Berber Doornbos-van der Meer

Periodontitis in established rheumatoid arthritis patients: a cross-sectional clinical, microbiological and serological study

IntroductionThe association between rheumatoid arthritis (RA) and periodontitis is suggested to be linked to the periodontal pathogen Porphyromonas gingivalis. Colonization of P. gingivalis in the oral cavity of RA patients has been scarcely considered. To further explore whether the association between periodontitis and RA is dependent on P. gingivalis, we compared host immune responses in RA patients with and without periodontitis in relation to presence of cultivable P. gingivalis in subgingival plaque.MethodsIn 95 RA patients, the periodontal condition was examined using the Dutch Periodontal Screening Index for treatment needs. Subgingival plaque samples were tested for presence of P. gingivalis by anaerobic culture technique. IgA, IgG and IgM antibody titers to P. gingivalis were measured by ELISA. Serum and subgingival plaque measures were compared to a matched control group of non-RA subjects.ResultsA higher prevalence of severe periodontitis was observed in RA patients in comparison to matched non-RA controls (27% versus 12%, p < 0.001). RA patients with severe periodontitis had higher DAS28 scores than RA patients with no or moderate periodontitis (p < 0.001), while no differences were seen in IgM-RF or ACPA reactivity. Furthermore, RA patients with severe periodontitis had higher IgG- and IgM-anti P. gingivalis titers than non-RA controls with severe periodontitis (p < 0.01 resp. p < 0.05), although subgingival occurrence of P. gingivalis was not different.ConclusionsSeverity of periodontitis is related to severity of RA. RA patients with severe periodontitis have a more robust antibody response against P. gingivalis than non-RA controls, but not all RA patients have cultivable P. gingivalis.

Increase in IL-21 producing T-cells in patients with systemic lupus erythematosus

IntroductionSystemic lupus erythematosus (SLE) is an autoimmune disease accompanied by a disturbed T-cell balance skewed towards effector T-cells, in particular Th17-cells. The novel cytokine interleukin-21 (IL-21) is suggested to be crucial for triggering T-cell responses towards IL-17 producing cells. Thus, we aimed to investigate the ability of T-cells to produce IL-21 and IL-17 in SLE patients.MethodsPeripheral blood of 34 SLE patients and 18 healthy controls (HC) was stimulated with phorbol myristate acetate (PMA) and calcium ionophore (Ca-Io). Percentages of IL-21- and IL-17A expressing T-cells were analysed by flow cytometry. The expression levels of the transcription factors B-cell lymphoma-6 (BCL-6) and factors retinoid-related orphan receptor (ROR-γt) were assessed in T-cells by real-time RT-PCR and flow cytometry. Additionally, IL-21 receptor (IL-21R) expression on B- and T-cells of patients and HC was analyzed.ResultsSignificantly increased percentages of IL-21 expressing CD4+ T-cells and CD8+ T-cells were found in SLE patients as compared to HC. The percentages of IL-21+ CD4+ T-cells and CD8+ T-cells correlated significantly with the percentages of IL-17A+ CD4+ T-cells and CD8+ T-cells, respectively. The relative expression of BCL-6 and ROR-γt did not differ between SLE patients and HC. IL-21R expression occurred mainly on B-cells and was not different comparing SLE patients and HC.ConclusionsThis study demonstrates an increased proportion of IL-21+ T-cells in SLE patients correlating with the proportion of IL-17+ T-cells. This suggests a pivotal role of IL-21 in the pathogenesis of SLE.

LRegulation of cytokine-induced HW-1 alpha expression in rheumatoid synovial fibroblasts

