Rheumatoid Arthritis: Pablos JL

Ferreirós-Vidal I, Barros F, Pablos JL, Carracedo A, Gómez-Reino JJ, Gonzalez A. · Hospital Clinico Universitario de Santiago, Santiago de Compostela, Spain. · Rheumatology (Oxford). · Pubmed #12810925 links to  free full text

Abstract: OBJECTIVE: To determine if the mutations in the CARD15/NOD2 gene predisposing to Crohn's disease (CD) contribute also to the genetic susceptibility to rheumatoid arthritis (RA). METHODS: The frequencies of the three commonest mutations of CARD15/NOD2 predisposing to CD (2104C > T, 2722G>C and 3020insC) were determined in 210 RA patients and 227 controls. RESULTS: Allelic frequencies of the CARD15/NOD2 mutations in RA patients (2104C>T, 2.8%; 2722G>C, 0.9%; and 3020insC, 2.4%) did not differ significantly from the controls (2104C>T, 5.3%; 2722G>C, 0.7%; and 3020insC, 1.1%). CONCLUSION: There was no evidence of association between the commonest CD CARD15/NOD2 mutations and RA susceptibility.

Dieguez-Gonzalez R, Calaza M, Perez-Pampin E, Balsa A, Blanco FJ, Cañete JD, Caliz R, Carreño L, de la Serna AR, Fernandez-Gutierrez B, Ortiz AM, Herrero-Beaumont G, Pablos JL, Narvaez J, Navarro F, Marenco JL, Gomez-Reino JJ, Gonzalez A. · Laboratorio de Investigacion 2 and Rheumatology Unit, Hospital Clinico Universitario de Santiago, Santiago de Compostela, Spain. · Arthritis Res Ther. · Pubmed #19292917 links to  free full text

Abstract: INTRODUCTION: Genome-wide association studies of rheumatoid arthritis (RA) have identified an association of the disease with a 6q23 region devoid of genes. TNFAIP3, an RA candidate gene, flanks this region, and polymorphisms in both the TNFAIP3 gene and the intergenic region are associated with systemic lupus erythematosus. We hypothesized that there is a similar association with RA, including polymorphisms in TNFAIP3 and the intergenic region. METHODS: To test this hypothesis, we selected tag-single nucleotide polymorphisms (SNPs) in both loci. They were analyzed in 1,651 patients with RA and 1,619 control individuals of Spanish ancestry. RESULTS: Weak evidence of association was found both in the 6q23 intergenic region and in the TNFAIP3 locus. The rs582757 SNP and a common haplotype in the TNFAIP3 locus exhibited association with RA. In the intergenic region, two SNPs were associated, namely rs609438 and rs13207033. The latter was only associated in patients with anti-citrullinated peptide antibodies. Overall, statistical association was best explained by the interdependent contribution of SNPs from the two loci TNFAIP3 and the 6q23 intergenic region. CONCLUSIONS: Our data are consistent with the hypothesis that several RA genetic factors exist in the 6q23 region, including polymorphisms in the TNFAIP3 gene, like that previously described for systemic lupus erythematosus.

Cañete JD, Celis R, Moll C, Izquierdo E, Marsal S, Sanmartí R, Palacín A, Lora D, de la Cruz J, Pablos JL. · Unitat d'Artritis, Servei de Reumatología, Hospital Clínic de Barcelona, Barcelona, Spain. · Ann Rheum Dis. · Pubmed #18495732 No free full text.

Abstract: OBJECTIVE: To investigate the clinical significance of lymphoid neogenesis (LN) in rheumatoid arthritis (RA), the clinicopathological correlates of this process and its evolution after anti-tumour necrosis factor (TNF)alpha therapy in a large series of synovial tissues were analysed. METHODS: Arthroscopic synovial biopsies from 86 patients with RA were analysed by immunohistochemistry. LN was defined as the presence of large aggregates of lymphocytes with T/B cell compartmentalisation and peripheral node addressin (PNAd) positive high endothelial venules. Clinical variables at baseline and after prospective follow-up were compared in LN positive and negative RA subsets. The evolution of LN and its correlation with the clinical course in a subgroup of 24 patients that underwent a second arthroscopic biopsy after anti-TNFalpha therapy was also analysed. RESULTS: LN was present in 49% of RA synovial tissues. Patients with LN had a significantly higher disease duration and a higher previous use of anti-TNFalpha agents. During prospective follow-up, the proportion of patients achieving good or moderate European League Against Rheumatism (EULAR) 28-joint Disease Activity Score (DAS28) responses was significantly lower in patients who were LN positive despite a significantly higher use of anti-TNFalpha agents. By multivariate logistic regression analysis, LN remained as an independent negative predictor of response to therapy. In the subgroup of patients rebiopsied after anti-TNFalpha therapy, reversal of LN features occurred in 56% of the patients and correlated with good clinical responses. CONCLUSIONS: Synovial LN in RA predicts a lower response to therapy. LN features can be reversed after a short period of anti-TNFalpha therapy in parallel to good clinical responses.

Arranz A, Gutiérrez-Cañas I, Carrión M, Juarranz Y, Pablos JL, Martínez C, Gomariz RP. · Departamento de Biología Celular, Facultad de Biología, Universidad Complutense de Madrid, 28040 Madrid, Spain. · Mol Immunol. · Pubmed #18452992 No free full text.

