Ulf Wagner

Chemokine Secretion of Rheumatoid Arthritis Synovial Fibroblasts Stimulated by Toll-Like Receptor 2 Ligands

To analyze the role of Toll-like receptors (TLR) in the pathogenesis of rheumatoid arthritis, we have assessed the effects of stimulation of cultured synovial fibroblasts by the TLR-2 ligand bacterial peptidoglycan. By using high density oligonucleotide microarray analysis we identified 74 genes that were up-regulated >2.5-fold. Fourteen CC and CXC chemokine genes were among the genes with the highest up-regulation. Quantitative real-time PCR analysis confirmed up-regulation of granulocyte chemotactic protein (GCP)-2, RANTES, monocyte chemoattractant protein (MCP)-2, IL-8, growth-related oncogene-2, and to a lesser extent, macrophage-inflammatory protein 1α, MCP-1, EXODUS, and CXCL-16. GCP-2, RANTES, and MCP-2 were detected in culture supernatants of synovial fibroblasts stimulated with peptidoglycan. Chemokine secretion induced by stimulation of rheumatoid arthritis synovial fibroblasts via TLR-2 was functionally relevant as demonstrated by chemotaxis assays. GCP-2 and MCP-2 expression, which have not been reported previously in rheumatoid arthritis, was demonstrated in synovial tissue sections of patients diagnosed with rheumatoid arthritis but not in those with osteoarthritis. Correspondingly, synovial fluid levels were significantly higher in patients diagnosed with rheumatoid arthritis as compared with osteoarthritis. Thus, we present evidence for an induction of chemokine secretion by activation of synovial fibroblasts via TLR-2, possibly contributing to the formation of inflammatory infiltrates characteristically found in rheumatoid arthritis joints.

Extracellular Ca2+ is a danger signal activating the NLRP3 inflammasome through G protein-coupled calcium sensing receptors.

Activation of the NLRP3 inflammasome enables monocytes and macrophages to release high levels of interleukin-1β during inflammatory responses. Concentrations of extracellular calcium can increase at sites of infection, inflammation or cell activation. Here we show that increased extracellular calcium activates the NLRP3 inflammasome via stimulation of G protein-coupled calcium sensing receptors. Activation is mediated by signalling through the calcium-sensing receptor and GPRC6A via the phosphatidyl inositol/Ca2+ pathway. The resulting increase in the intracellular calcium concentration triggers inflammasome assembly and Caspase-1 activation. We identified necrotic cells as one source for excess extracellular calcium triggering this activation. In vivo, increased calcium concentrations can amplify the inflammatory response in the mouse model of carrageenan-induced footpad swelling, and this effect was inhibited in GPRC6A−/− mice. Our results demonstrate that G-protein-coupled receptors can activate the inflammasome, and indicate that increased extracellular calcium has a role as a danger signal and amplifier of inflammation.

Pain and fatigue in adult patients with rheumatoid arthritis : association with demographic factors, disease related factors, body awareness, emotional and psychosocial factors

OBJECTIVE Circulating monocytes contain a subpopulation of CD14+CD16+ cells; this subpopulation of cells has been described to be proinflammatory and to have an increased frequency in rheumatoid arthritis (RA). New evidence suggests that this subpopulation can be further subdivided into CD14(dim) CD16+ and CD14(bright) CD16+ cells. The aim of this study was to determine which of the two CD16+ monocyte subpopulations is expanded in patients with RA and to investigate their possible role in disease pathogenesis. METHODS The frequencies of monocyte subpopulations in the peripheral blood of healthy donors and patients with RA were determined by flow cytometry. Monocyte subpopulations were sorted and cocultured with CD4+ T cells. Cytokines were determined in the supernatant, and Th17 cell frequencies were measured by flow cytometry. RESULTS In comparison with the other monocyte subpopulations, CD14(bright) CD16+ cells showed higher HLA-DR and CCR5 expression and responded with higher tumor necrosis factor production to direct cell contact with preactivated T cells. They were observed at increased frequencies in the peripheral blood of patients with RA, while CD14(dim) CD16+ monocyte frequencies were not increased. CD14(bright) CD16+ cells were extremely potent inducers of Th17 cell expansion in vitro. Their frequency in the peripheral blood of patients with RA correlated closely with Th17 cell frequencies determined ex vivo. CONCLUSION This study is the first to provide a link between the increased frequency of the CD14(bright) CD16+ monocyte subpopulation in RA and the expansion of Th17 cells, which are likely to have a role in the pathogenesis of autoimmunity.

Post-thymic in vivo proliferation of naive CD4 + T cells constrains the TCR repertoire in healthy human adults

In spite of thymic involution early in life, the numbers of naive CD4+ T cells only slowly decline in ageing humans implying peripheral post‐thymic naive CD4+ T cell expansion. This proliferation may compensate for continuous activation and death of naive CD4+ T cells but may also have negative consequences for protective immunity. Here we show that naive CD4+ T cells that have proliferated in the periphery are characterized by a highly restricted oligoclonal TCR repertoire. Additionally these cells, which constitute the majority of naive CD4+ T cells in the elderly, display signatures of recent TCR engagement. Our results demonstrate for the first time that peripheral post‐thymic proliferation of naive CD4+ T cells in healthy human individuals causes a significant contraction of the peripheral TCR repertoire. This age‐dependent deterioration of CD4+ T cell immunity could entail ageing‐associated autoimmunity, increased susceptibility to infection or cancer and decreased efficiency of vaccination.

