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.

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.

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.

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.

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