis University, Medical School, Budapest, Hungary, d Department of Orthopedic Surgery, Szeged,Hungary We have shown earlier that certain synovial fluid exoglycosidases are predictors of rheumatoid arthritis and are capable of depleting the articular cartilage in glycosamino-glycans. In the current study we investigated the expression of several glycosidases and glycosidase-like molecules including hexosaminidase (Hex), glucuronidase (Gus), hyaluronidase (Hyal), klotho and the chitinase-like human cartilage glycoprotein 39 (Hc-gp39) in synovial fluid and membrane samples as well as synovial fibroblast strains of patients with osteoarthritis (OA) and rheumatoid arthritis (RA). The gene expression of the chitinase like Hc-gp 39 was by far the highest among the tested genes both in synovial fibroblasts and synovial membrane samples. HexA gene was characterized by the second strongest gene expression, followed by the expression of HexB, GusB, Hyal1 and KLOTHO in a decreasing sequence of order. The only significant difference was found in the gene expression of Hyal1 of the RA and OA patients. Synovial membrane homogenates were characterized by high b-D-Nacetyl-glucosaminidase, b-D-N-acetyl-galactosaminidase and b-Dglucuronidase expression as compared to the synovial fluid samples. We found that while synovial fibroblasts appeared the primary sources of the b-D-N-acetyl-glucosaminidase and b-D-N-acetyl-galactosaminidase enzymes, they produced relatively low amounts of b-D-glucuronidase. There was no significant difference in the activities associated with the synovial membrane and synovial fibroblast of OA and RA patients. Using fluorescent substrates of b-D-glucuronidase we found stronger enzyme activities in OA fibroblasts as compared to those isolated from patients with RA. Furthermore, we found that b-D-glucuronidase activity was associated with microparticles found in the supernatants of synovial fibroblast of both RA and OA patients. We also tested if cytokines, implicated in the pathomechanism of RA, regulated the expression of the above enzymes. While IL-17 had no effect, TNF-alpha markedly upregulated the expression of the KLOTHO gene.
AB05 Proinflammatory cytokines (IL-15, TNFa, IL-6 and IL-1b) in rheumatoid arthritis bone marrow preferentially promote activation of T-cells* E. Warnawin, A. Radzikowska, T. Burakowski, W. Maslinski Institute of Rheumatology, Warsaw, Poland
Joint Bone Spine 75 (2008) 242e249 http://france.elsevier.com/direct/BONSOI/
Abstracts 4th Meeting of Young Rheumatologists Across Europe (MYRACE) Vienna/Semmering, Austria, 17-19 January 2008 AB01 Difference between phenotypes of bone marrow and peripheral blood leukocytes indicate rheumatoid arthritis bone marrow as an important site for cell activation and proliferation
Acknowledgements: This work was partially supported by funds from the European Community’s FP6 Project 018661 Autocure.
A. Radzikowskaa, E. Warnawina, T. Burakowskib, E. Kontnyb, A. Gru¨tzkaua, A. Radbrucha, W. Maslinskia a Department of Pathophysiology and Immunology, Institute of b Rheumatology, Warsaw, Poland, Deutches, RheumaForschungzentrum, Berlin, Germany
AB02
Objectives: Although the pathogenesis of RA is still unknown, recent data suggest bone marrow compartment is an important site contributing to the initiation and perpetuation of chronic inflammation that may spread to the joint. The aim of the present study was to compare phenotypes of bone marrow and peripheral blood leukocytes isolated from rheumatoid arthritis (RA) and osteoarthritis (OA) patients that may indicate whether bone marrow is implicated in the activation and propagation of mature leukocytes. Methods: Mononuclear cells were isolated from peripheral blood and bone marrow of patients with RA and OA undergoing hip replacement. Sodium citrate was used as an anticoagulant. To assess cell phenotypes, specific monoclonal antibodies against: CD3, CD19, CD20, CD4, CD8, CD16, CD56, CD38, CD27, CD25, CD69, CD14, CD123, CD11c, HLA-DR, BDCA-1 and BDCA-2 were used for staining, followed by flow cytometric analysis. Results: Preliminary results indicate that RA bone marrow compartment contained different proportions of activated CD4+ and CD8+ T cell, B-cells, NK-cells and macrophages than paired peripheral blood. Unlike in peripheral blood from RA and OA, where relatively small differences in cell phenotypes were detected, composition of bone marrow mononuclear cells differed substantially between these two diseases. Conclusions: Phenotypic differences between leukocytes from bone marrow and peripheral blood indicate that in RA bone marrow microenvironment provides stronger signals leading to activation and maturation of leukocytes than in OA. Thus, these data support notion that bone marrow actively participates in the pathogenesis of RA.
