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.

Peripheral CD4CD8 double positive (DP) T cells have been reported to play a role in several autoimmune diseases, virus infections and cancer. In rheumatoid arthritis (RA), both CD4 and CD8 single positive (SP) T cells are known to be involved in the pathogenesis, but the role of peripheral CD4CD8 DP T cells has not been investigated in detail. Anti cyclic citrullinated antibodies (ACPA) positive and ACPA negative RA patients, patients with systemic lupus erythematodes (SLE) and age matched healthy donors (HD) were enrolled in the analysis. The frequencies and phenotype of DP T cells in PBMC were investigated. In addition, DP T cells were quantified in biopsies from rheumatoid synovium. After in vitro restimulation, the cytokine production of DP T cells was investigated in cultures of PBMC. CMV specific cytokine secretion as well as proliferation was analyzed following antigen specific restimulation after an appropriate culture duration. DP T cells were found more frequently in RA patients than in healthy controls or patients with SLE. These DP T cells express αβ TCRs, are of a memory phenotype and share features of both CD4 as well as CD8 SP T cells. Importantly, DP T cells were found to also be present in the rheumatoid synovium. Further characterization of DP T cells from RA patients revealed increased production of IL-21 and IL-4, implying a possible role as T helper cells. In addition, DP T cells in RA seem to contribute to the inflammatory process, because they produce significantly more IFNγ than counterparts from HD and are increased in CMV+ RA patients. Given their capacity to produce a variety of cytokines (IL4, IL21 and IFNγ), their association with ACPA positive RA and their presence in the synovium, we suggest an important role of double positive T cells in the pathogenesis of rheumatoid arthritis.

Objective. Monocytes are a major source of proinflammatory cytokines in rheumatoid arthritis (RA), and inhibitors of monocytic cytokines are highly efficient agents for treatment of the disease. The aim of this study was to analyze the effects of a therapeutic antitumor necrosis factor ␣ (anti-TNF␣) antibody on monocytes from patients with RA and healthy control subjects.Methods. Peripheral blood monocytes from patients with RA and healthy control subjects were incubated in the presence of anti-TNF␣ antibody or IgG. Annexin V staining, caspase activation, poly(ADP-ribose) polymerase cleavage, and DNA staining with propidium iodide were used to analyze apoptosis. The signaling events elicited in monocytes by infliximab were analyzed by Western blotting and electromobility shift assay.Results. Peripheral blood monocytes from patients with RA were characterized by increased expression of transmembrane TNF␣, spontaneous in vitro production of interleukin-1␤ (IL-1␤), and a decreased rate of spontaneous ex vivo apoptosis. Incubation with infliximab induced significantly increased apoptosis in monocytes from patients with RA but not in monocytes from healthy control subjects. This apoptosis was triggered by reverse signaling of transmembrane TNF after ligation by infliximab and was independent of caspase activation. Instead, transmembrane TNF reverse signaling inhibited the constitutive NF-B activation in RA monocytes, suppressed IL-1␤ secretion, and normalized spontaneous in vitro apoptosis. This normalization was reversible by the addition of exogenous IL-1␤.Conclusion. This study demonstrates that outside-to-inside signaling through transmembrane TNF after ligation by infliximab inhibits constitutive NF-B activation and suppresses spontaneous IL-1␤ production by monocytes from patients with RA. Besides the induction of monocyte apoptosis, this inhibition could also contribute to the therapeutic effects observed during treatment with TNF␣ inhibitors.

IntroductionPeripheral blood monocytes are no longer regarded as a homogeneous cell population, but can be differentiated both phenotypically and functionally into various subpopulations. In rheumatoid arthritis, the subpopulation of CD14bright/CD16+ monocyte is expanded and prone towards generation of Th17 cells. CD56+ monocytes represent a different subpopulation, which is also expanded in conditions associated with autoimmunity like inflammatory bowel diseases. The aim of the study was the quantification and functional characterization of the CD56+ monocyte subset in rheumatoid arthritis (RA).MethodsFrequencies of peripheral blood monocyte subpopulations were analyzed by flow cytometry in 86 healthy controls and 75 RA patients. In 16 patients, anti-tumor necrosis factor (TNF) therapy was initiated, and the CD56+ monocyte frequency was monitored longitudinally. Lipopolysaccharide (LPS)-induced cytokine production of CD56+ and CD56– monocytes was determined by intracellular staining or cytokine secretion assays.ResultsIn healthy individuals, 8.6% ± 0.6 of the monocytes co-expressed CD56, with the majority of CD56+ monocytes being CD14bright (7.9% ± 0.5), while only a minor population was CD14dim (0.7% ± 0.1). We found a strong positive correlation between an individual’s age and the frequency of CD56+ monocytes. Upon stimulation with LPS, CD56+ monocytes became more frequently positive for TNF, IL-10 and IL-23 than CD56– monocytes. In addition, CD56+ monocytes spontaneously produced more reactive oxygen intermediates than CD56- monocytes. In RA patients, the frequency of CD56+ monocytes was significantly higher than in healthy controls (12.2% ± 0.9 vs. 7.9% ± 0.5, p = 0.0002), and this difference most pronounced in RA patients below 40 years of age (11.1% ± 1.6 vs. 4.1% ± 0.4, P < 0.0001). Treatment of the patients with an anti-TNF blocking agent significantly reduced CD56+ monocyte frequencies (baseline 12.4% vs. 24 weeks treatment 8.0%, P = 0.0429), and the magnitude of this decrease was found to correlate with the change in disease activity under the therapy.ConclusionThe CD14bright/CD56+ monocyte subset is expanded in aging individuals as well as in patients with RA. The pro-inflammatory production of cytokines and reactive oxygen species as well as the elimination of those cells in patients with a good response towards TNF inhibiting agents indicates a possible contribution of those monocytes in the inflammatory response in RA.

The association of anti-CCP antibodies with DRB1*01 and with SE-positive DRB1*04 alleles implies a functional role for the SE sequence motif. The determination of SE-positive DRB1*04 alleles and of anti-CCP antibody positivity facilitates the prediction of disease course and prognosis at the time of initial presentation.