Association of PTPN22 1858 single-nucleotide polymorphism with rheumatoid arthritis in a German cohort: higher frequency of the risk allele in male compared to female patients

The functional single-nucleotide polymorphism (SNP) of the gene PTPN22 is a susceptibility locus for rheumatoid arthritis (RA). The study presented here describes the association of the PTPN22 1858T allele with RA in a German patient cohort; 390 patients with RA and 349 controls were enrolled in the study. For 123 patients, clinical and radiographic documentation over 6 years was available from the onset of disease. Genotyping of the PTPN22 1858 SNP was performed using an restriction fragment length polymorphism PCR-based genotyping assay. The odds ratio to develop RA was 2.57 for carriers of the PTPN22 1858T allele (95% confidence interval (CI) 1.85–3.58, p < 0.001), and 5.58 for homozygotes (95% CI 1.85–16.79). The PTPN22 1858T allele was significantly associated not only with rheumatoid factor (RF) and anti-cyclic citrullinated peptide (CCP) positive RA, but also with RF and anti-CCP negative disease. The frequency of the PTPN22 1858T allele was increased disproportionately in male patients (53.8% compared to 33.0% in female patients, p < 0.001), and the resulting odds ratio for male carriers was increased to 4.47 (95% CI 2.5–8.0, p < 0.001). Moreover, within the male patient population, the rare allele was significantly associated with the HLA-DRB1 shared epitope (p = 0.01). No significant differences in disease activity or Larsen scores were detected. The results provide further evidence that the PTPN22 1858T allele is associated with RA irrespective of autoantibody production. The increased frequency of the risk allele in male patients and its association with the shared epitope indicate that the genetic contribution to disease pathogenesis might be more prominent in men.

Interaction between Transmembrane TNF and TNFR1/2 Mediates the Activation of Monocytes by Contact with T Cells

Monocytes and monocytic cells produce proinflammatory cytokines upon direct cell contact with activated T cells. In the autoimmune disease rheumatoid arthritis, the pivotal role of TNF-α implies that the interaction between transmembrane TNF-α (mTNF) and the TNF receptors (TNFR1 and TNFR2) might participate in the T cell contact-dependent activation of monocytes. Accordingly, treatment of rheumatoid arthritis by administration of a TNF-α-blocking Ab was found to significantly decrease TNF-α production by monocytes. Several lines of evidence indicated that signaling through TNFR1/2 and through mTNF (reverse signaling) is involved in TNF-α production by monocytes after T cell contact: 1) blocking mTNF on activated T cells leads to a significant reduction in TNF-α production; 2) down-regulation of TNFR1/2 on monocytes by transfection with small interfering RNA results in diminished TNF-α production; 3) blocking or down-regulating TNFR2 on activated T cells inhibits TNF-α production, indicating that mTNF on the monocyte surface mediates signaling; 4) ligation of mTNF on monocytes by surface TNFR2 transfected into resting T cells induces TNF-α production due to reverse signaling by mTNF; and 5) ligation of mTNF on monocytes by a soluble TNFR2:Ig receptor construct induces TNF-α production due to reverse signaling. In conclusion, we identified mTNF and TNFR1/2 as interaction partners contributing to TNF-α production in monocytes. Both pathways initiated by mTNF-TNFR interaction are likely to be inhibited by treatment with anti-TNF-α Abs.

Cytokine-induced human IFN-γ–secreting effector-memory Th cells in chronic autoimmune inflammation

T-helper (Th) cells activated by cytokines in the absence of T-cell receptor ligation are suspected to participate in inflammatory processes by production of interferon-gamma (IFN-gamma). Still, the relevance of such a mechanism has not been addressed in humans. Here we demonstrate that a subset of human effector-memory Th cells expressing functional interleukin-12R (IL-12R), IL-18Ralpha, and CCR5 ex vivo can be induced to secrete IFN-gamma by cytokines signaling via the IL-2R common gamma-chain in combination with IL-12 and IL-18. Cytokine-driven IFN-gamma production depends on JAK3- and p38 mitogen-activated kinase signals and is sensitive to suppression by CD25(++) regulatory T cells. Contrary to IFN-gamma(+) Th cells induced upon antigen-specific stimulation, their cytokine-activated counterparts characteristically lack expression of costimulator 4-1BB (CD137). Strikingly, the majority of Th cells infiltrating inflamed joints of rheumatoid arthritis patients is equipped with receptors prerequisite for cytokine-induced IFN-gamma secretion. Among these cells, we detected a substantial fraction that secretes IFN-gamma directly ex vivo but lacks 4-1BB expression, indicating that cytokine-induced IFN-gamma(+) Th cells operate in autoimmune inflammation. Our data provide a rationale for how human effector-memory Thcells can participate in perpetuating inflammatory processes in autoimmunity even in the absence of T-cell receptor ligation.

