[Show abstract][Hide abstract] ABSTRACT: Rheumatoid arthritis (RA) is a chronic inflammatory disease whose pathogenesis and severity correlates with the presence of macrophage-derived pro-inflammatory cytokines within the inflamed synovium. Macrophage-derived cytokines fuel the pathological processes in RA and are targets of clinically successful therapies. However, although macrophage polarization determines cytokine production, the polarization state of macrophages in RA joints remains poorly defined. To dissect the molecular basis for the tissue-damaging effects of macrophages in RA joints, we undertook the phenotypic and transcriptomic characterization of ex vivo isolated CD14+ RA synovial fluid (RA-SF) macrophages. Flow cytometry and gene profiling indicated that RA-SF macrophages express pro-inflammatory polarization markers (MMP12, EGLN3, CCR2), lack expression of markers associated to homeostatic and anti-inflammatory polarization (IGF1, HTR2B), and exhibit a transcriptomic profile that resembles the activin A-dependent gene signature of pro-inflammatory in vitro generated macrophages. In fact, high levels of Smad-activating activin A were found in RA-SF and, accordingly, the Smad signaling pathway was activated in ex vivo isolated RA-SF macrophages. In vitro experiments on monocytes and macrophages indicated that RA-SF promote the acquisition of pro-inflammatory markers (INHBA, MMP12, EGLN3, CCR2), but led to a significant reduction in the expression genes associated to homeostasis and inflammation resolution (FOLR2, SERPINB2, IGF1, CD36), thus confirming pro-inflammatory polarization ability of RA-SF. Importantly, the macrophage polarizing ability of RA-SF was inhibited by an anti-activin A neutralizing antibody, thus demonstrating that activin A mediates the pro-inflammatory macrophage polarizing ability of RA-SF. Moreover, and in line with these findings, multicolor immunofluorescence evidenced that macrophages within RA synovial membranes (RA-SM) also express pro-inflammatory polarization markers whose expression is activin A-dependent. Altogether, our results demonstrate that macrophages from RA synovial fluids and membranes exhibit an MMP12+ EGLN3+ CCR2+ pro-inflammatory polarization state whose acquisition is partly dependent on activin A from the synovial fluid.
The Journal of Pathology 10/2014; 235(3). DOI:10.1002/path.4466 · 7.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Introduction
Synovial fibroblasts (SF) undergo phenotypic changes in rheumatoid arthritis (RA) that contribute to inflammatory joint destruction. This study was undertaken to evaluate the clinical and functional significance of ectopic podoplanin (gp38) expression by RA SF.
Expression of gp38 and its CLEC2 receptor was analyzed by immunohistochemistry in synovial arthroscopic biopsies from RA patients and normal and osteoarthritic controls. Correlation between gp38 expression and RA clinicopathological variables was analyzed. In patients rebiopsied after anti-TNF-α therapy, changes in gp38 expression were determined. Platelet-SF coculture and gp38 silencing in SF were used to analyze the functional contribution of gp38 to SF migratory and invasive properties, and to SF platelet crosstalk.
gp38 was abundantly but variably expressed in RA, and it was undetectable in normal synovial tissues. Among clinicopathologigal RA variables, significantly increased gp38 expression was only found in patients with lymphoid neogenesis (LN), and RF or ACPA autoantibodies. Cultured synovial but not dermal fibroblasts showed strong constitutive gp38 expression that was further induced by TNF-α. In RA patients, anti-TNF-α therapy significantly reduced synovial gp38 expression. In RA synovium, CLEC2 receptor expression was only observed in platelets. gp38 silencing in cultured SF did not modify their migratory and invasive properties but reduced the expression of IL-6 and IL-8 genes induced by SF-platelet interaction.
In RA, synovial expression of gp38 is strongly associated to LN and it is reduced after anti-TNF-α therapy. Interaction between gp38 and CLEC2 platelet receptor is feasible in RA synovium in vivo and can specifically contribute to gene expression by SF.
