RGS16 restricts the pro-inflammatory response of monocytes.

Immune cells express powerful and harmful effectors that require tight regulation. Heterotrimeric G proteins are critical mediators in translating extracellular signals into cell responses, which need a fine-tuned regulation for the control of cell activation. Regulator of G-protein signalling 16 (RGS16) has been identified as a key factor of G protein-mediated activation in lymphocytes, modulating inflammatory and survival responses of various cell types. However, data about the expression of this regulatory protein in monocytes are scarce, and it has remained unclear whether activation and migration of these cells are regulated by RGS16. In this study, the impact of RGS16 on the production of inflammatory cytokines by activated human monocytes was investigated in vitro using the human promonocytic cell line THP-1 as a model. Gain and loss of function experiments showed that RGS16 overexpression reduces the expression of pro-inflammatory cytokines IL-1ß, IL-6, IL-8 and TNFα, while RGS16 knockdown by RNAi upregulates IL-1ß, IL-6 and TNFα but not IL-8. RGS16 knockdown was also shown to enhance Pam3-mediated induction of the anti-inflammatory cytokine IL-10. Our results indicate that RGS16 restricts the activation-induced pro-inflammatory profile in myeloid cells.

Assessment of ability of murine and human anti-lipid A monoclonal antibodies to bind and neutralize lipopolysaccharide.

The use of monoclonal antibodies (mAbs) directed to lipid A for the therapy of gram-negative sepsis is controversial. In an attempt to understand their biologic basis of action, we used a fluid-phase radioimmunoassay to measure binding between bacterial lipopolysaccharide (LPS) and two IgM mAbs directed to lipid A that are being evaluated for the treatment of gram-negative bacterial sepsis. Both antibodies bound 3H-LPS prepared from multiple strains of gram-negative bacteria when large excesses of antibody were used, although binding was modest and only slightly greater than control preparations. We also studied the ability of each anti-lipid A antibody to neutralize some of the biological effects of LPS in vitro. Despite large molar excesses, neither antibody neutralized LPS as assessed by the limulus lysate test, by a mitogenic assay for murine splenocytes, or by the production of cytokines interleukin (IL)-1, IL-6, or tumor necrosis factor from human monocytes in culture medium or in whole blood. Our experiments do not support the hypothesis that either of these anti-lipid A mAbs function by neutralizing the toxic effects of LPS.

Interleukin 1 is released by murine macrophages during apoptosis induced by Shigella flexneri.

Peritoneal macrophages undergoing apoptosis induced by Shigella flexneri infection release the inflammatory cytokine interleukin 1 (IL-1), but not IL-6 or tumor necrosis factor alpha (TNF alpha). Wild type shigella causes a very fast and significant release of IL-1 from prestimulated peritoneal macrophages, before the cell's integrity is compromised. Both IL-1 alpha and IL-1 beta are released, IL-1 beta in its mature processed form. IL-1 is released from presynthesized cytoplasmic pools. These results demonstrate that bacteria-induced apoptosis of macrophages may play an active role in vivo by releasing IL-1, which in turn mediates an early inflammatory response in epithelial tissues.

Dysregulation of in vitro cytokine production by monocytes during sepsis.

The production by monocytes of interleukin-1 alpha (IL-1 alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF alpha) in intensive care unit (ICU) patients with sepsis syndrome (n = 23) or noninfectious shock (n = 6) is reported. Plasma cytokines, cell-associated cytokines within freshly isolated monocytes and LPS-induced in vitro cytokine production were assessed at admission and at regular intervals during ICU stay. TNF alpha and IL-6 were the most frequently detected circulating cytokines. Despite the fact that IL-1 alpha is the main cytokine found within monocytes upon in vitro activation of cells from healthy individuals, it was very rarely detected within freshly isolated monocytes from septic patients, and levels of cell-associated IL-1 beta were lower than those of TNF alpha. Cell-associated IL-1 beta and TNF alpha were not correlated with corresponding levels in plasma. Upon LPS stimulation, we observed a profound decrease of in vitro IL-1 alpha production by monocytes in all patients, and of IL-1 beta, IL-6, and TNF alpha in septic patients. This reduced LPS-induced production of cytokines was most pronounced in patients with gram-negative infections. Finally, monocytes from survival patients, but not from nonsurvival ones recovered their capacity to produce normal amounts of cytokines upon LPS stimulation. In conclusion, our data indicate an in vivo activation of circulating monocytes during sepsis as well as in noninfectious shock and suggest that complex regulatory mechanisms can downregulate the production of cytokines by monocytes during severe infections.