Abstract: Since recent evidences point out the potential involvement of Toll-like receptors (TLRs) in the therapeutic effect of vasoactive intestinal peptide (VIP), the purpose of this study is to elucidate the role of VIP as a negative regulator of TLR-signaling. To this aim, we analyzed in fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) or osteoarthritis (OA), the expression profile of TLR-pathway related molecules, as well as the alterations induced by LPS stimulation in RA-FLS and the effect of VIP treatment. Cultured FLS were obtained from patients with RA or OA. RA-FLS were next stimulated with lipopolysaccharide (LPS) in presence or absence of VIP. The gene expression profiling of molecules involved in LPS-mediated TLR4-signaling was studied by cRNA microarray analysis. Twenty three molecules involved in TLR signaling resulted over-expressed at mRNA level in basal RA-FLS compared to OA-FLS. Moreover, in RA-FLS, 23 of the analyzed genes were found to be up-regulated by LPS stimulation whereas 30 were not affected. VIP down-regulated the LPS-induced RNA expression of molecules involved in TLR signaling pathway. Up-regulation of RNA expression of CD14, MD2, TRAM, TRIF, IRAK4, TAB2, TRAF6 and TBK1 was corroborated by RT-PCR as well as the VIP regulatory effect. Increased protein levels of TRAF6, TBK1 and pIRAK1 after exposure to LPS, and the inhibitory effect of VIP, were described by Western blotting. As functional consequences, it was observed the VIP-induced impaired production of IL-6 and RANTES/CCL5 after LPS stimulation. In conclusion, VIP acts as a negative modulator of the TLR4-signaling by overturning the production of several checkpoints molecules of the cascade and thus, widening its potential therapeutic effects.

Juarranz Y, Gutiérrez-Cañas I, Santiago B, Carrión M, Pablos JL, Gomariz RP. · Departamento de Biología Celular, Facultad de Biología, Universidad Complutense de Madrid, Madrid, Spain. · Arthritis Rheum. · Pubmed #18383383 links to  free full text

Abstract: OBJECTIVE: Vasoactive intestinal peptide (VIP) has shown potent antiinflammatory effects in murine arthritis and ex vivo in human rheumatoid arthritis (RA) synovial cells. To investigate the potential endogenous participation of this system in the pathogenesis of RA, we analyzed the expression and regulation of VIP and its functional receptors in human fibroblast-like synoviocytes (FLS) from patients with osteoarthritis (OA) and patients with RA. METHODS: The expression of VIP was studied by reverse transcription-polymerase chain reaction (RT-PCR), enzyme immunoassay, and immunofluorescence in cultured FLS, and by immunohistochemical analysis in synovial tissue. The expression and function of the potential VIP receptors in FLS were studied by RT-PCR, determination of intracellular cAMP production, cell membrane adenylate cyclase (AC) activity, and interleukin-6, CCL2, and CXCL8 production in response to VIP or specific agonists and antagonists. RESULTS: VIP expression was detected in human FLS at the messenger RNA and protein levels, and it was significantly decreased in RA FLS compared with OA FLS. VIP receptor type 1 (VPAC1) was the dominant AC-coupled receptor in OA FLS, in contrast with RA FLS, in which VPAC2 was dominant. Tumor necrosis factor alpha-treated OA FLS reproduced the VIP and VPAC receptor expression pattern of RA FLS. The antagonistic effects of VIP on FLS proinflammatory factor production were reproduced by VPAC1- and VPAC2-specific agonists in OA FLS and RA FLS, respectively. CONCLUSION: VIP expression is down-regulated in RA and in tumor necrosis factor alpha-treated FLS, suggesting that down-regulation of this endogenous antiinflammatory factor may contribute to the pathogenesis of RA. In RA FLS, VPAC2 mediates the antiinflammatory effects of VIP, suggesting that VPAC2 agonists may be an alternative to VIP as antiinflammatory agents.

Gutiérrez-Cañas I, Juarranz Y, Santiago B, Martínez C, Gomariz RP, Pablos JL, Leceta J. · Departamento de Biología Celular, Facultad de Biología, Universidad Complutense de Madrid, 28040 Madrid, Spain. · Brain Behav Immun. · Pubmed #17951026 No free full text.

Abstract: Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease whose pathogenesis is not completely understood. Unbalanced Th1/Th2 T-cell polarization has been suggested to play a pathogenetic role and therefore, modulation of T-cell polarization is a potential therapeutic target. Vasoactive intestinal peptide (VIP) is a broadly distributed peptide that exerts anti-inflammatory and immunomodulatory effects, in the collagen-induced arthritis (CIA) murine model of RA, and ex vivo, in synovial cells from RA patients. In the present study, we have found that polyclonal stimulation of peripheral blood lymphocytes (PBL) from RA patients produces higher levels of inflammatory mediators and lower levels of Th1 cytokines than PBL from healthy controls; moreover, VIP has negligible effects on inflammatory mediators and Th1 cytokines produced by PBL from healthy controls but favours Th2 profile and enhanced IL-10 production after stimulation. VIP increases the levels of IL-10 and IL-4 in the supernatant of human CD4(+)CD45RA(+) cells cultured in a non-conditioned or a Th2-conditioned situation. In contrast, VIP does not modify the production of these cytokines in a Th1-conditioned medium. In summary, VIP can differentially modify the functional capacity of human lymphocytes by inducing Th2/Treg differentiation depending on their previous phenotype.