PLoS ONE 06/2014; 9(6):e99607. DOI:10.1371/journal.pone.0099607 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Synovial fibroblasts (SF) or fibroblast-like synoviocytes are the major resident cellular component of joint synovial membrane. Numerous studies support the hypothesis that SF play an important role in the pathogenesis of rheumatoid arthritis (RA). In the RA synovial membrane, SF increase in number (hyperplasia) and exhibit an altered phenotype that persists in culture in the absence of external stimuli. These abnormalities are associated with the activation of specific signalling pathways that promote cell growth and the expression of multiple factors such as cytokines, chemokines, growth factors, adhesion molecules, and matrix degradation enzymes. The activation and expansion of SF appear to contribute to the recruitment, retention and activation of inflammatory cells, new blood vessel formation (angiogenesis), and bone and cartilage destruction. The relative contribution of SF to these processes is very important in animal models but has not been determined in human RA due to the lack of treatment interventions specifically targeting these cells. The identification of the molecular pathways involved in the altered phenotype of rheumatoid SF and their pathophysiological contribution are the basis for the development of new therapeutic alternatives for chronic inflammation and joint damage not targeting the immune system.
Seminarios de la Fundación Española de Reumatología 10/2013; 14(4):121–128. DOI:10.1016/j.semreu.2013.06.001
[Show abstract][Hide abstract] ABSTRACT: Objective:
[corrected] Systemic sclerosis (SSc) is an autoimmune disease characterised by progressive fibrosis. Although SSc shares pathogenetic features with other autoimmune diseases, the participation of profibrotic Th2 cytokines is unique to SSc, but the mechanisms of Th2 skewing are unknown. We have analysed the expression and function of thymic stromal lymphopoietin (TSLP), a central regulator of Th2-mediated allergic inflammation, in human SSc, primary lung fibrosis and in a mouse model of scleroderma.
TSLP expression was analysed by immunohistochemistry in human SSc skin, primary lung fibrosis and mouse bleomycin-induced skin fibrosis, and by quantitative RT-PCR in mouse skin and cultured fibroblasts. The regulation of TSLP expression by specific toll-like receptors (TLR)-2, -3 and -4 agonists was analysed in human dermal fibroblast cultures. The role of TSLP in skin fibrosis and local cytokine expression was analysed in TSLP receptor (TSLPR)-deficient mice.
TSLP was overexpressed by epithelial cells, mast cells and fibroblasts in human SSc skin and lung fibrosis, and in the bleomycin model of scleroderma. In cultured human and mouse skin fibroblasts, TSLP expression was inducible by activation of TLR, particularly TLR3. In TSLPR-deficient mice, bleomycin-induced fibrosis was significantly reduced in parallel with significantly reduced local expression of IL-13.
These data provide the first evidence of TSLP overexpression in SSc and other non-allergic fibrotic conditions, and demonstrate a profibrotic role that is potentially meditated by specific changes in the local cytokine milieu. Thus, modulating TSLP may have antifibrotic therapeutic implications.
Annals of the rheumatic diseases 02/2013; 72(12). DOI:10.1136/annrheumdis-2012-202279 · 10.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Changes in rheumatoid arthritis synovial fibroblast (RASF) gene expression are usually defined by a comparison to osteoarthritis synovial fibroblasts (OASFs). This study was undertaken to analyse the transcriptome of OASFs as compared to RASFs and healthy synovial fibroblasts (HSFs).
The authors used microarray messenger RNA expression profiling of synovial fibroblasts cultured from osteoarthritis (OA), rheumatoid arthritis and normal synovial tissues. Quantitative real-time PCR of selected genes was performed to validate microarray data. Analysis of variance, Student t test and the Benjamini-Hochberg multiple testing correction method for multiple testing correction were used to determine the statistical significance of the changes between the three groups.
Larger numbers of transcripts showed a differential expression in OASFs versus the other groups, rather than in RASFs versus HSFs. Cluster analysis confirmed that the differences between the three groups were mostly due to the differences between OA and the other groups. Functional classification identified a significant number of genes related to growth factor activities, cell adhesion, neurotransmission and Ras signalling that are differentially expressed in OASFs. Classical proinflammatory factors or proteases involved in cartilage degradation were not found to be overexpressed in OASFs.
Cultured OASFs display a more homogeneous transcriptomic profile than RASFs when compared to HSFs. This supports the participation of synovial fibroblasts in the pathogenesis of OA and may reflect global defects in the mesenchyma-derived lineages of the different tissues in OA joints. These data support individual heterogeneity among RASFs and advise against the use of OASFs as controls.
