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.

IntroductionIn vitro apoptosis of peripheral monocytes in rheumatoid arthritis (RA) is disturbed and influenced by cytokine production and transmembrane TNF (tmTNF) reverse signaling. The goal of the study was the analysis of the predictive value of the rate of in vitro apoptosis for the therapeutic response to anti-TNF treatment.MethodsSpontaneous and tmTNF reverse signaling-induced apoptosis were determined in vitro in monocytes from 20 RA patients prior to initiation of therapeutic TNF inhibition with etanercept, and the subsequent clinical response was monitored.ResultsSpontaneous in vitro apoptosis was significantly reduced in RA patients compared to controls. Deficiency in spontaneous apoptosis was associated with an insufficient therapeutic response according to the European League Against Rheumatism (EULAR) response criteria and less reduction of the disease activity determined by disease activity score (DAS) 28. High susceptibility to reverse signaling-induced apoptosis was also associated with less efficient reduction in the DAS28. Of note, a strong negative correlation between the two apoptotic parameters was discernible, possibly indicative of two pathogenetically relevant processes counter-regulating each other.tmTNF reverse signaling induced in vitro production of soluble IL1-RI and IL-1RII only in monocytes not deficient in spontaneous apoptosis, and the levels of soluble IL1-RII were found to be predictive of a good clinical response to Etanercept.ConclusionAlthough tmTNF reverse signaling is able to induce apoptosis of RA monocytes in vitro, this process appears to occur in vitro preferentially in patients with suboptimal therapeutic response. Resistance to spontaneous in vitro apoptosis, in contrast, is a predictor of insufficient response to treatment.

BackgroundTreatment with TNF inhibitors is very efficient in the majority of the patients with rheumatoid arthritis (RA), but it does not achieve a sufficient treatment response in 40–50% of the cases. Goal of the study was to assess functional ex vivo-tests of RA monocytes as prognostic parameters of the subsequent treatment response.Methods20 anti-TNF naïve RA patients were enrolled in a prospective, open-label trial, and Etanercept therapy was initiated. Prior to treatment, reverse signaling was induced in peripheral blood monocytes by tmTNF crosslinking via TNFR2:Ig construct Etanercept in a standardized ex vivo-assay. Released cytokine and cytokine receptor concentrations were determined as parameters of the monocyte response.ResultsCrosslinking of tmTNF and consecutive reverse signaling led to production of pro- and anti-inflammatory cytokines and of soluble cytokine decoy receptors such as sTNFR1 and sIL-1R2. Several of the measured concentrations were found to correlate with the treatment response according to the EULAR criteria. The correlation was most pronounced in sTNFR1 concentrations (r = −0.657, p = 0.0031), which also predicted a good clinical response with the highest sensitivity and specificity according to EULAR criteria.ConclusionsHerein we propose that the tmTNF crosslinking-triggered shedding of soluble decoy receptors and production of anti-inflammatory cytokines could contribute to the clinical efficacy of TNF inhibitors, and that in vitro quantification of this secretion by RA monocytes prior to treatment can be used to predict the clinical response. Further development of such standardized tests could be a step towards personalized medicine by providing rheumatologists with a rational choice for first line biological therapy in patients with RA.

Objective Dysregulated apoptosis of monocytes is a pathogenic feature of rheumatoid arthritis (RA). The aim of this study was to investigate the role of TRAIL and TRAIL‐induced apoptosis in patients with RA. Methods Cell surface expression and serum concentrations of TRAIL were determined in 63 patients with RA, and TRAIL‐induced monocyte apoptosis was quantified. Surface expression of TRAILR‐1, TRAILR‐2, TRAILR‐3, TRAILR‐4, CXCR1, and CXCR2 was determined, and intracellular signal transduction was investigated. In 8 patients with RA, clinical and laboratory parameters of disease activity were investigated longitudinally, before and after initiation of treatment with tumor necrosis factor (TNF) inhibitors. Results Serum concentrations of both TRAIL and interleukin‐8 (IL‐8) were increased in patients with RA, while cell surface expression of the TRAIL receptors TRAILR‐1, TRAILR‐2, TRAILR‐3, and TRAILR‐4 was diminished. TRAIL‐induced monocyte apoptosis was significantly decreased in RA due to increased TRAIL‐induced IL‐8 secretion by RA monocytes. The combined effect of TRAIL and IL‐8 on monocytes resulted in activation of antiapoptotic pathways, including p42/44 MAPK and p38. Susceptibility to TRAIL‐induced apoptosis was restored in RA monocytes after 3 months of TNF inhibition. Conclusion In RA, circulating monocytes with the potential to produce proinflammatory cytokines appear to have defects in several pathways of apoptosis induction, among which is a deficiency in TRAIL‐induced apoptosis. Although this resistance to apoptosis might contribute to perpetuation of the disease, it remains to be determined whether specific induction of apoptosis could be therapeutically beneficial.