García S, Bodaño A, Pablos JL, Gómez-Reino JJ, Conde C. · Research Laboratory and Rheumatology Unit, Hospital Clínico Universitario de Santiago de Compostela, Spain. · Ann Rheum Dis. · Pubmed #17890271 No free full text.

Abstract: OBJECTIVES: To investigate the effect of poly(ADP-ribose) polymerase (PARP) inhibition on the production of inflammatory mediators and proliferation in tumour necrosis factor (TNF)-stimulated fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA). METHODS: Cultured FLS from patients with RA were treated with two PARP inhibitors, 3,4-dihydro-5-[4-1(1-piperidinyl)buthoxy]-1(2H)-isoquinolinona (DPQ) or 4-amino-1,8-naphthalimida (ANI) before TNF stimulation. PARP-1 expression was also suppressed in RA FLS by small interfering RNA (siRNA) transfection. Expression and secretion of inflammatory mediators were analysed by quantitative polymerase chain reaction and by enzyme-linked immunosorbent assay, respectively. Proliferation of RA FLS was also determined. Mitogen-activated protein kinase (MAPK) activity was analysed by western blot assay and activator protein (AP)-1 and nuclear factor (NF)kappaB binding by electrophoretic mobility shift assay. RESULTS: We show, for the first time, that PARP inhibition either with specific inhibitors or by siRNA transfection significantly reduced TNF-induced cytokine and chemokine expression in FLS from patients with RA. PARP inhibitors also decreased TNF-induced RA FLS proliferation. PARP inhibition reduced TNF-induced JNK phosphorylation and AP-1 and NF kappaB binding activities were partially impaired by treatment with PARP inhibitors or by PARP-1 knockdown. CONCLUSION: PARP inhibition reduces the production of inflammatory mediators and the proliferation of RA FLS (in response to TNF), suggesting that PARP inhibitors could have therapeutic benefits in RA.

Cañete JD, Santiago B, Cantaert T, Sanmartí R, Palacin A, Celis R, Graell E, Gil-Torregrosa B, Baeten D, Pablos JL. · Servicio de Reumatología, Hospital 12 de Octubre, 28041 Madrid, Spain. · Ann Rheum Dis. · Pubmed #17223654 No free full text.

Abstract: BACKGROUND: Ectopic lymphoid neogenesis (LN) occurs in rheumatoid synovium, where it is thought to drive local antigen-dependent B cell development and autoantibody production. This process involves the expression of specific homing chemokines and the development of high endothelial venules (HEV). OBJECTIVE: To investigate whether these mechanisms occur in psoriatic arthritis (PsA) synovium, where autoantibodies have not been described and the organisation and function of B cells is not clear, and to analyse their clinical correlates. METHODS: Arthroscopic synovial biopsy specimens from patients with PsA before and after tumour necrosis factor alpha blockade were characterised by immunohistochemical analysis for T/B cell segregation, peripheral lymph node addressin (PNAd)-positive HEV, and the expression of CXCL13, CCL21 and CXCL12 chemokines in relation to the size of lymphoid aggregates. RESULTS: Lymphoid aggregates of variable sizes were observed in 25 of 27 PsA synovial tissues. T/B cell segregation was often observed, and was correlated with the size of lymphoid aggregates. A close relationship between the presence of large and highly organised aggregates, the development of PNAd+ HEV, and the expression of CXCL13 and CCL21 was found. Large organised aggregates with all LN features were found in 13 of 27 tissues. LN in PsA synovitis was not related to the duration, pattern or severity of the disease. The synovial LN pattern remained stable over time in persistent synovitis, but a complete response to treatment was associated with a regression of the LN features. CONCLUSIONS: LN occurs frequently in inflamed PsA synovial tissues. Highly organised follicles display the characteristic features of PNAd+ HEV and CXCL13 and CCL21 expression, demonstrating that the microanatomical bases for germinal centre formation are present in PsA. The regression of LN on effective treatment indicates that the pathogenic and clinical relevance of these structures in PsA merits further investigation.

Juarranz Y, Gutiérrez-Cañas I, Arranz A, Martínez C, Abad C, Leceta J, Pablos JL, Gomariz RP. · Departamento de Biología Celular, Facultad de Biología, UCM, 28040 Madrid, Spain. · Ann N Y Acad Sci. · Pubmed #16888192 No free full text.

Abstract: It has been demonstrated that VIP produces beneficial effects both in a murine model of rheumatoid arthritis and in human rheumatoid synovial fibroblasts through the modulation of proinflammatory mediators. Toll-like receptors (TLRs) play a key role in the immediate recognition of microbial surface components by immune cells prior to the development of adaptative microbe-specific immune responses. In this study, we demonstrate that VIP decreases lipopolysaccharide (LPS) and TNF-alpha-induced expression of TLR4 and its correlation with the production of CCL2 and CXCL8 chemokines in human synovial fibroblasts from patients with rheumatoid arthritis and osteoarthritis. Our results add a new step for the use of VIP, as a promising candidate, for the treatment of rheumatoid arthritis.

Paya M, Segovia JC, Santiago B, Galindo M, del Rio P, Pablos JL, Ramírez JC. · Unidad de Investigación, Laboratorio de Reumatologia, Hospital 12 de Octubre, Avda. Cordoba S/N, Madrid 28049, Spain. · J Virol Methods. · Pubmed #16839616 No free full text.