Reprogramming of circulatory cells in sepsis and SIRS.

Immune status is altered in patients with sepsis or non-infectious systemic inflammatory response syndrome (SIRS). Reduced ex-vivo TNF production by endotoxin-activated monocytes has been regularly reported. This observation is reminiscent of the phenomenon of endotoxin tolerance, and the term 'leukocyte reprogramming' well defines this phenomenon. This review will outline that the hyporesponsiveness of circulating leukocytes is not a generalized phenomenon in sepsis and SIRS. Indeed, the nature of the insult (i.e. infectious versus non-infectious SIRS; under anesthesia [surgery] or not [trauma, burn]), the nature of the activator used to trigger leukocytes (i.e. different Toll-like receptor ligands or whole bacteria), the nature of the cell culture (i.e. isolated monocytes versus peripheral blood mononuclear cells versus whole blood assays), and the nature of the analyzed cytokines (e.g. IL-1beta versus IL-1ra; TNF versus IL-10) have a profound influence on the outcome of the response.

Ganglioside identification on human monocyte membrane with Clostridium perfringens delta-toxin.

Clostridium perfringens delta-toxin was first described as a hemolysin with a restricted lytic spectrum. A selective cytotoxicity of the delta-toxin was then found on rabbit leukocytes: peritoneal and alveolar macrophages were uniformly killed, whereas thymocytes were essentially resistant. The toxin was shown to be specific for GM2 ganglioside or a GM2-like structure. In the present study we report the interaction of delta-toxin with human monocytes. A specific, saturable, and irreversible binding of 125I-delta-toxin was demonstrated. Binding was inhibited by preincubation of the radiolabeled toxin with GM2 and with high amount of GM1 ganglioside. As judged by dye exclusion, no cytotoxicity was observed on freshly isolated monocytes, but when added at the beginning of a culture of human adherent cells, the cytotoxic effect was detected after 48 hours of culture. Taken together, these data indicate the presence of monosialoganglioside(s) at the surface of human monocytes, and suggest a possible reorganisation of such structure into the cell membrane when monocytes mature in vitro toward macrophage-like cells.

Increased production of tumor necrosis factor-alpha in whole blood cultures from children with primary malnutrition.

Because low tumor necrosis factor-alpha (TNF-alpha) production has been reported in malnourished children, in contrast with high production of TNF-alpha in experimental protein-energy malnutrition, we reevaluated the production of TNF-alpha in whole blood cultures from children with primary malnutrition free from infection, and in healthy sex- and age-matched controls. Mononuclear cells in blood diluted 1:5 in endotoxin-free medium released TNF-alpha for 24 h. Spontaneously released TNF-alpha levels (mean +/- SEM), as measured by enzyme immunoassay in the supernatants of unstimulated 24-h cultures, were 10,941 +/- 2,591 pg/ml in children with malnutrition (N = 11) and 533 +/- 267 pg/ml in controls (N = 18) (P < 0.0001). TNF-alpha production was increased by stimulation with lipopolysaccharide (LPS), with maximal production of 67,341 +/- 16,580 pg/ml TNF-alpha in malnourished children and 25,198 +/- 2,493 pg/ml in controls (P = 0.002). In control subjects, LPS dose-dependently induced TNF-alpha production, with maximal responses obtained at 2000 ng/ml. In contrast, malnourished patients produced significantly more TNF-alpha with 0.02-200 ng/ml LPS, responded maximally at a 10-fold lower LPS concentration (200 ng/ml), and presented high-dose inhibition at 2000 ng/ml. TNF-alpha production a) was significantly influenced by LPS concentration in control subjects, but not in malnourished children, who responded strongly to very low LPS concentrations, and b) presented a significant, negative correlation (r = -0.703, P = 0.023) between spontaneous release and the LPS concentration that elicited maximal responses in malnourished patients. These findings indicate that malnourished children are not deficient in TNF-alpha production, and suggest that their cells are primed for increased TNF-alpha production.