The metastasis-associated protein S100A4 is a member of a large family of calcium-binding proteins that appears to play regulatory roles in cancer promoting activities. S100A4 protein regulates cell motility, proliferation, apoptosis, and stimulation of angiogenesis as well as remodeling of the extracellular matrix. It has been demonstrated that proliferating synovial fibroblasts in rheumatoid arthritis (RA) up-regulate expression of S100A4 mRNA. We have previously shown increased expression of S100A4 protein at the sites of joint destruction in RA synovial tissue. The aim of our current study was to examine the involvement of S100A4 in the pathogenesis of RA. Immunohistochemical and immunofluoresence studies were performed to analyze cell-specific distribution of S100A4 protein in RA and osteoarthritis (OA) synovial tissues. Western blotting was used to quantify the activation of the tumor suppressor protein p53 in synovial fibroblasts. Plasma concentrations of S100A4 from RA and OA patients were measured by sandwich ELISA. As we previously shown that S100A4 oligomer is involved in the regulation of several matrix degrading enzymes, in this study we could demonstrate that it can also modulate the transcriptional activation function of the tumor suppressor protein p53 in RA synovial fibroblasts. Moreover, we have identified increased expression of S100A4 in RA compared to OA synovial tissue and several S100A4 producing cells such as synovial fibroblasts, macrophages, mast cells, neutrophils, fraction of T-cells, endothelial cells and pericytes, but not B-cells, within the synovium. The local up-regulation of S100A4 was accompanied by high plasma and synovial fluid concentrations of the S100A4 protein in RA patients. Unlike other S100 proteins including S100A8/9 and S100A12, S100A4 plasma concentrations were not modified by TNF blocking therapy in active RA patients. Taken together, it can be speculated that increased S100A4 protein in circulation and locally
S1297-319X(08)00007-9
The link between metastasis-associated protein S100A4 and rheumatoid arthritis* L. Sˇenolt
Abstracts / Joint Bone Spine 75 (2008) 242e249
at sites of inflammation, particularly at sites of joint destruction, might be implicated in the process of aggressive fibroblast behavior contributing to the pathogenesis of rheumatoid arthritis. Keywords: S100A4; rheumatoid arthritis; apoptosis; matrix degrading enzymes; synovial fibroblasts
243
*
This work was supported by grant MRTN-CT-2004-005693 while Toma´sˇ Dallos, Monika Krivosˇ´ıkova´ and Elizabeth Za´nˇova´ were Marie-Curie fellows at the Department of Pathophysiology and Immunology of the Institute of Rheumatology, Warsaw, Poland.
AB04 Synovial glycosidases in joint diseases
*
This work was supported by Czech Ministry of Health - NR/9082-4 and Pfizer EU ARTICULUM Fellowship.