Annals of the rheumatic diseases 02/2012; 71(2):275-80. DOI:10.1136/annrheumdis-2011-200281 · 10.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: CXCL12γ is an alternative splicing isoform of CXCL12 with enhanced affinity for heparan sulfate (HS) proteoglycans. This study was undertaken to investigate the distribution and potential function of CXCL12γ in rheumatoid arthritis (RA) synovium and normal lymphoid tissue, where its immobilization to HS may be relevant in pathologic or homeostatic immune cell migration and activation.
Expression of CXCL12 or CXCL12γ was immunodetected in RA and normal synovium, lymphoid tissue, and cultured cells with anti-pan-CXCL12 or anti-CXCL12γ-specific monoclonal antibodies. CXCL12α and CXCL12γ messenger RNA expression was analyzed by quantitative reverse transcription-polymerase chain reaction. Binding of wild-type CXCL12 isoforms or their HS binding-defective mutants to monocyte-derived dendritic cells (DCs) was analyzed by flow cytometry. The effect of DC-bound CXCL12α and CXCL12γ on T cell activation was analyzed in DC/T cell allogeneic cultures.
CXCL12γ expression was increased in RA compared to normal synovium and preferentially located in endothelia and DC-SIGN-positive cells. This distribution was also observed in lymphoid organs. Surface-bound CXCL12γ was detected in a fraction of freshly isolated DCs. Monocyte-derived DCs, but not monocytes, showed a high capacity to bind CXCL12γ in an HS-dependent manner. Surface-bound CXCL12α and CXCL12γ on monocyte-derived DCs were potent inhibitors of allogeneic T cell activation, in contrast to the T cell-stimulatory effects of soluble CXCL12 proteins.
CXCL12γ shows a specific and similar distribution in RA synovium and lymphoid tissue, consistent with its higher HS binding affinity. Presentation of CXCL12 to T cells on membrane HS in DCs can play a distinct regulatory role in T cell activation.
[Show abstract][Hide abstract] ABSTRACT: Synovial fibroblast (SF) hyperplasia contributes to the pathogenesis of rheumatoid arthritis (RA), but quantitative information on this process is scarce. This study was undertaken to evaluate the fibroblast-specific marker Hsp47 as a quantitative marker for SFs and to analyze its clinicopathologic correlates and evolution after anti-tumor necrosis factor α (anti-TNFα) therapy.
Synovial biopsy samples were obtained from 48 patients with RA and 20 controls who were healthy or had osteoarthritis (OA). Twenty-five RA patients who had active disease at the time of biopsy underwent a second biopsy after anti-TNFα therapy. Immunolabeling for Hsp47, inflammatory cells, and vascular cell markers was performed. Hsp47-positive lining and sublining fractional areas were quantified, and their correlation with clinicopathologic variables was analyzed.
In normal and diseased synovial tissue, Hsp47 was specifically and uniformly expressed by lining, sublining, and perivascular fibroblasts. Lining SF area was significantly increased in both RA and late OA tissue compared to normal tissue. Sublining SF area was increased in RA tissue but not in late OA tissue compared to normal tissue. Lining SF area was positively correlated with macrophage density, Disease Activity Score in 28 joints, and RA disease duration. In contrast, sublining SF area was negatively correlated with RA disease duration and activity. A significant reduction in lining SF area but not sublining SF area was observed after anti-TNFα therapy.
Our findings indicate that Hsp47 is a reliable marker for quantifying SFs in human synovial tissue. Our data suggest that lining and sublining SFs undergo different dynamics during the course of the disease. Lining SF expansion parallels the activity and temporal progression of RA and can be partially reversed by anti-TNFα therapy.
[Show abstract][Hide abstract] ABSTRACT: CXCL12 is a constitutively expressed chemokine with important homeostatic functions. Increased CXCL12 expression has been observed in several inflammatory conditions, including rheumatoid arthritis (RA). This study was undertaken to identify potential mechanisms of regulation of CXCL12 gene expression by human fibroblasts under normal or inflammatory conditions.