Abstract: Fibroblast like synoviocytes are the main resident cells in normal joints and are known to play a major role in the pathogenesis of rheumatoid arthritis. Efficient gene targeting of fibroblast like synoviocytes (FLS) is a major goal of current ex vivo gene therapy approaches for the treatment of rheumatoid arthritis. However, there is a need to improve viral systems capable of delivering genes to human rheumatoid fibroblasts and attempts have been made to develop a protocol for high efficiency, reproducible gene transfer using a replication-defective retrovirus vector. The effects of different experimental conditions were examined as well as those related to cellular and viral features on the efficiency of transducing the retroviral-driven expression of enhanced green fluorescent protein (EGFP) to FLS harvested from patients with rheumatoid arthritis. The optimal method established involved a double round of infection by centrifugation with a resting period of 4h between rounds. This approach led to the transduction of 30-70% of FLS obtained from nine patients with rheumatoid arthritis. Consistent transduction efficiencies were achieved in repeat assays such that it could be inferred that the variations observed were attributable to the specific characteristics of each cell line. This simple protocol renders a consistent and reproducible efficiency of rheumatoid fibroblast transduction and makes stable gene targeting using non-replicating retrovirus derived vectors an affordable option for the treatment of rheumatoid arthritis.

Orozco G, Sánchez E, González-Gay MA, López-Nevot MA, Torres B, Pascual-Salcedo D, Balsa A, Pablos JL, García A, González-Escribano MF, Martín J. · Instituto de Parasitología y Biomedicina, Granada, Spain. · J Rheumatol. · Pubmed #16821265 No free full text.

Abstract: OBJECTIVE: To replicate the association reported in Japanese individuals of functional SLC22A4 and RUNX1 polymorphisms with rheumatoid arthritis (RA), and to test the possible role in this trait of a functional variant of the SUMO4 gene that was shown to be associated with another related autoimmune disease, type 1 diabetes (T1D). METHODS: Our study population consisted of 886 patients with RA and 987 healthy controls. All subjects were of Spanish Caucasian origin. We conducted a case-control association study with 6 single-nucleotide polymorphisms (SNP) spanning the SLC22A4 gene. SNP mapping in the RUNX1 gene associated with RA in a Japanese population and a SUMO4 polymorphism associated with T1D were also studied. RESULTS: No statistically significant differences between patients with RA and healthy controls were observed when comparing the distribution of the genotypes or alleles of any of the SLC22A4 polymorphisms tested. Similarly, no evidence of association between RA and the SLC22A4 haplotype previously reported to be associated in a Japanese population was found. With regard to the RUNX1 and SUMO4 SNP, we did not observe statistically significant differences in the distribution of genotypes or alleles between patients with RA and healthy controls. CONCLUSION: These results suggest that the SLC22A4, RUNX1, and SUMO4 polymorphisms analyzed do not confer a relevant role in susceptibility to RA in the Spanish population.

Costas J, Perez-Pampin E, Ferreiros-Vidal I, Torres M, Phillips C, Vicario JL, Pablos JL, Carracedo A, Gomez-Reino JJ, Gonzalez A. · Research Laboratory 2, Rheumatology Unit, and National Genotyping Center, Hospital Clinco Universitario de Santiago, 15706 Santiago de Compostela, Spain. · J Rheumatol. · Pubmed #16755651 No free full text.

Abstract: OBJECTIVE: To explore the role of single nuclear polymorphisms (SNP) in 2 candidate genes, SUMO4 and MAP3K7IP2, in susceptibility to rheumatoid arthritis (RA). METHODS: Two cohorts from different Spanish towns totalling 635 patients with RA and 826 controls were studied. Six SNP were genotyped by matrix assisted laser desorption-ionization time-of-flight (MALDI-TOF) with the MassARRAY SNP genotyping system. RESULTS: We found no association with susceptibility to RA for any of the SNP including a previously described functional variant in the SUMO4 gene (163A-->G). RA susceptibility was independent of the haplotypes defined by the 6 SNP and there was also no association with clinical features of RA. Conclusion. SUMO4 and MAP3K7IP2 SNP did not significantly influence predisposition to and features of RA, in contrast to previous genetic and functional evidence that suggested their involvement.

Palao G, Santiago B, Galindo MA, Rullas JN, Alcamí J, Ramirez JC, Pablos JL. · Servicio de Reumatología, Hospital 12 de Octubre, Madrid, Spain. · Arthritis Rheum. · Pubmed #16646028 links to  free full text