[Differential modulation of TLR2 and TLR4-induced TNF production by murin haemorrhagic shock]. / Modulation différentielle des effets de la stimulation des récepteurs Toll-like 2 et 4 par l'état de choc hémorragique.

OBJECTIVE: To investigate the influence of haemorrhagic shock in mice on ex vivo TNF production by whole blood cells (WBC) stimulated through Toll-like receptors (TLR) 4 and 2. STUDY DESIGN AND ANIMALS: Experimental study using BALB/c male mice. METHODS: Haemorrhage (0,026+/-0,003 ml/g) by transparietal cardiac puncture under general anaesthesia. Measurement of left intraventricular pressure through a direct subcostal cardiac puncture. Possible restitution of shed blood volume (SBV) in retroorbital venous plexus, 60 minutes following haemorrhage. Lethal exsanguination 120 minutes following general anaesthesia (Control group), cardiac puncture (Sham group), blood sample (Haemorrhage group), or 60 minutes following SBV retransfusion (SBV group). Cultures (24 hours) of whole blood from the exsanguination, alone or with Escherichia coli endotoxin (LPS, TLR 4) or with heat-killed Staphylococcus aureus Cowan (SAC, TLR 2). Assessment of TNF levels in the cultures supernatant (Elisa). RESULTS: Hemorrhage (approximately 30% of calculated blood volume) resulted in arterial hypotension (-50%) which was reversed by SBV retransfusion. TNF production by LPS-stimulated WBC was reduced by haemorrhage (approximately -50%) with or without SBV retransfusion. TNF production by SAC-stimulated WBC remained unchanged. CONCLUSION: The reduction of proinflammatory cytokines production by WBC stimulated with pathogen-associated molecular patterns is not a generalized phenomenon following murin haemorrhagic shock. It depends on the used stimulus and studied signalling pathways.

Immunodepression in sepsis and SIRS assessed by ex vivo cytokine production is not a generalized phenomenon: a review.

Sepsis and non-infectious systemic inflammatory response syndrome (SIRS) are paradoxically associated with an exacerbated production of cytokines, as assessed by their presence in biological fluids, and a diminished ability of circulating leukocytes to produce cytokine upon in vitro activation. In this review, we depict that the observed cellular hyporeactivity is not a global phenomenon and that some signalling pathways are unaltered and allow the cells to respond normally to certain stimuli. Furthermore, we illustrate that during sepsis and SIRS, cells derived from tissues are either fully responsive to ex vivo stimuli or even primed, in contrast to cells derived from hematopoietic compartments (blood, spleen, etc.) which are hyporeactive. In addition to cytokine production, nuclear factor-kappa B (NF-kappa B) status within leukocytes can be used as a useful marker of hypo- or hyper-reactivity. We illustrate that the immune-depression reported in sepsis and SIRS patients, often revealed by a diminished capacity of leukocytes to respond to lipopolysaccharide, is not a generalized phenomenon and that SIRS is associated with a compartmentalized responsiveness which involves either anergic or primed cells.

Variable estimates of cytokine levels produced by commercial ELISA kits: results using international cytokine standards.