Abstract:  The transcription factor hypoxia‐inducible factor (HIF)‐1 plays a central physiological role in oxygen and energy homeostasis, and is activated during hypoxia by stabilization of the subunit HIF‐1α. Activation can also occur by proinflammatory cytokines during inflammation. Hypoxia, as well as proinflammatory cytokines, plays an important role in the synovia in rheumatoid arthritis (RA) patients. Expression of HIF‐1α has been demonstrated in RA synovial lining layer. The aim of the study was to investigate the regulation of the intracellular signal transduction pathways, involved in the expression of HIF‐1α, and in the expression of genes regulated by HIF‐1α in rheumatoid synovial fibroblasts (RSF). RSF were cultured under proinflammatory conditions (IL‐1β and TNF‐α stimulation) and under chemical hypoxia (CoCl2 treatment). Expression of HIF‐1α was analyzed in nuclear extracts by Western blotting. The effect of inhibitors of the PI3K and the ERK pathway on HIF‐1α protein expression was measured. mRNA expression of HIF‐1α, COX‐2, vascular endothelial growth factor (VEGF), and stromal cell‐derived factor (SDF)‐1 was determined by real‐time RT‐PCR, and protein production of VEGF and SDF‐1 by ELISA. Treatment of the synovial fibroblasts with 150 mM CoCl2 as well as stimulation with 10 ng/mL IL‐1β or TNF‐α resulted in strong protein expression of HIF‐1α, measured with Western blotting. Pretreatment with the MEK1/2 inhibitor PD98059 as well as the PI3K inhibitor LY294002 resulted in inhibition of the cytokine‐induced HIF‐1α expression. Furthermore, it was shown that cytokine‐induced mRNA expression of HIF‐1α was inhibited by the PI3K inhibitor. We found that cytokine stimulation induced VEGF mRNA and protein production, but no significant effect of kinase inhibition was found on VEGF production in cytokine‐stimulated RSF. Both the ERK pathway and the PI3K pathway are involved in the cytokine‐induced HIF‐1α expression in RSF and in the expression of proangiogenic factors.

Strong inhibition of TNF-α production and inhibition of IL-8 and COX-2 mRNA expression in monocyte-derived macrophages by RWJ 67657, a p38 mitogen-activated protein kinase (MAPK) inhibitor

In inflammatory processes, the p38 mitogen-activated protein kinase (MAPK) signal transduction route regulates production and expression of cytokines and other inflammatory mediators. Tumor necrosis factor α (TNF-α) is a pivotal cytokine in rheumatoid arthritis and its production in macrophages is under control of the p38 MAPK route. Inhibition of the p38 MAPK route may inhibit production not only of TNF-α, but also of other inflammatory mediators produced by macrophages, and indirectly of inflammatory mediators by other cells induced by TNF-α stimulation. Here we investigate the effects of RWJ 67657, a p38 MAPK inhibitor, on mRNA expression and protein production of TNF-α and other inflammatory mediators, in monocyte-derived macrophages. A strong inhibition of TNF-α was seen at pharmacologically relevant concentrations of RWJ 67657, but also inhibition of mRNA expression of IL-1β, IL-8, and cyclooxygenase-2 was shown. Furthermore, it was shown that monocyte-derived macrophages have a high constitutive production of matrix metalloproteinase 9, which is not affected by p38 MAPK inhibition. The results presented here may have important implications for the treatment of rheumatoid arthritis.

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

BackgroundMacrophages 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.MethodsDifferentiated 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.ResultsHIF-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.ConclusionsExpression 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.

Antibodies against Porphyromonas gingivalis in seropositive arthralgia patients do not predict development of rheumatoid arthritis

Clinical studies point towards an association between periodontitis and rheumatoid arthritis (RA).1 ,2 A pathogenic role is suggested for Porphyromonas gingivalis .3 P gingivalis may contribute to the pathogenesis of RA by breaking immune tolerance through formation of (bacterial and human) citrullinated proteins, leading to anticitrullinated protein antibody production (ACPA).4 ,5 Since ACPA production precedes RA development6 and because P gingivalis IgG antibodies are long-term stable in untreated periodontitis patients,7 we investigated whether anti- P gingivalis antibody levels are prognostic for development of RA, by assessing these antibodies in a cohort of 289 adults at risk for RA. Patients with arthralgia and seropositivity for IgM-rheumatoid factor (IgM-RF) and/or ACPA were selected from a prospective follow-up study on arthritis development.8 They are further referred to as seropositive arthralgia patients (SAP); their median follow-up was 30 months (IQR 13–49). Baseline sera were used for measurement of ACPA, IgM-RF, C-reactive protein (CRP) and HLA-DRB1 SE carrier status.8 IgA, IgG and IgM antibody levels against P gingivalis were determined by in-house ELISA with a pooled lysate of clinical isolates of P gingivalis as …

Decreased CXCR1 and CXCR2 expression on neutrophils in anti-neutrophil cytoplasmic autoantibody-associated vasculitides potentially increases neutrophil adhesion and impairs migration