Abstract: OBJECTIVE: The expansion of an aggressive population of fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) synovium occurs despite their expression of functional death receptors and exposure to death receptor ligands. FLS can survive Fas challenge because of the constitutive expression of FLIP apoptosis inhibitor. We investigated whether Fas signaling plays a pathogenetic role by activating a nonapoptotic proinflammatory program in RA FLS. METHODS: Cultured RA FLS were stimulated with an agonistic anti-Fas antibody in the presence or absence of the caspase inhibitor Z-VAD-FMK or after RNA interference with a short hairpin RNA expression plasmid directed against FLIP. NF-kappaB and activator protein 1 (AP-1) activation was studied by electrophoretic mobility shift assays and p65 immunofluorescence analysis, and expression of messenger RNA (mRNA) for monocyte chemoattractant protein 1, interleukin-8, IkappaB alpha, and matrix metalloproteinases (MMPs) 1, 9, and 13 was examined by reverse transcription-polymerase chain reaction. Chemotactic activity of Fas-activated FLS-conditioned media was studied in Transwell migration assays. RESULTS: Fas stimulation activated NF-kappaB and AP-1, and this response required caspase activity, since Z-VAD-FMK inhibitor precluded it. FLIP was processed to p43 protein after Fas stimulation in a caspase-dependent manner, and inhibition of FLIP expression resulted in reduced Fas-triggered NF-kappaB activation. Fas stimulation increased expression of mRNA for IkappaB alpha, MMPs, and chemokines, and Fas-activated RA FLS displayed increased chemotactic activity for monocytic cells. CONCLUSION: Fas triggering may contribute to the proinflammatory features of RA FLS by activating NF-kappaB and AP-1 and by expression of relevant target genes, such as MMPs and chemokines. Fas proinflammatory signaling is dependent upon caspase activity and FLIP expression. These data implicate FLIP as a potentially important molecular switch that turns the Fas signaling away from apoptosis and toward induction of a proinflammatory phenotype in RA FLS.

Santiago B, Baleux F, Palao G, Gutiérrez-Cañas I, Ramírez JC, Arenzana-Seisdedos F, Pablos JL. · Servicio de Reumatología y Unidad de Investigación, Hospital 12 de Octubre, Avda, de Córdoba s/n, 28041 Madrid, Spain. · Arthritis Res Ther. · Pubmed #16507142 links to  free full text

Abstract: The chemokine CXCL12 (also known as stromal cell-derived factor, SDF-1) is constitutively expressed by stromal resident cells and is involved in the homeostatic and inflammatory traffic of leukocytes. Binding of CXCL12 to glycosaminoglycans on endothelial cells (ECs) is supposed to be relevant to the regulation of leukocyte diapedesis and neoangiogenesis during inflammatory responses. To improve our understanding of the relevance of this process to rheumatoid arthritis (RA), we have studied the mechanisms of presentation of exogenous CXCL12 by cultured RA ECs. RA synovial tissues had higher levels of CXCL12 on the endothelium than osteoarthritis (OA) tissues; in both, CXCL12 colocalized to heparan sulfate proteoglycans (HSPGs) and high endothelial venules. In cultured RA ECs, exogenous CXCL12alpha was able to bind in a CXCR4-independent manner to surface HSPGs. Desulfation of RA EC HSPGs by pretreatment with sodium chlorate, or by replacing in a synthetic CXCL12alpha the residues Lys24 and Lys27 by Ser (CXCL12alpha-K2427S), decreased or abrogated the ability of the chemokine to bind to RA ECs. Ex vivo, synovial ECs from patients with either OA or RA displayed a higher CXCL12-binding capacity than human umbilical vein ECs (HUVECs), and in HUVECs the binding of CXCL12 was increased on exposure to tumor necrosis factor-alpha or lymphotoxin-alpha1beta2. Our findings indicate that CXCL12 binds to HSPGs on ECs of RA synovium. The phenomenon relates to the interaction of HSPGs with a CXCL12 domain with net positive surface charge located in the first beta strand, which encompasses a canonical BXBB HSPG-binding motif. Furthermore, we show that the attachment of CXCL12 to HSPGs is upregulated by inflammatory cytokines. Both the upregulation of a constitutive chemokine during chronic inflammation and the HSPG-dependent immobilization of CXCL12 in EC surfaces are potential sites for therapeutic intervention.

Gutiérrez-Cañas I, Juarranz Y, Santiago B, Arranz A, Martinez C, Galindo M, Payá M, Gomariz RP, Pablos JL. · Servicio de Reumatología, Hospital 12 de Octubre, Avda. de Córdoba s/n 28041, Madrid, Spain. · Rheumatology (Oxford). · Pubmed #16319097 links to  free full text

Abstract: OBJECTIVES: Vasoactive intestinal peptide (VIP) has demonstrated therapeutic effects in arthritis by inhibiting both innate and acquired immune responses. We investigated the potential effects of VIP in the regulation of Toll-like receptor (TLR) expression and function in synovial fibroblasts from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS: Cultured fibroblast-like synoviocytes (FLS) were obtained from patients with RA and OA. The effects of VIP on basal or TNF-alpha or lipopolysaccharide (LPS)-induced TLR2, TLR4 and MyD88 expression and its effects on TLR4-mediated CCL2 and CXCL8 chemokine production were studied by reverse transcription-polymerase chain reaction, western blotting and enzyme-linked immunosorbent assay. RESULTS: TLR2, TLR4 and MyD88 mRNA expression was increased in RA FLS compared with OA FLS. The largest increase was observed for TLR4 and there was also overexpression at the protein level in RA FLS. TLR4 and MyD88 mRNA and proteins were induced by LPS and TNF-alpha in RA FLS. VIP down-regulated the induced but not the constitutive expression of TLR4 and MyD88 in RA FLS. VIP treatment decreased CCL2 and CXCL8 chemokine production in response to TLR4 activation with LPS in RA FLS. CONCLUSIONS: We demonstrate that VIP down-regulates LPS and TNF-alpha activation of TLR4 expression and the TLR4 functional response in terms of proinflammatory chemokine production. These studies suggest that the pleiotropic anti-inflammatory actions of VIP involve inhibitory effects on TLR4 expression and signalling.