Commercially available ELISA kits now make it possible to measure cytokines in biological samples and cell culture supernatants. We have compared the levels of IL-1 beta, IL-6, IL-8 and TNF-alpha in various pathological plasma and synovial fluids, and in supernatants of human monocytes activated by lipopolysaccharide (LPS). Measurements were performed using ELISA kits from different companies. A wide variation in values was obtained when measurements were deduced from the standard curves formed with the standard provided by the manufacturers. We also performed calibration curves for all ELISA kits, using the international standards provided by the NIBSC (UK). The coefficients of variation were then significantly improved for IL-6 and IL-8 measurements but not for IL-1 beta and TNF alpha assays. However, despite this attempt to obtain uniform measurements, none of the kits gave similar values for individual samples. These results suggest that the nature of the different pairs of monoclonal antibodies employed in each ELISA does not permit comparable recognition of cytokines in samples. Further work with the various kits is required to establish whether (i) denaturation of the recognized epitope within the natural cytokine, (ii) fragmentation of the cytokine following enzymatic cleavage, (iii) depolymerization, (iv) binding of cytokines to undefined ligands, (v) variable glycosylation of the natural cytokines (vi) recognition of precursor forms, interferes with the measurements.

Cytokine production by murine cells activated by erythrogenic toxin type A superantigen of Streptococcus pyogenes.

The mode of pathogenic action of the Steptococcus pyogenes superantigen erythrogenic toxin type A (ETA) in causing toxic shock-like syndrome in humans is thought to be mediated by massive release of cytokines by patients immune cells. The cytokine-inducing capacity of ETA as an extracellular protein was compared with that of lipopolysaccharide (LPS), a component of cell wall of gram-negative bacteria. Peritoneal macrophages and splenocytes of BALB/c and C3H/HeJ mice were stimulated by ETA and LPS. Tumor necrosis factor (TNF), interleukin 3 (IL-3) and interleukin 6 (IL-6) activities in the supernatants of stimulated cells were evaluated. In contrast to LPS, ETA induced only low amounts of IL-6 and no detectable TNF activities in peritoneal macrophage supernatants. ETA-triggered BALB/c and C3H/HeJ splenocytes produced great amounts of IL-6. ETA triggered the production of IL-3 by both mice strains splenocytes in a dose dependent manner. The amounts of IL-3 in supernatants were comparable to those induced by concanavalin A. The simultaneous presence of ETA and LPS in macrophage and splenocyte cultures induced a slight enhancement above an additive value after 72-96 h. Challenge of BALB/c mice with ETA 6 h before the harvest of peritoneal macrophages led to an enhanced production of IL-6 upon stimulation with ETA as well as with LPS. Splenocytes of nude BALB/c mice did not produce IL-6 upon stimulation with ETA, whereas LPS-induced IL-6 production was similar in these mice and in their littermates. The pathogenic effect of ETA on host's immune cells could most likely be explained as a consequence of T cell activation. The results confirm also that LPS- and ETA-induced shock is mediated by different cell types.

Ex vivo T-lymphocyte derived cytokine production in SIRS patients is influenced by experimental procedures.

Ex vivo production of interleukin-2 (IL-2), IL-4, IL-5, and IL-10 by peripheral blood mononuclear cells (PBMC) was studied in 13 septic patients with infectious systemic inflammatory response syndrome (SIRS) and 13 patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) (noninfectious SIRS). We have investigated the levels of cytokines after activation by either concanavalin A (ConA), phytohemagglutinin (PHA), or anti-CD3 antibodies. In whole blood assays, ConA-induced IL-10 was significantly reduced in both groups of patients compared with healthy controls. In sepsis patients, IL-2, IL-5, and IL-10 productions by isolated PBMC were diminished on ConA-induced activation but not in response to PHA and anti-CD3; in CPB patients, only anti-CD3-induced IL-10 production was significantly reduced. Our data indicate that subtle modifications of the reactivity of circulating cells occur during infectious and noninfectious SIRS. Production of both Th1 and Th2 cytokines can be down-regulated; however, the nature of the SIRS, of the cell population, and of the activator may influence the observation.

Influence of gender and age on course of infection and cytokine responses in mice with disseminated Cryptococcus neoformans infection.