Synovial fibroblasts (SF) were cultured from RA and osteoarthritis (OA) synovial tissues. CXCL12 mRNA expression was analysed by real time quantitative RT-PCR in RA-SF under different growth conditions, and exposed to hypoxia or to different pro-inflammatory factors. A 5'CXCL12 -1.4 kb promoter region fragment was cloned in a luciferase reporter plasmid and its activity analysed in human fibroblasts.
CXCL12 mRNA expression was not constitutively increased in RA- compared to OA-SF. LPS, pro-inflammatory cytokines or growth factors did not induce CXCL12 mRNA expression in SF. Hypoxia and growth arrest by either serum starvation or confluent growth induced CXCL12 mRNA and protein expression in SF. Constitutive and induced expression of CXCL12 in fibroblasts was regulated at the transcriptional level by specific regions of the -1.4 kb promoter.
Pro-inflammatory factors and cytokines do not up-regulate CXCL12 gene expression in SF. Growth arrest and hypoxia are potentially important inducers of CXCL12 expression in human fibroblasts and operate by regulating transcriptional activity of the promoter.
[Show abstract][Hide abstract] ABSTRACT: Hypoxia is a prominent feature in rheumatoid arthritis (RA) synovium. However, its contribution to the pathogenesis of RA remains unclear. We undertook this study to systematically characterize the changes in gene expression induced by hypoxia in synovial fibroblasts.
We used microarray expression profiling in paired normoxic and hypoxic cultures of healthy synovial fibroblasts (HSFs) and RA synovial fibroblasts (RASFs). We used Student's paired t-test with Benjamini and Hochberg multiple testing correction to determine statistical significance. Validation of microarray data was performed by quantitative real-time reverse transcription-polymerase chain reaction analysis of selected genes. Biologic pathways differentially modulated by hypoxia in RASFs or HSFs were identified using unsupervised Ingenuity Pathways Analysis.
Hypoxia induced significant changes in the expression of a large group of genes in both HSFs and RASFs. In RASFs, we observed a lower number of hypoxia-regulated genes and partial differences in their functional categories. The number of differentially expressed genes in RASFs compared with HSFs was significantly increased by hypoxia. Multiple gene sets involved in energy metabolism, intracellular signal transduction, angiogenesis, and immune and inflammatory pathways were significantly modified, the last in both proinflammatory and antiinflammatory directions.
These data demonstrate that hypoxia induces significant changes in gene expression in HSFs and RASFs and identify differences between RASF and HSF profiles. The hypoxia-induced gene expression program in synovial fibroblasts identifies new factors and pathways relevant to understanding their contribution to the pathogenesis of chronic arthritis.
[Show abstract][Hide abstract] ABSTRACT: Angiogenesis is considered an important factor in the pathogenesis of Rheumatoid Arthritis (RA) where it has been proposed as a therapeutic target. In other settings, active angiogenesis is characterized by pathologic, immature vessels that lack periendothelial cells. We searched for the presence of immature vessels in RA synovium and analyzed the dynamics of synovial vasculature along the course of the disease, particularly after therapeutic response to TNF antagonists.
Synovial arthroscopic biopsies from RA, osteoarthritis (OA) and normal controls were analyzed by double labeling of endothelium and pericytes/smooth muscle mural cells to identify and quantify mature/immature blood vessels. To analyze clinicopathological correlations, a cross-sectional study on 82 synovial biopsies from RA patients with variable disease duration and severity was performed. A longitudinal analysis was performed in 25 patients with active disease rebiopsied after anti-TNF-alpha therapy. We found that most RA synovial tissues contained a significant fraction of immature blood vessels lacking periendothelial coverage, whereas they were rare in OA, and inexistent in normal synovial tissues. Immature vessels were observed from the earliest phases of the disease but their presence or density was significantly increased in patients with longer disease duration, higher activity and severity, and stronger inflammatory cell infiltration. In patients that responded to anti-TNF-alpha therapy, immature vessels were selectively depleted. The mature vasculature was similarly expanded in early or late disease and unchanged by therapy.
RA synovium contains a significant fraction of neoangiogenic, immature blood vessels. Progression of the disease increases the presence and density of immature but not mature vessels and only immature vessels are depleted in response to anti-TNFalpha therapy. The different dynamics of the mature and immature vascular fractions has important implications for the development of anti-angiogenic interventions in RA.