Rueda B, González-Gay MA, López-Nevot MA, García A, Fernández-Arquero M, Balsa A, Pablos JL, Pascual-Salcedo D, de la Concha EG, González-Escribano MF, Martín J. · Instituto de Parasitología y Biomedicina, Granada, Spain. · Hum Immunol. · Pubmed #16216669 No free full text.

Abstract: The vascular endothelial growth factor (VEGF) is one of the most important pro-angiogenic mediators related to inflammation-associated synovial angiogenesis. The aim of this study was to asses the role of -1154 G-->A (rs1570360) and -634 G-->C (rs2010963) VEGF gene functional variants with rheumatoid arthritis (RA). The population under study was composed of a total of 753 unrelated RA patients and 801 healthy controls. The VEGF -1154 G-->A and -634 G-->C polymorphism genotyping was performed by real-time polymerase chain reaction technology, using TaqMan 5' allelic discrimination assay. No evidence of association was observed between the -1154 G-->A and the -634 G-->C VEGF polymorphisms, or inferred VEGF haplotypes with RA susceptibility or clinical manifestations. Our results suggest that the analyzed VEGF promoter polymorphisms may not play a relevant role in RA pathogenesis in our population.

Carreira PE, Gonzalez-Crespo MR, Ciruelo E, Pablos JL, Santiago B, Gomez-Camara A, Gomez-Reino JJ. · Servicio de Reumatoloía, Hospital 12 de Octubre, Madrid, Spain. · Arthritis Rheum. · Pubmed #16200608 links to  free full text

Abstract: OBJECTIVE: To assess whether an interleukin-1 receptor antagonist gene (IL1RN) polymorphism is associated with disease susceptibility and/or severity in a Spanish population of patients with rheumatoid arthritis (RA). METHODS: An 86-bp variable-number tandem repeat polymorphism within IL1RN intron 2 was analyzed by polymerase chain reaction in genomic DNA obtained from 247 unrelated patients with RA (group A) and 287 healthy control subjects. The polymorphism analysis was repeated in a second group of 194 patients with RA (group B). Clinical information from patients in group A was used to compare activity and severity data in patients stratified according to the different alleles or genotypes. Odds ratios (ORs) with 95% confidence intervals (95% CIs) were used to determine the strength of the association of the different alleles or genotypes with RA activity or severity. RESULTS: In the control group, the allelic frequencies were 76% for IL1RN*1 (4 repeats), 21% for IL1RN*2 (2 repeats), 3% for IL1RN*3 (5 repeats), and 0.3% for IL1RN*4 (3 repeats). In group A patients with RA, both the frequency (OR 1.47, 95% CI 1.1-1.96, P = 0.007) and carriage rate (OR 1.6, 95% CI 1.1-2.2, P = 0.01) of allele IL1RN*2 were significantly increased. The increased frequency of IL1RN*2 was confirmed in group B patients with RA (OR 1.44, 95% CI 1.1-1.97, P = 0.01). In patients with RA, homozygosity for IL1RN*2 was associated with an increased number of affected articular areas during the first year of followup but not with other parameters of disease activity or severity. CONCLUSION: Our results suggest that IL1RN has a role in determining susceptibility to RA in the Spanish population.

Pablos JL, Santiago B, Tsay D, Singer MS, Palao G, Galindo M, Rosen SD. · Servicio de Reumatología y Unidad de Investigación, Hospital 12 de Octubre, 28041 Madrid, Spain. · BMC Immunol. · Pubmed #15752429 links to  free full text

Abstract: BACKGROUND: The recruitment of lymphocytes to secondary lymphoid organs relies on interactions of circulating cells with high endothelial venules (HEV). HEV are exclusive to these organs under physiological conditions, but they can develop in chronically-inflamed tissues. The interaction of L-selectin on lymphocytes with sulfated glycoprotein ligands on HEV results in lymphocyte rolling, which represents the initial step in lymphocyte homing. HEV expression of GlcNAc6ST-2 (also known as HEC-GlcNAc6ST, GST-3, LSST or CHST4), an HEV-restricted sulfotransferase, is essential for the elaboration of L-selectin functional ligands as well as a critical epitope recognized by MECA-79 mAb. RESULTS: We examined the expression of GlcNAc6ST-2 in relationship to the MECA-79 epitope in rheumatoid arthritis (RA) synovial vessels. Expression of GlcNAc6ST-2 was specific to RA synovial tissues as compared to osteoarthritis synovial tissues and localized to endothelial cells of HEV-like vessels and small flat-walled vessels. Double MECA-79 and GlcNAc6ST-2 staining showed colocalization of the MECA-79 epitope and GlcNAc6ST-2. We further found that both TNF-alpha and lymphotoxin-alphabeta induced GlcNAc6ST-2 mRNA and protein in cultured human umbilical vein endothelial cells. CONCLUSION: These observations demonstrate that GlcNAc6ST-2 is induced in RA vessels and provide potential cytokine pathways for its induction. GlcNAc6ST-2 is a novel marker of activated vessels within RA ectopic lymphoid aggregates. This enzyme represents a potential therapeutic target for RA.

