Heterogeneous expression pattern of interleukin 17A (IL-17A), IL-17F and their receptors in synovium of rheumatoid arthritis, psoriatic arthritis and osteoarthritis: possible explanation for nonresponse to anti-IL-17 therapy?

Accumulating evidence suggests an important role for interleukin 17 (IL-17) in the pathogenesis of several inflammatory diseases, including rheumatoid arthritis (RA) and psoriatic arthritis (PsA). Accordingly, clinical trials aimed at blocking IL-17 have been initiated, but clinical results between patients and across different diseases have been highly variable. The objective was to determine the variability in expression of IL-17A, IL-17F and their receptors IL-17RA and IL-17RC in the synovia of patients with arthritis. Synovial biopsies were obtained from patients with RA (n = 11), PsA (n = 15) and inflammatory osteoarthritis (OA, n = 14). For comparison, synovia from noninflamed knee joints (n = 7) obtained from controls were included. Frozen sections were stained for IL-17A, IL-17F, IL-17RA and IL-17RC and evaluated by digital image analysis. We used confocal microscopy to determine which cells in the synovium express IL-17A and IL-17F, double-staining with CD4, CD8, CD15, CD68, CD163, CD31, von Willebrand factor, peripheral lymph node address in, lymphatic vessel endothelial hyaluronan receptor 1, mast cell tryptase and retinoic acid receptor-related orphan receptor γt (RORγt). IL-17A, IL-17F, IL-17RA and IL-17RC were abundantly expressed in synovial tissues of all patient groups. Whereas IL-17RA was present mostly in the synovial sublining, IL-17RC was abundantly expressed in the intimal lining layer. Digital image analysis showed a significant (P  < 0.05) increase of only IL-17A in arthritis patients compared to noninflamed control tissues. The expression of IL-17A, IL-17F and their receptors was similar in the different patient groups, but highly variable between individual patients. CD4+ and CD8+ cells coexpressed IL-17A, and few cells coexpressed IL-17F. IL-17A and IL-17F were not expressed by CD15+ neutrophils. Mast cells were only occasionally positive for IL-17A or IL-17F. Interestingly, IL-17A and IL-17F staining was also observed in macrophages, as well as in blood vessels and lymphatics. This staining probably reflects receptor-bound cytokine staining. Many infiltrated cells were positive for the transcription factor RORγt. Colocalisation between RORγt and IL-17A and IL-17F indicates local IL-17 production. Increased expression of IL-17A is not restricted to synovial tissues of RA and PsA patients; it is also observed in inflammatory OA. The heterogeneous expression levels may explain nonresponse to anti-IL-17 therapy in subsets of patient

Impaired lymph node stromal cell function during the earliest phases of rheumatoid arthritis.

BACKGROUND: Systemic autoimmunity can be present years before clinical onset of rheumatoid arthritis (RA). Adaptive immunity is initiated in lymphoid tissue where lymph node stromal cells (LNSCs) regulate immune responses through their intimate connection with leucocytes. We postulate that malfunctioning of LNSCs creates a microenvironment in which normal immune responses are not properly controlled, possibly leading to autoimmune disease. In this study we established an experimental model for studying the functional capacities of human LNSCs during RA development. METHODS: Twenty-four patients with RA, 23 individuals positive for autoantibodies but without clinical disease (RA risk group) and 14 seronegative healthy control subjects underwent ultrasound-guided inguinal lymph node (LN) biopsy. Human LNSCs were isolated and expanded in vitro for functional analyses. In analogous co-cultures consisting of LNSCs and peripheral blood mononuclear cells, αCD3/αCD28-induced T-cell proliferation was measured using carboxyfluorescein diacetate succinimidyl ester dilution. RESULTS: Fibroblast-like cells expanded from the LN biopsy comprised of fibroblastic reticular cells (gp38+CD31-) and double-negative (gp38-CD31-) cells. Cultured LNSCs stably expressed characteristic adhesion molecules and cytokines. Basal expression of C-X-C motif chemokine ligand 12 (CXCL12) was lower in LNSCs from RA risk individuals than in those from healthy control subjects. Key LN chemokines C-C motif chemokine ligand (CCL19), CCL21 and CXCL13 were induced in LNSCs upon stimulation with tumour necrosis factor-α and lymphotoxin α1β2, but to a lesser extent in LNSCs from patients with RA. The effect of human LNSCs on T-cell proliferation was ratio-dependent and altered in RA LNSCs. CONCLUSIONS: Overall, we developed an experimental model to facilitate research on the role of LNSCs during the earliest phases of RA. Using this innovative model, we show, for the first time to our knowledge, that the LN stromal environment is changed during the earliest phases of RA, probably contributing to deregulated immune responses early in disease pathogenesis

