Increased frequency of circulating IL-21 producing Th-cells in patients with granulomatosis with polyangiitis (GPA).

INTRODUCTION: The present study aimed to explore a possible role for IL-21 producing Th-cells in the immunopathogenesis of granulomatosis with polyangiitis (GPA). METHODS: Peripheral blood from 42 GPA patients in remission and 29 age-matched healthy controls (HCs) were stimulated in vitro, and the frequencies of IL-21 producing Th-cells were determined by flow cytometry. Since Th17-cells produce a low level of IL-21, IL-17 was also included in the analysis. Given that IL-21 is a hallmark cytokine for T follicular helper cells (T(FH)), we next evaluated the expression of their key transcription factor BCL-6 by RT-PCR and flow cytometry. To investigate the effect of IL-21 on autoantibody-production, PBMCs from GPA patients were stimulated in vitro with BAFF/IL-21 and total IgG and ANCA levels were measured in supernatants. In addition, the expression of IL-21-receptor on B-cells was analyzed. RESULTS: Percentages of IL-21 producing Th-cells were significantly elevated in GPA-patients compared to HCs, and were restricted to ANCA-positive patients. The expression of BCL-6 was significantly higher in ANCA-positive GPA-patients, as compared with ANCA-negative patients and HCs. IL-21 enhanced the production of IgG and ANCA in vitro in stimulated PBMCs from GPA patients. No difference was found in the expression of the IL-21-receptor on B-cells between ANCA-negative patients, ANCA-positive patients, and HCs. CONCLUSION: The increased frequency of circulating IL-21 producing Th-cells in ANCA-positive GPA patients and the stimulating capacity of IL-21 on ANCA-production suggest a role for these cells in the immunopathogenesis of GPA. Blockade of IL-21 could constitute a new therapeutic strategy for GPA

Expression and regulation of HIF-1alpha in macrophages under inflammatory conditions; significant reduction of VEGF by CaMKII inhibitor

<p>Abstract</p> <p>Background</p> <p>Macrophages expressing the pro-angiogenic transcription factor hypoxia-inducible factor (HIF)-1alpha have been demonstrated in rheumatoid arthritis (RA) in the synovial tissue. Aim of the present study was to investigate intracellular signal transduction regulation of pro-inflammatory HIF-1 alpha expression in macrophages to identify possible new intervention strategies. We investigated the effects of CaMKII-inhibitors amongst other kinase inhibitors, on HIF-1 alpha expression and downstream production of pro-angiogenic factors in macrophages.</p> <p>Methods</p> <p>Differentiated THP-1 cells and synovial fluid (SF) macrophages were stimulated with 1 μg/ml LPS with or without pretreatment with specific inhibitors of the ERK pathway (PD98059), the PI3K pathway (LY294002), and the CaMKII pathway (KN93 and SMP-114). mRNA and protein expression of HIF-1 alpha, VEGF, MMP-9, and IL-8 was measured in cell lysates and cell supernatants.</p> <p>Results</p> <p>HIF-1 alpha protein expression in LPS-stimulated THP-1 macrophages could be blocked by ERK- and PI3K-inhibitors, but also by the CaMKII inhibitor KN93. THP-1 and SF macrophages produced high levels of VEGF, IL-8, and MMP-9, and VEGF protein production was significantly inhibited by PI3K-inhibitor, and by both CaMKII inhibitors. LPS stimulation in an hypoxic environment did not change VEGF levels, suggesting that LPS induced VEGF production in macrophages is more important than the hypoxic induction.</p> <p>Conclusions</p> <p>Expression of HIF-1 alpha and downstream effects in macrophages are regulated by ERK-, PI3K, but also by CaMKII pathways. Inhibition of HIF-1α protein expression and significant inhibition of VEGF production in macrophages was found using CaMKII inhibitors. This is an unknown but very interesting effect of the CaMKII inhibitor SMP-114, which has been in clinical trial as DMARD for the treatment of RA. This effect may contribute to the anti-arthritic effects of SMP-114.</p

Differential influence of p38 mitogen activated protein kinase (MAPK) inhibition on acute phase protein synthesis in human hepatoma cell lines

BACKGROUND: Inhibition of intracellular signal transduction is considered to be an interesting target for treatment in inflammation. p38 MAPK inhibitors, especially, have been developed and are now in phase II clinical trials for rheumatoid arthritis (RA). OBJECTIVE: To investigate the influence of p38 MAPK inhibition on acute phase protein (APP) production, which is dependent on both JAK/STAT and p38 MAPK pathways. METHODS: The effects of p38 MAPK inhibition on APP production and mRNA expression in four human hepatoma cell lines was investigated, after stimulation with interleukin (IL)6 and/or IL1β or tumour necrosis factor α. RESULTS: Two out of four cell lines produced C reactive protein (CRP), especially after combined IL6 and IL1β stimulation. CRP production was significantly inhibited by the p38 MAPK specific inhibitor RWJ 67657 at 1 μmol/l, which is pharmacologically relevant. Fibrinogen production was also inhibited at 1 μmol/l in all cell lines. Serum amyloid A (SAA) was produced in all four lines. In contrast with CRP, SAA production was not inhibited by RWJ 67657 at 1 μmol/l. CONCLUSION: Production and mRNA expression of CRP and fibrinogen, but not SAA production and mRNA expression, were significantly inhibited by p38 MAPK specific inhibitor in hepatoma cell lines. For p38 MAPK inhibitor treatment in RA SAA might be a better marker of disease activity than CRP and fibrinogen, because SAA is not directly affected by p38 MAPK inhibition