Joven B, González N, Aguilar F, Santiago B, Galindo M, Alcamí J, Pablos JL. · Hospital 12 de Octubre, Madrid, Spain. · Arthritis Rheum. · Pubmed #15641073 links to  free full text

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Palao G, Santiago B, Galindo M, Payá M, Ramirez JC, Pablos JL. · Hospital 12 de Octubre, Madrid, Spain. · Arthritis Rheum. · Pubmed #15457448 links to  free full text

Abstract: OBJECTIVE: Hyperplasia of fibroblast-like synoviocytes (FLS) contributes to chronic inflammation and joint destruction in rheumatoid arthritis (RA). FLICE-inhibitory protein (FLIP) is an antiapoptotic protein that might prevent apoptotic elimination of FLS in response to death ligands such as tumor necrosis factor alpha (TNFalpha) or Fas ligand, which are present in RA synovium. Previous studies on FLIP expression by osteoarthritis (OA) and RA FLS have shown variable results, and the specific role of FLIP as an apoptosis inhibitor in these cells remains unclear. We undertook this study to investigate the expression and antiapoptotic function of FLIP in FLS. METHODS: We studied the expression of FLIP by immunohistochemistry and immunoblotting in synovial tissues or cultured FLS from RA and OA patients. FLS apoptosis was induced by an agonistic anti-Fas monoclonal antibody and FLS were then quantified. We studied the effects of cycloheximide (CHX), TNFalpha, and FLIP antisense oligonucleotide on FLIP expression and FLS apoptotic susceptibility. RESULTS: FLIP(L) was the isoform mainly expressed in lining synoviocytes and cultured FLS. Synovial tissues and cultured FLS from OA and RA tissues displayed similar patterns and levels of expression of FLIP. Fas-induced apoptosis was variable in different FLS lines, but differences between OA and RA groups were not detected. TNFalpha induced increases in FLIP(L) and FLIP(S) expression and protected RA FLS from apoptosis, while CHX induced the opposite effects. Down-regulation of FLIP by antisense oligonucleotide strongly sensitized RA FLS to Fas-mediated apoptosis. CONCLUSION: Apoptosis susceptibility and FLIP expression are similar in OA and RA FLS. Down-regulation of FLIP sensitizes RA FLS to Fas-mediated apoptosis and may be a valuable tool for targeting RA FLS hyperplasia.

Juarranz MG, Santiago B, Torroba M, Gutierrez-Cañas I, Palao G, Galindo M, Abad C, Martinez C, Leceta J, Pablos JL, Gomariz RP. · Departamento de Biología Celular, Facultad de Biología, Universidad Complutense de Madrid, Spain. · Rheumatology (Oxford). · Pubmed #14657510 links to  free full text

Abstract: OBJECTIVE: Vasoactive intestinal peptide (VIP) has demonstrated beneficial effects in several murine models of immune-mediated inflammation by inhibiting both the inflammatory and the autoimmune components of the disease. We investigate its potential to modulate the release of proinflammatory cytokines and chemokines by human synovial cells from patients with rheumatoid arthritis (RA). METHODS: Fresh suspensions of synovial tissue cells (STC) or cultured fibroblast-like synoviocytes (FLS) were obtained from patients with RA or osteoarthritis (OA). The effects of VIP on basal or tumour necrosis factor alpha (TNF-alpha)-stimulated production of CCL2 (MCP-1, monocyte chemotactic protein 1), CXCL8 [interleukin (IL)-8], IL-6 and TNF-alpha were studied by specific ELISAs (enzyme-linked immunosorbent assays). The mRNAs for CCL2, CXCL8 and IL-6 in FLS were analysed by real-time reverse transcription-polymerase chain reaction. RESULTS: VIP at 10 nm down-regulated chemokine production by STC and FLS from RA and OA patients. VIP also down-regulated the expression of mRNAs for CCL2, CXCL8 and IL-6. The effects of VIP were more clearly detected in RA samples and after stimulation with TNF-alpha. CONCLUSION: Our observations confirm that the proposed anti-inflammatory actions of VIP in murine models also apply to human synovial cells ex vivo. Further studies are encouraged to evaluate the use of VIP as a potential therapy for chronic inflammatory joint diseases.

Pablos JL, Santiago B, Galindo M, Torres C, Brehmer MT, Blanco FJ, García-Lázaro FJ. · Servicio de Reumatología y Unidad de Investigación, Hospital 12 de Octubre, 28041 Madrid, Spain. · J Immunol. · Pubmed #12574387 links to  free full text

Abstract: CXCL12 (stromal cell-derived factor-1) is a potent CXC chemokine that is constitutively expressed by stromal resident cells. Although it is considered a homeostatic rather than an inflammatory chemokine, CXCL12 has been immunodetected in different inflammatory diseases, but also in normal tissues, ant its potential functions and regulation in inflammation are not well known. In this study, we examined the cellular sources of CXCL12 gene expression and the mechanism and effects of its interactions with endothelial cells in rheumatoid arthritis synovium. We show that CXCL12 mRNA was not overexpressed nor induced in cultured rheumatoid synoviocytes, but it specifically accumulated in the rheumatoid hyperplastic lining layer and endothelium. CXCL12 gene expression was restricted to fibroblast-like synoviocytes, whereas endothelial cells did not express CXCL12 mRNA, but displayed the protein on heparitinase-sensitive factors. CXCL12 colocalized with the angiogenesis marker alpha(v)beta(3) integrin in rheumatoid endothelium and induced angiogenesis in s.c. Matrigel plugs in mice. The angiogenic activity of rheumatoid synovial fluid in vivo was abrogated by specific immunodepletion of CXCL12. Our results indicate that synoviocyte-derived CXCL12 accumulates and it is immobilized on heparan sulfate molecules of endothelial cells, where it can promote angiogenesis and inflammatory cell infiltration, supporting a multifaceted function for this chemokine in the pathogenesis of rheumatoid arthritis.