PLoS ONE 12/2009; 4(12):e8131. DOI:10.1371/journal.pone.0008131 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hyperplasia and phenotypic changes in fibroblasts are often observed in chronic inflammatory lesions, and yet the autonomous pathogenic contribution of these changes is uncertain. The purpose of this study was to analyze the intrinsic ability of fibroblasts from chronically inflamed synovial tissue to drive cell recruitment and angiogenesis.
Fibroblasts from patients with rheumatoid arthritis (RA) or osteoarthritis (OA), as well as fibroblasts from healthy synovial tissue and healthy skin, were cultured and subcutaneously engrafted into immunodeficient mice. Cell infiltration and angiogenesis were analyzed in the grafts by immunohistochemical studies. The role of vascular endothelial growth factor (VEGF), CXCL12, and hypoxia-inducible transcription factor 1alpha (HIF-1alpha) in these processes was investigated using specific antagonists or small interfering RNA (siRNA)-mediated down-regulation of HIF-1alpha in fibroblasts.
Inflammatory (OA and RA) synovial fibroblasts, compared with healthy dermal or synovial tissue fibroblasts, induced a significant enhancement in myeloid cell infiltration and angiogenesis in immunodeficient mice. These activities were associated with increased constitutive and hypoxia-induced expression of VEGF, but not CXCL12, in inflammatory fibroblasts compared with healthy fibroblasts. VEGF and CXCL12 antagonists significantly reduced myeloid cell infiltration and angiogenesis. Furthermore, targeting of HIF-1alpha expression by siRNA or of HIF-1alpha transcriptional activity by the small molecule chetomin in RA fibroblasts significantly reduced both responses.
These results demonstrate that chronic synovial inflammation is associated with stable fibroblast changes that, under hypoxic conditions, are sufficient to induce inflammatory cell recruitment and angiogenesis, both of which are processes relevant to the perpetuation of chronic inflammation.
[Show abstract][Hide abstract] ABSTRACT: The CXCL12gamma chemokine arises by alternative splicing from Cxcl12, an essential gene during development. This protein binds CXCR4 and displays an exceptional degree of conservation (99%) in mammals. CXCL12gamma is formed by a protein core shared by all CXCL12 isoforms, extended by a highly cationic carboxy-terminal (C-ter) domain that encompass four overlapped BBXB heparan sulfate (HS)-binding motifs. We hypothesize that this unusual domain could critically determine the biological properties of CXCL12gamma through its interaction to, and regulation by extracellular glycosaminoglycans (GAG) and HS in particular. By both RT-PCR and immunohistochemistry, we mapped the localization of CXCL12gamma both in mouse and human tissues, where it showed discrete differential expression. As an unprecedented feature among chemokines, the secreted CXCL12gamma strongly interacted with cell membrane GAG, thus remaining mostly adsorbed on the plasmatic membrane upon secretion. Affinity chromatography and surface plasmon resonance allowed us to determine for CXCL12gamma one of the higher affinity for HS (K(d) = 0.9 nM) ever reported for a protein. This property relies in the presence of four canonical HS-binding sites located at the C-ter domain but requires the collaboration of a HS-binding site located in the core of the protein. Interestingly, and despite reduced agonist potency on CXCR4, the sustained binding of CXCL12gamma to HS enabled it to promote in vivo intraperitoneal leukocyte accumulation and angiogenesis in matrigel plugs with much higher efficiency than CXCL12alpha. In good agreement, mutant CXCL12gamma chemokines selectively devoid of HS-binding capacity failed to promote in vivo significant cell recruitment. We conclude that CXCL12gamma features unique structural and functional properties among chemokines which rely on the presence of a distinctive C-ter domain. The unsurpassed capacity to bind to HS on the extracellular matrix would make CXCL12gamma the paradigm of haptotactic proteins, which regulate essential homeostatic functions by promoting directional migration and selective tissue homing of cells.
PLoS ONE 07/2008; 3(7):e2543. DOI:10.1371/journal.pone.0002543 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.
Annals of the rheumatic diseases 06/2008; 68(5):751-6. DOI:10.1136/ard.2008.089284 · 10.38 Impact Factor