Harmonization of the intracellular cytokine staining assay

Active immunotherapy for cancer is an accepted treatment modality aiming to reinforce the T-cell response to cancer. T-cell reactivity is measured by various assays and used to guide the clinical development of immunotherapeutics. However, data obtained across different institutions may vary substantially making comparative conclusions difficult. The Cancer Immunotherapy Immunoguiding Program organizes proficiency panels to identify key parameters influencing the outcome of commonly used T-cell assays followed by harmonization. Our successes with IFNγ-ELISPOT and peptide HLA multimer analysis have led to the current study on intracellular cytokine staining (ICS). We report the results of three successive panels evaluating this assay. At the beginning, 3 out of 9 participants (33 %) were able to detect >6 out of 8 known virus-specific T-cell responses in peripheral blood of healthy individuals. This increased to 50 % of the laboratories in the second phase. The reported percentages of cytokine-producing T cells by the different laboratories were highly variable with coefficients of variation well over 60 %. Variability could partially be explained by protocol-related differences in background cytokine production leading to sub-optimal signal-to-noise ratios. The large number of protocol variables prohibited identification of prime guidelines to harmonize the assays. In addition, the gating strategy used to identify reactive T cells had a major impact on assay outcome. Subsequent harmonization of the gating strategy considerably reduced the variability within the group of participants. In conclusion, we propose that first basic guidelines should be applied for gating in ICS experiments before harmonizing assay protocol variables

Intrinsic healing response of the human anterior cruciate ligament: an histological study of reattached ACL remnants

A reattachment of the tibial remnant of the torn anterior cruciate ligament (ACL) to the posterior cruciate ligament is sometimes observed during surgery and apparently implies that the human ACL does have a healing response. The aim of this study was to investigate whether this reattachment tissue has similar histological characteristics of a healing response as the medial collateral ligament (MCL), which can heal spontaneously. Standard histology and immunostaining of α-smooth muscle actin and collagen type 3 was performed. The results shows that the reattached tissue has typical characteristics of a healing response: there attached ACL remnant could not be released by forceful traction; microscopy showed that the collagen fibers of the reattached tissue were disorganized with no preferred direction; increased neovascularization; the presence of lipid vacuoles; the mean number of cells within the biopsy tissue was 631±269 cells per mm2; and 68±20% was expressing α-SMA; semi-quantitative analysis of collagen type 3 expression showed that collagen type 3 had an high expression with an average score of 3. In conclusion, this study shows that the human proximal 1/3 ACL has an intrinsic healing response with typical histological characteristics similar to the MC

B-cell imaging with zirconium-89 labelled rituximab PET-CT at baseline is associated with therapeutic response 24 weeks after initiation of rituximab treatment in rheumatoid arthritis patients