Santiago B, Galindo M, Rivero M, Brehmer MT, Mateo I, Pablos JL. · Unidad de Investigación and Servicio de Reumatología, Hospital 12 de Octubre, 28041 Madrid, Spain. · Rheumatol Int. · Pubmed #12111085 No free full text.

Abstract: OBJECTIVE: The objective was to study the potential role of the chemokine receptor CCR5 in the chemoattraction of lymphocytes by rheumatoid arthritis synovial fluid (RA-SF). METHODS: The expression of the CCR5 receptor was studied by flow cytometry. Chemotaxis of peripheral blood lymphocytes in response to RA-SF was analyzed on transmigration chambers. Chemotaxis of immortalized lymphocytes from individuals homozygous for the Delta32 deletion of the CCR5 gene (CCR5-/-) was analyzed. The effect of a neutralizing anti-CCR5 antibody on the migration of CCR5+/+ cells was also studied. RESULTS: We confirmed an increase in the proportion of CCR5-expressing lymphocytes in RA-SF and a preferential migration of CCR5+ lymphocytes toward RA-SF in vitro. CCR5-/- lymphocytes showed decreased chemotactic responses to the chemokine MIP-1beta but not to RA-SF. The chemotactic responses of CCR5+/+ lymphocytes to RA-SF were not modified by anti-CCR5 neutralizing antibody. CONCLUSIONS: We confirm a preferential accumulation of CCR5-expressing lymphocytes into RA-SF. However, the chemotactic responses of lymphocytes to RA-SF were not dependent on a functional CCR5 receptor, suggesting that CCR5 is a marker of a lymphocyte subset rather than a specific mediator of chemotactic responses to chemokines in RA-SF.

Rivero M, Santiago B, Galindo M, Brehmer MT, Pablos JL. · Servicio de Reumatología, Unidad de Investigación, Hospital 12 de Octubre, Madrid, Spain. · Clin Exp Rheumatol. · Pubmed #12102475 No free full text.

Abstract: OBJECTIVE: In T cells, cyclooxygenase-1 is constitutively expressed, and cyclooxygenase-2 is induced during activation but their functions are not well known. Although exogenous prostaglandins are potent inhibitors of T cell activation, both immunoactivation and immunosuppression have been attributed to cyclooxygenases inhibitors (NSAIDs). Understanding the functions of the cyclooxygenases on T cells is relevant to the therapeutic use of NSAIDs on T cell mediated rheumatic diseases such as rheumatoid arthritis. In this study, we analyze whether cyclooxygenases play a significant role in T cell functions. METHODS: Activation, proliferation, and Fas induced apoptosis were analyzed in T cells treated with non-selective (indomethacin) or cyclooxygenase-2 selective (dimethyl-furanone) inhibitors. Intracellular peroxidation was studied in activated T cells by dihydrorhodamine 123 fluorescence analysis of cells treated with COX-2 antisense or control oligonucleotides. COX-2 expression was analyzed by RT-PCR analysis. RESULTS: Our data show that neither non-selective or selective cyclooxygenase-2 inhibition modify T cell activation, proliferation or apoptosis susceptibility. Furthermore, inhibition of cyclooxygenase-2 expression by antisense oligonucleotides lacks significant effects on T lymphocytes and does not modify their peroxydative capacity. CONCLUSIONS: According to these data, cyclooxygenases do not seem to play a relevant role in T cells functions in vitro. Therefore, the use of either cyclooxygenase-2 selective or non-selective NSAIDs in patients with autoimmune inflammatory diseases is not expected to induce direct immunomodulatory effects through direct effects on T cells.

Gómez-Reino JJ, Pablos JL, Carreira PE, Santiago B, Serrano L, Vicario JL, Balsa A, Figueroa M, de Juan MD. · Hospital Universitario 12 de Octubre, Madrid, Spain. · Arthritis Rheum. · Pubmed #10323455 links to  free full text

Abstract: OBJECTIVE: To investigate whether the pathogenesis of rheumatoid arthritis (RA) is associated with the functional chemokine receptor CCR5, which is the primary CC chemokine receptor expressed by T cells in rheumatoid synovium, and its nonfunctional receptor, delta32CCR5, which is generated by the homozygous 32-basepair deletion (delta32) in the CCR5 gene. METHODS: The frequency of the CCR5 genotype was compared among 673 patients with RA, 113 patients with systemic lupus erythematosus (SLE), and 815 control subjects. The CCR5 genotype was studied by polymerase chain reaction amplification of the region flanking the delta32 deletion (delta32CCR5). RESULTS: Frequencies of the wild-type CCR5 alleles (0.929, 0.907, and 0.942, respectively) and delta32CCR5 alleles (0.071, 0.093, and 0.058, respectively) in controls, SLE patients, and RA patients did not differ significantly. However, none of the RA patients had the homozygous delta32CCR5 genotype, compared with a frequency of 0.009 in controls (P = 0.014 by Fisher's exact test; chi2 = 4.12 with Yates' correction, P = 0.042) and 0.027 in SLE patients (P = 0.003 by Fisher's exact test; chi2 = 11.63 with Yates' correction, P = 0.0006). CONCLUSION: The results suggest that the CCR5 receptor plays an important role in RA and may be a suitable target for therapy.