IntroductionIn anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides (AAV), persistent inflammation within the vessel wall suggests perturbed neutrophil trafficking leading to accumulation of activated neutrophils in the microvascular compartment. CXCR1 and CXCR2, being major chemokine receptors on neutrophils, are largely responsible for neutrophil recruitment. We speculate that down-regulated expression of CXCR1/2 retains neutrophils within the vessel wall and, consequently, leads to vessel damage.MethodsMembrane expression of CXCR1/2 on neutrophils was assessed by flow cytometry. Serum levels of interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-α), angiopoietin 1 and angiopoietin 2 from quiescent and active AAV patients and healthy controls (HC) were quantified by ELISA. Adhesion and transendothelial migration of isolated neutrophils were analyzed using adhesion assays and Transwell systems, respectively.ResultsExpression of CXCR1 and CXCR2 on neutrophils was significantly decreased in AAV patients compared to HC. Levels of IL-8, which, as TNFα, dose-dependently down-regulated CXCR1 and CXCR2 expression on neutrophils in vitro, were significantly increased in the serum of patients with active AAV and correlated negatively with CXCR1/CXCR2 expression on neutrophils, even in quiescent patients. Blocking CXCR1 and CXCR2 with repertaxin increased neutrophil adhesion and inhibited migration through a glomerular endothelial cell layer.ConclusionsExpression of CXCR1 and CXCR2 is decreased in AAV, potentially induced by circulating proinflammatory cytokines such as IL-8. Down-regulation of these chemokine receptors could increase neutrophil adhesion and impair its migration through the glomerular endothelium, contributing to neutrophil accumulation and, in concert with ANCA, persistent inflammation within the vessel wall.

Differential Expression of Granulopoiesis Related Genes in Neutrophil Subsets Distinguished by Membrane Expression of CD177

Objective Differential gene expression in CD177+ and CD177− neutrophils was investigated, in order to detect possible differences in neutrophil function which could be related to the pathogenesis of ANCA-associated Vasculitides (AAV). Methods Neutrophils were isolated from healthy controls (HC) with high, negative or bimodal CD177 expression, and sorted into CD177+ and CD177− subpopulations. Total RNA was screened for expression of 24,000 probes with Illumina Ref-8 Beadchips. Genes showing differential expression between CD177+ and CD177− subsets in microarray analysis were re-assessed using quantitative-PCR. CD177 expression on neutrophil precursors in bone marrow was analyzed using quantitative PCR and flowcytometry. Results The proportion of CD177+ cells increased during neutrophil maturation in bone marrow. Fold change analysis of gene expression profile of sorted CD177+ and CD177− neutrophils resulted in 14 genes with fold change (fc) >3 difference in expression. Interestingly, 10 of these genes have been reported to change significantly in expression during neutrophil maturation, and most of these genes were granule protein (GP) coding genes. mRNA expression levels measured by RT-PCR of a number of these GP, and of PR3 and MPO were higher in the CD177− neutrophil subset in HC, however, particular granule protein amounts were comparable between CD177+ and CD177− neutrophil subsets. AAV patients had higher amounts of CD177+ neutrophils, but contrary to neutrophils from HC expression of GP-genes was increased, possibly due to activation. Conclusion The neutrophil population can be distinguished by membrane expression of CD177 into subsets that are different in expression of GP mRNA but not in GP protein production. GP gene expression is also elevated in AAV patients, which is not explained by skewed distribution of CD177+ and CD177− subsets but may be associated with neutrophil activation during on-going inflammation.

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

IntroductionThe present study aimed to explore a possible role for IL-21 producing Th-cells in the immunopathogenesis of granulomatosis with polyangiitis (GPA).MethodsPeripheral 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 (TFH), 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.ResultsPercentages 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.ConclusionThe 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.

Role for CaMKII Inhibition in Rheumatoid Arthritis: Effects on HIF-1-Induced VEGF Production by Rheumatoid Synovial Fibroblasts

Background and aims: The present study was designed to investigate the effects of a novel CaMKII inhibitor SMP‐114, which was effective in a collagen‐induced arthritis model in rats, on rheumatoid synovial fibroblasts. Methods: Rheumatoid synovial fibroblasts (RSF) were cultured under pro‐inflammatory (IL‐1β stimulation) and hypoxic conditions. Protein and mRNA expression of HIF‐1α and the effects of inhibitors of the CaMKII‐, PI3K‐, and ERK pathway on VEGF and IL‐6 production were analyzed. Results: Stimulation under hypoxic conditions induced higher expression of HIF‐1α and VEGF mRNA than treatment with either IL‐1β or hypoxia alone. However, incubation with a specific CaMKII inhibitor did not result in reduced VEGF production in RSF. Conclusions: CaMKII activation has been reported to be involved in HIF‐1α stabilization, but incubation with SMP‐114, a specific CaMKII inhibitor, had no effect on HIF‐1‐induced VEGF production by rheumatoid synovial fibroblasts.