Th1-Induced CD106 Expression Mediates Leukocytes Adhesion on Synovial Fibroblasts from Juvenile Idiopathic Arthritis Patients

<div><p>This study tested the hypothesis that subsets of human T helper cells can orchestrate leukocyte adhesion to synovial fibroblasts (SFbs), thus regulating the retention of leukocytes in the joints of juvenile idiopathic arthritis (JIA) patients. Several cell types, such as monocytes/macrophages, granulocytes, T and B lymphocytes, SFbs and osteoclasts participate in joint tissue damage JIA. Among T cells, an enrichment of classic and non-classic Th1 subsets, has been found in JIA synovial fluid (SF), compared to peripheral blood (PB). Moreover, it has been shown that IL-12 in the SF of inflamed joints mediates the shift of Th17 lymphocytes towards the non-classic Th1 subset. Culture supernatants of Th17, classic and non-classic Th1 clones, have been tested for their ability to stimulate proliferation, and to induce expression of adhesion molecules on SFbs, obtained from healthy donors. Culture supernatants of both classic and non-classic Th1, but not of Th17, clones, were able to induce CD106 (VCAM-1) up-regulation on SFbs. This effect, mediated by tumor necrosis factor (TNF)-α, was crucial for the adhesion of circulating leukocytes on SFbs. Finally, we found that SFbs derived from SF of JIA patients expressed higher levels of CD106 than those from healthy donors, resembling the phenotype of SFbs activated in vitro with Th1-clones supernatants. On the basis of these findings, we conclude that classic and non-classic Th1 cells induce CD106 expression on SFbs through TNF-α, an effect that could play a role in leukocytes retention in inflamed joints.</p></div

SFbs derived from synovial fluid of JIA patients express higher level of CD106 than normal SFbs.

<p><b>A</b>. MNCs from PB and SF of JIA patients were polyclonally stimulated with PMA plus ionomycin for 6h the last 4h in presence of BFA and then evaluated for intracellular cytokines production. Columns represent mean ± SE of % of CD3+CD4+ T cells of seven JIA patients producing the indicated cytokine and expressing CD161. * p < 0.05; ** p < 0.01, *** p < 0.001 PB versus SF; <b>B</b> SFbs obtained from healthy subjects and from SF of JIA patients were evaluated by phase contrast microscope (magnification 200X, one representative experiment out of seven and of this one field out of five is shown, scale bar = 100 μm and <b>(C)</b> were characterized by flow cytometry for surface expression of CD106, CD29, CD90, CD106, CD73. Columns represent the mean ± SE of % of SFbs from six healthy donors and from seven JIA patients positive for the indicated markers. *** p < 0.001 JIA versus healthy SFbs; <b>D-E</b> CFSE-labelled leukocytes derived from PB of healthy donors were cultured for 2h on healthy- or JIA-derived SFbs, in presence or absence of anti-CD106 neutralizing mAb. Leucocytes adhesion on SFbs was evaluated by fluorescence microscope analysis by average of adherent leucocytes counted in five different random fields (<b>D</b>, columns represent mean ± SE of number of adherent leukocytes of three different experiments, *** p < 0.001 stimulated condition versus ctrl or indicated by bar) and by confocal microscopy analysis (<b>E</b>, magnification 400x, one representative experiment out of three and of this one field out of five is shown). Statistical analysis was performed by using the Student t-test (two groups) or the ANOVA test (several groups).</p

Effects of culture supernatants of Th cells clones on SFbs.

<p>SFbs from healthy donors were cultured in presence of medium (CTRL) or culture supernatants of unstimulated or anti-CD3/CD28 stimulated T helper cells of different phenotypes (classic and non-classic Th1 and Th17). <b>A</b>. SFbs vitality was evaluated by WST-1 assay after 24h and 48h of culture. Columns represent mean ± SE of % of viable SFbs compared to control condition (defined as 100% and shown as line in the Fig) in three experiments. Statistical analysis was performed by using the ANOVA test. <b>B</b>. After 48h SFbs morphology was evaluated by phase contrast microscope (magnification 200X). One representative experiment out of six is shown (one field out of five). Scale bar in all images = 100μm.</p