BACKGROUND: B cells are key players in the pathogenesis of rheumatoid arthritis (RA). Although successful in 50-60% of patients with RA, anti-B-cell therapy given as rituximab could be more efficient by identifying potential responders prior to treatment. Positron emission tomography (PET) using radiolabeled rituximab for B-cell imaging might provide the means to fulfil this unmet clinical need. The objective of this study was to investigate the association between biodistribution of zirconium-89 ((89)Zr)-rituximab on PET-computed tomography (CT) and clinical response in patients with RA. METHODS: We included 20 patients with RA who were starting rituximab treatment. At the first intravenous (i.v.) therapeutic dose, patients were also injected with (89)Zr-rituximab, followed by PET-CT. European League Against Rheumatism (EULAR) response criteria were applied to determine response at week 24. PET-CT was analyzed visually and quantitatively. Lymph node (LN) biopsies were performed at 0 and 4 weeks to correlate B-cell counts with imaging data. RESULTS: PET-positive hand joints (range 1-20) were observed in 18/20 patients. Responders had significantly higher (89)Zr-rituximab uptake in PET-positive hand joints than non-responders (median target-to-background (T/B)) ratios (IQR) were 6.2 (4.0-8.8) vs. 3.1 (2.2-3.9), p = 0.02). At T/B ≥4.0, positive and negative predictive values for clinical response were respectively 90% and 75%. Quantitative (89)Zr-rituximab hand joint uptake on PET correlated inversely with CD22(+) B-cell count in LN tissue at 4 weeks of treatment (r = 0.6, p = 0.05). In addition, the CD22(+) B-cell count in LN correlated positively with quantitative LN PET data at baseline, supporting the specificity of B-cell imaging on PET. CONCLUSIONS: Non-invasive B-cell imaging by (89)Zr-rituximab PET-CT has promising clinical value to select RA responders to rituximab at baseline. (89)Zr-rituximab PET-CT may also hold promise for monitoring anti-B-cell therapies in other B-cell driven autoimmune diseases, such as systemic lupus erythematosus and Sjögren's disease

Serum Vaspin Levels Are Associated with the Development of Clinically Manifest Arthritis in Autoantibody-Positive Individuals.

We have previously shown that overweight may increase the risk of developing rheumatoid arthritis (RA) in autoantibody positive individuals. Adipose tissue could contribute to the development of RA by production of various bioactive peptides. Therefore, we examined levels of adipokines in serum and synovial tissue of subjects at risk of RA.Fifty-one individuals positive for immunoglobulin M rheumatoid factor (IgM-RF) and/or anti-citrullinated protein antibodies (ACPA), without arthritis, were included in this prospective study. Levels of adiponectin, vaspin, resistin, leptin, chemerin and omentin were determined in baseline fasting serum samples (n = 27). Synovial tissue was obtained by arthroscopy at baseline and we examined the expression of adiponectin, resistin and visfatin by immunohistochemistry.The development of clinically manifest arthritis after follow-up was associated with baseline serum vaspin levels (HR1.5 (95% CI 1.1 to 2.2); p = 0.020), also after adjustment for overweight (HR1.7 (95% CI 1.1 to 2.5); p = 0.016). This association was not seen for other adipokines. Various serum adipokine levels correlated with BMI (adiponectin r = -0.538, leptin r = 0.664; chemerin r = 0.529) and systemic markers of inflammation such as CRP levels at baseline (adiponectin r = -0.449, omentin r = -0.557, leptin r = 0.635, chemerin r = 0.619, resistin r = 0.520) and ESR (leptin r = 0.512, chemerin r = 0.708), p-value<0.05. Synovial expression of adiponectin, resistin and visfatin was not associated with development of clinically manifest arthritis.In this exploratory study, serum adipokines were associated with an increased inflammatory state in autoantibody-positive individuals at risk of developing RA. Furthermore, serum vaspin levels may assist in predicting the development of arthritis in these individuals

Distinctive expression of T cell guiding molecules in human autoimmune lymph node stromal cells upon TLR3 triggering

Infections are implicated in autoimmunity. Autoantibodies are produced in lymphoid tissue where lymph node stromal cells (LNSCs) regulate lymphocyte function. Infections can alter the interaction between LNSCs and lymphocytes resulting in defective immune responses. In rheumatoid arthritis (RA) autoantibody production precedes clinical disease allowing identification of at risk individuals. We investigated the ability of human LNSCs derived from RA, RA-risk and healthy individuals to sense and respond to pathogens. Human LNSCs cultured directly from freshly collected lymph node biopsies expressed TLR1-9 with exception of TLR7. In all donors TLR3 triggering induced expression of ISGs, IL-6 and adhesion molecules like VCAM-1 and ICAM-1. Strikingly, T cell guiding chemokines CCL19 and IL-8 as well as the antiviral gene MxA were less induced upon TLR3 triggering in autoimmune LNSCs. This observed decrease, found already in LNSCs of RA-risk individuals, may lead to incorrect positioning of lymphocytes and aberrant immune responses during viral infection