Clonally expanded CD4+CD28null T cells in rheumatoid arthritis use distinct combinations of T cell receptor BV and BJ elements

Clonally expanded, autoreactive CD4+CD28null cells can be found in the peripheral blood of patients with rheumatoid arthritis and have been shown to be associated with severeextra‐articular disease manifestations. We investigated the size of the CD4+CD28null compartment and the TCR β chain repertoire of expanded CD4+ clonotypes in 94 rheumatoid arthritis patients by complementarity‐determining region 3 (CDR3) length analysis (spectratyping) in the BV6 and BV14 TCR families, with primers specific for three arbitrarily chosen β chain joining elements (BJ1S2, BJ2S3 and BJ2S7). The spectratyping results showed a strong correlation of the size of the CD4+CD28null compartment with the detected number of BV14 clonotypes, whereas no association with BV6 oligoclonality was found. Only clones using the BV14–BJ1S2 and BV14–BJ2S3 combinations contributed to this correlation, however, whereas BV14–BJ2S7 clones did not. This preferential correlation implies a role for the TCR β chain in stimulating clonal outgrowth and argues against the previously suggested superantigenic stimulation of in‐vivo‐expanded clones. Instead, since no evidence for shared antigen specificity could be detected, clonal expansion of T cells in rheumatoid arthritis might be influenced by the BJ elements because of changes in the flexibility of the protein backbone of the β‐chain.

Toll-Like Receptor 4 Is Involved in Inflammatory and Joint Destructive Pathways in Collagen-Induced Arthritis in DBA1J Mice

In rheumatoid arthritis, a significant proportion of cytokine and chemokine synthesis is attributed to innate immune mechanisms. TLR4 is a prominent innate receptor since several endogenous ligands known to activate the innate immune system bind to it and may thereby promote joint inflammation. We generated TLR4 deficient DBA1J mice by backcrossing the TLR4 mutation present in C3H/HeJ strain onto the DBA1J strain and investigated the course of collagen-induced arthritis in TLR4 deficient mice in comparison to wild type littermates. The incidence of collagen- induced arthritis was significantly lower in TLR4 deficient compared to wild type mice (59 percent vs. 100 percent). The severity of arthritis was reduced in the TLR4 deficient mice compared to wild type littermates (mean maximum score 2,54 vs. 6,25). Mice deficient for TLR4 were virtually protected from cartilage destruction, and infiltration of inflammatory cells was reduced compared to wt mice. In parallel to the decreased clinical severity, lower anti-CCP antibody concentrations and lower IL-17 concentrations were found in the TLR4 deficient mice. The study further supports the role of TLR4 in the propagation of joint inflammation and destruction. Moreover, since deficiency in TLR4 led to decreased IL-17 and anti-CCP antibody production, the results indicate a link between TLR4 stimulation and the adaptive autoimmune response. This mechanism might be relevant in human rheumatoid arthritis, possibly in response to activating endogenous ligands in the affected joints.

Negative association of the chemokine receptor CCR5 d32 polymorphism with systemic inflammatory response, extra-articular symptoms and joint erosion in rheumatoid arthritis.

IntroductionChemokines and their receptors control immune cell migration during infections as well as in autoimmune responses. A 32 bp deletion in the gene of the chemokine receptor CCR5 confers protection against HIV infection, but has also been reported to decrease susceptibility to rheumatoid arthritis (RA). The influence of this deletion variant on the clinical course of this autoimmune disease was investigated.MethodsGenotyping for CCR5d32 was performed by PCR and subsequent electrophoretic fragment length determination. For the clinical analysis, the following extra-articular manifestations of RA were documented by the rheumatologist following the patient: presence of rheumatoid nodules, major organ vasculitis, pulmonary fibrosis, serositis or a Raynauds syndrome. All documented CRP levels were analyzed retrospectively, and the last available hand and feet radiographs were analyzed with regards to the presence or absence of erosive disease.ResultsAnalysis of the CCR5 polymorphism in 503 RA patients and in 459 age-matched healthy controls revealed a significantly decreased disease susceptibility for carriers of the CCR5d32 deletion (Odds ratio 0.67, P = 0.0437). Within the RA patient cohort, CCR5d32 was significantly less frequent in patients with extra-articular manifestations compared with those with limited, articular disease (13.2% versus 22.8%, P = 0.0374). In addition, the deletion was associated with significantly lower average CRP levels over time (median 8.85 vs. median 14.1, P = 0.0041) and had a protective effect against the development of erosive disease (OR = 0.40, P = 0.0047). Intriguingly, homozygosity for the RA associated DNASE2 -1066 G allele had an additive effect on the disease susceptibility conferred by the wt allele of CCR5 (OR = 2.24, P = 0.0051 for carrier of both RA associated alleles)ConclusionsThe presence of CCR5d32 significantly influenced disease susceptibility to and clinical course of RA in a German study population. The protective effect of this deletion, which has been described to lead to a decreased receptor expression in heterozygous patients, underlines the importance of chemokines in the pathogenesis of RA.