José L. Pablos

Stromal-Cell Derived Factor Is Expressed by Dendritic Cells and Endothelium in Human Skin

Stromal-cell derived factor or SDF-1 is a CXC chemokine constitutively expressed by stromal bone marrow cell cultures that binds to the G-protein-coupled receptor CXCR4. SDF-1/CXCR4 represents a unique, nonpromiscuous ligand/receptor pair that plays an essential role in prenatal myelo- and lymphopoiesis as well as in cardiovascular and neural development. SDF-1 prevents entry of CXCR4-dependent (X4) HIV viruses in T lymphocytes, by binding and internalizing CXCR4. The expression pattern of SDF-1 protein in normal tissues is not known. Here we describe an analysis of SDF-1 mRNA and protein in normal and inflamed skin by in situ hybridization and immunohistochemistry, using a novel anti-SDF-1 monoclonal antibody. We also describe the expression pattern of CXCR4 receptor by immunohistochemistry. Our results show that SDF-1 protein and mRNA are normally expressed by endothelial cells, pericytes, and either resident or explanted CD1a+ dendritic cells. Epithelial cells of sweat glands but not keratinocytes also express SDF-1. In various inflammatory skin diseases, a large number of mononuclear cells and fibroblasts in close contact with CXCR4-positive lymphocytic infiltrates also express SDF-1. CXCR4 was also detected in many different normal cell types, including endothelial and epithelial cells, which points to a role for SDF-1/CXCR4 cell signaling in vascular and epithelial homeostasis. The demonstration of SDF-1 expression in dendritic and endothelial cells provides new insights into the mechanisms of normal and pathological lymphocyte circulation and makes it possible to envisage a role for locally secreted SDF-1 in the selective incapacity of mucosal dendritic cells to support and propagate infection by X4 HIV isolates.

Synoviocyte-Derived CXCL12 Is Displayed on Endothelium and Induces Angiogenesis in Rheumatoid Arthritis

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 αvβ3 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.

Ectopic lymphoid neogenesis in psoriatic arthritis

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 α 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.

Chemokine expression by systemic sclerosis fibroblasts: abnormal regulation of monocyte chemoattractant protein 1 expression.

OBJECTIVE Chemokines are important mediators in the chemoattraction of leukocytes to sites of inflammation. This study investigated the potential contribution of systemic sclerosis (SSc) fibroblasts to chemokine production and its potential relevance to the pathogenesis of SSc. METHODS The expression of messenger RNA (mRNA) for different C-C and C-X-C chemokines by SSc and normal fibroblasts was studied by RNase protection assay. Monocyte chemoattractant protein 1 (MCP-1) protein production was analyzed by enzyme-linked immunosorbent assay. The chemotactic effect of fibroblast-derived MCP-1 on monocytic cells was analyzed in a transmigration assay. Nuclear factor kappaB (NF-kappaB) and activator protein 1 (AP-1) activation in fibroblasts was studied by electromobility shift analysis. MCP-1 expression in SSc skin sections was studied by immunohistochemistry. RESULTS Among all chemokine genes studied, only MCP-1 and interleukin-8 mRNA were expressed by nonstimulated normal and SSc fibroblasts. SSc fibroblasts displayed increased constitutive expression of MCP-1 mRNA and protein and showed a blunted response to oxidative stress. Increased MCP-1 production was associated with higher chemotactic activity for monocytic cells. Increased NF-kappaB or AP-1 activation was not responsible for the constitutive overexpression of MCP-1 by SSc fibroblasts. In SSc skin sections, MCP-1 expression was detected in fibroblasts, keratinocytes, and mononuclear cells, whereas it was undetectable in normal skin. CONCLUSION SSc fibroblasts display a specific pattern of chemokine gene expression that is characterized by constitutively increased and abnormally regulated expression of MCP-1 in vitro. MCP-1 is also expressed in lesional skin and can participate in the pathogenesis of SSc.

Clinical significance of synovial lymphoid neogenesis and its reversal after anti-tumour necrosis factor α therapy in rheumatoid arthritis

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)α 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-TNFα 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-TNFα 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-TNFα 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-TNFα 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-TNFα therapy in parallel to good clinical responses.

