Lipopolysaccharide (LPS) from Gram-negative bacteria is one of the most potent innate immune-activating stimuli known. Here we review the current understanding of LPS effects on human monocyte and macrophage function. We provide an overview of LPS signal transduction with attention given to receptor cooperativity and species differences in LPS responses, as well as the role of tyrosine phosphorylation and lysine acetylation in signalling. We also review LPS-regulated transcription, with emphasis on chromatin remodeling and primary versus secondary transcriptional control mechanisms. Finally, we review the regulation and function of LPS-inducible cytokines produced by human monocytes and macrophages including TNFα, the IL-1 family, IL-6, IL-8, the IL-10 family, the IL-12 family, IL-15 and TGFβ.
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.
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.
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.
Objective. The cytokine tumor necrosis factor (TNF) plays a central role in the pathogenesis of rheumatoid arthritis (RA), but its disease-specific effector mechanisms have not been fully elucidated. This study was undertaken to investigate the role of TNF in T cell accumulation and migration in the synovitic joints of RA patients.Methods. Vital tissue sections from rheumatoid synovium were generated using a horizontally oscillating microtome and were coincubated with fluorescencelabeled CD4؉ T cells. Migration was detected by fluorescence and confocal microscopy. Migrating T cells were recovered from the tissue and analyzed for phenotype. Chemotaxis of CD4؉ T cells from RA patients in response to increasing concentrations of TNF was analyzed in Transwell experiments.Results. CD4؉ T cells from RA patients migrated into the tissue sections in significantly higher numbers than T cells from healthy controls. Migrating CD4؉ T cells differed from nonmigrating ones in their increased expression of TNF receptor type I (TNFRI), which was expressed on a fraction of circulating CD4؉ T cells from RA patients, but not from controls. CD4؉ T cells from the peripheral blood of RA patients were also found to migrate along TNF concentration gradients ex vivo. Accordingly, blockade of either TNF or TNFRI nearly abrogated in vitro T cell migration in synovial tissue.Conclusion. Our findings indicate that the interaction of TNF with TNFRI is pivotal for T cell migration in synovial tissue in vitro, and thereby suggest a relevant role of the cytokine for in vivo T cell trafficking to synovitic joints.
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