AB03 Bone marrow derived mesenchymal stromal cells provide survival signals to B-cells in vitro e no major role for BAFF* T. Dallosa,c, M. Krivosˇ´ıkova´b,c, M. Chora˛zy-Massalskac, E. Warnawinc, E. Za´nˇova´c,d, W. Rudnickac, A. Radzikowskac, W. Maslin´skic a 2nd Department of Paediatrics, Comenius University, Bratislava, Slovak Republic, b Institute of Immunology, Medical Faculty, Comenius University, Bratislava, Slovak Republic, c Department of Pathophysiology and Immunology, Institute of Rheumatology, Warsaw, Poland, d National Institute of Rheumatic Diseases, Piesˇˇtany, Slovak Republic Background: Mesenchymal stromal cells (MSCs) are a unique cell type that has strong anti-proliferative effects on co-cultured activated T and B-cells in vitro. Based on our observation of significant differences between rheumatoid arthritis (RA) and osteoarthritis (OA) bone marrow B-cell compartments, we hypothesized that RA bone marrow MSCs may contribute to the pathogenesis of RA by enhancing B-cell survival. Objectives: To compare the effect of RA and OA bone marrow derived MSCs (RA-MSCs, OA-MSCs) on the survival of healthy donor purified B-cells. Methods: RA-MSCs (n¼7) and OA-MSCs (n¼5) were isolated from patients undergoing hip replacement surgery, and cultured in vitro for 2-5 passages. Washed cells were co-cultured with CD20+ B-cells for 60 hours in 17 different co-culture experiments. Cell survival was analyzed using 7-amino-actinomycin D (7AAD) labelling and flow-cytometric analysis and compared to the survival of B-cells cultured without MSCs (n¼8). Expression of B-cell activating factor (BAFF) mRNA and protein was determined by RT-PCR and flow-cytomery after labelling with BAFF-specific antibodies. Results: We observed that the presence of both RA-MSCs and OAMSCs in the cultures significantly enhanced B-cell survival (70,117,28% and 54,6511,83% viable cells, respectively) as compared to controls (35,1313,83%, p < 0,001, Kruskal-Wallis ANOVA), the effect being more prominent in RA-MSCs (p < 0,05, Tukey-Kramer test). Both RA-MSCs and OA-MSCc displayed expression of BAFF mRNA and protein. We did not observe a convincing enhancement of BAFF mRNA expression by TNF-a. Blocking BAFF signalling by specific BAFF and BAFF-R antibodies, reduced the survival of B-cells by 20%, but did not abrogate the positive effect of MSCs on B-cell survival. Conclusions: MSC interaction with B-cells may provide additional stimuli for lymphocyte survival via an as yet unidentified factor and therefore contribute to the pathogenesis of RA. BAFF, though produced by MSCs, is of minor importance in this setting. Further studies to identify the molecular basis of our observation are warranted.
M. Pa´szto´ia, G. Nagyc, P. Ge´herc, T. Lakatosc, K. To´thd, P. Po´czaa, M. Mercedesza, A. Falusa,b, E.I. Buzasa a Department of Genetics, Cell and Immunobiology, Semmelweis University, Medical School, Budapest, Hungary, b Inflammation Biology and Immunogenomics Research Group, Hungarian Academy of Sciences-Semmelweis University, c Department of Rheumatology, Semmelweis University, Medical School, Budapest, Hungary, d Department of Orthopedic Surgery, Szeged,Hungary We have shown earlier that certain synovial fluid exoglycosidases are predictors of rheumatoid arthritis and are capable of depleting the articular cartilage in glycosamino-glycans. In the current study we investigated the expression of several glycosidases and glycosidase-like molecules including hexosaminidase (Hex), glucuronidase (Gus), hyaluronidase (Hyal), klotho and the chitinase-like human cartilage glycoprotein 39 (Hc-gp39) in synovial fluid and membrane samples as well as synovial fibroblast strains of patients with osteoarthritis (OA) and rheumatoid arthritis (RA). The gene expression of the chitinase like Hc-gp 39 was by far the highest among the tested genes both in synovial fibroblasts and synovial membrane samples. HexA gene was characterized by the second strongest gene expression, followed by the expression of HexB, GusB, Hyal1 and KLOTHO in a decreasing sequence of order. The only significant difference was found in the gene expression of Hyal1 of the RA and OA patients. Synovial membrane homogenates were characterized by high b-D-Nacetyl-glucosaminidase, b-D-N-acetyl-galactosaminidase and b-Dglucuronidase expression as compared to the synovial fluid samples. We found that while synovial fibroblasts appeared the primary sources of the b-D-N-acetyl-glucosaminidase and b-D-N-acetyl-galactosaminidase enzymes, they produced relatively low amounts of b-D-glucuronidase. There was no significant difference in the activities associated with the synovial membrane and synovial fibroblast of OA and RA patients. Using fluorescent substrates of b-D-glucuronidase we found stronger enzyme activities in OA fibroblasts as compared to those isolated from patients with RA. Furthermore, we found that b-D-glucuronidase activity was associated with microparticles found in the supernatants of synovial fibroblast of both RA and OA patients. We also tested if cytokines, implicated in the pathomechanism of RA, regulated the expression of the above enzymes. While IL-17 had no effect, TNF-alpha markedly upregulated the expression of the KLOTHO gene.
AB05 Proinflammatory cytokines (IL-15, TNFa, IL-6 and IL-1b) in rheumatoid arthritis bone marrow preferentially promote activation of T-cells* E. Warnawin, A. Radzikowska, T. Burakowski, W. Maslinski Institute of Rheumatology, Warsaw, Poland