Nontropical pyomyositis in adults

Pyomyositis (PMS) is a primary infection of striated muscle. Recent scanty reports suggest that non-tropical PMS may differ from classical tropical PMS. To address this question, 12 cases of nontropical PMS seen at two hospitals between 1976 and 1992 were reviewed and an English-literature search of similar cases was conducted. Both the series and reported cases are pooled together and herein reported. The age distribution of the 97 patients showed 30-50 and 60-70-year peaks, with a 3:1 (male-female) ratio. Fever, high erythrocyte sedimentation rate, and muscle stiffness or inflammation were present in more than 75% of patients. Muscles of the thigh (54%), back (13%), buttock (11%), arm (9%), or chest wall (4%) were involved. Staphylococci (61%), gram-negative bacilli (16%), streptococci (12%), and fungi (2%) were isolated from muscle specimens. Human immunodeficiency virus infection, diabetes mellitus, hemopoietic disorders, and other conditions with defective neutrophil function were present in 64 patients (66%). Drainage of pus and antibiotic therapy were the standard treatments. The mortality rate reached 10%. Analysis of patients classified by the comorbid condition showed differences in age, causative microorganisms, clinical features, and death rate. It is concluded that several clinical presentations of nontropical PMS are at variance with that of tropical PMS.

A HEV-restricted sulfotransferase is expressed in rheumatoid arthritis synovium and is induced by lymphotoxin-α/β and TNF-α in cultured endothelial cells

BackgroundThe 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.ResultsWe 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-α and lymphotoxin-αβ induced GlcNAc6ST-2 mRNA and protein in cultured human umbilical vein endothelial cells.ConclusionThese 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.

Hypoxia induces expression of the chemokines monocyte chemoattractant protein-1 (MCP-1) and IL-8 in human dermal fibroblasts

Hypoxia is an important factor in the pathophysiology of vascular and inflammatory diseases. Leucocyte infiltration, as a consequence of adhesion molecule up‐regulation and chemokine release, is a prominent feature of these diseases. The objective of our study was to investigate the potential role of resident fibroblasts in hypoxia‐induced chemotactic responses. We show that MCP‐1 and IL‐8 mRNA are specifically induced by hypoxia in dermal fibroblasts. This response is paralleled by increased NF‐κB p65/p50 binding activity, and it is inhibited by pretreatment with N‐acetyl‐L‐cysteine. MCP‐1 secreted by fibroblasts is chemotactic for monocytic cells and this activity is significantly increased by hypoxia. Chemotactic index correlates with MCP‐1 protein levels and is significantly decreased by neutralizing anti‐MCP‐1 MoAb. These findings demonstrate the ability of resident fibroblasts to mediate chemotaxis of leucocytes through the release of chemokines in response to hypoxia. Our data point to MCP‐1 as an important component in this response, and therefore it may be a potential target in inflammatory responses associated with hypoxia.

Clinical significance of synovial lymphoid neogenesis and its reversal after anti-TNF-α therapy in rheumatoid arthritis

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)α 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-TNFα 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-TNFα 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-TNFα 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-TNFα 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-TNFα therapy in parallel to good clinical responses.

Keratinocyte apoptosis and p53 expression in cutaneous lupus and dermatomyositis

Keratinocyte apoptosis may be induced by ultraviolet‐B radiation and represents a potential source of fragmented autoantigens in autoimmune diseases. This study investigates whether excessive keratinocyte apoptosis occurs in the skin lesions of cutaneous lupus (CLE) and dermatomyositis (DM) and the potential mechanisms responsible for this phenomenon. Skin biopsies have been studied from 19 patients with CLE and DM, eight with scleroderma, and five healthy controls. Apoptosis was detected by in situ end‐labelling of fragmented DNA. The expression of Bcl‐2, PCNA, p53, and Ki‐67 proteins was studied by immunohistochemistry. In DM and CLE skin, the number of apoptotic keratinocytes was significantly increased (p=0·008) compared with normal skin. In both diseases, a large accumulation of apoptotic keratinocytes and apoptotic bodies was present in the disrupted basal zone. Unlike normal skin, a large number of keratinocytes, particularly those morphologically apoptotic, expressed p53 protein. A significant increase in the number of proliferating Ki‐67 positive (p=0·0007) and PCNA‐positive (p=0·0008) nuclei was also observed. In both CLE and DM, exaggerated and inappropriate keratinocyte apoptosis occurs. It is associated with increased expression of p53 and PCNA. This suggests that normal solar radiation alone or in combination with additional local factors induces DNA damage and excessive keratinocyte apoptosis in these autoimmune diseases of the skin. Apoptosis can mediate the severe epidermal lesions observed in both diseases and the release of fragmented autoantigens into the dermis. Copyright