OBJECTIVE: To study the influence of gender and age on the course of infection and the cytokine response in a murine model of disseminated cryptococcosis. METHODS: The course of the infection (survival and fungal load in blood and tissues) as well as pro-inflammatory and anti-inflammatory cytokine responses in plasma and organs were compared according to gender and age in outbred mice previously infected with Cryptococcus neoformans NIH52D. RESULTS: Although survival and fungal load were similar in male and female mice, the expression of all cytokines in plasma and of tumour necrosis factor-alpha and interferon-gamma in spleen was significantly increased in female mice compared to male mice in two independent experiments. Young male mice had a significantly shortened survival, were significantly more infected and had predominant tumour necrosis factor-alpha and interferon-gamma responses in comparison with older male mice. CONCLUSION: Host factors should be taken into account when studying the immune response to experimental C. neoformans infection. Our data support epidemiological and clinical data showing differences in susceptibility to cryptococcosis according to gender and age.

Comparison of the allergenic potency of spores and mycelium of Cladosporium.

The allergenic potency of spore and mycelium extracts of Cladosporium was estimated by RAST, RAST inhibition and PCA tests. Spores contained a concentration of allergens higher than mycelia. Results of PCA tests suggested that spores contained specific allergens. However, in a comparative study of extracts from different species of Cladosporium animal and human models gave different estimates of the allergenic potency of the different species. In spite of these variations it was shown that extracts from spores of Cladosporium contained the highest amount of Cladosporium allergens.

Inhibition of lipopolysaccharide-induced monocyte interleukin 1 secretion by gangliosides.

Gangliosides known to be potent immunosuppressors are shown to inhibit the secretion of interleukin 1 (IL 1) by human monocytes stimulated with lipopolysaccharide (LPS). The inhibitory activity was observed either with mono-, di- or trisialogangliosides. The inhibition of LPS-induced IL 1 secretion was obtained in the presence of indomethacin, indicating that the ganglioside inhibitory capacity was not due to prostaglandin induction. Indeed the inhibitory activity seems to be mediated by an interaction with LPS molecules, preventing their ability to deliver a signal to monocytes for IL 1 secretion.

Presence of interleukin 3-like activity in the supernatants of lipopolysaccharide-stimulated mouse splenocytes.

An Interleukin 3 (IL 3) activity was found in the supernatants of mouse spleen cells stimulated by lipopolysaccharide (LPS). The IL 3 activity was maximum in the supernatants of 5 to 7 days cultures. IL 3 activity was assessed by the capacity of the spleen cell supernatants to allow the growth of the FDC-P2 cell line, a strictly IL 3 dependent cell line. Polymyxin B could inhibit the IL 3 induction by LPS.

IL-10 and IL-4 synergize with TNF-alpha to induce IL-1ra production by human neutrophils.

The anti-inflammatory properties of IL-4, IL-10, IL-13 and TGF-beta are associated with their ability to repress the production of pro-inflammatory cytokines and to favour the release of interleukin-1 receptor antagonist (IL-1ra). Here, we investigate their actions on activated human polymorphonuclear cells (PMN). IL-4 and TGF-beta were able to increase the production of IL-1ra, however only IL-4 were able to further increase IL-1ra production in the presence of LPS. When IL-1ra production by PMN was induced by tumour necrosis factor-alpha (TNF-alpha), IL-10 and IL-4 both amplified its release and its presence as a cell-associated form. In conclusion, IL-10 which was unable to induce IL-1ra by itself or to amplify the LPS-induced production by PMN, was able to increase its release when TNF-alpha, is the triggering signal. IL-4 was active in the different combinations tested; IL-13 and TGF-beta did not further modulate LPS- and TNF-alpha-induced IL-1ra production by PMN.

Regulation by anti-inflammatory cytokines (IL-4, IL-10, IL-13, TGFbeta)of interleukin-8 production by LPS- and/ or TNFalpha-activated human polymorphonuclear cells.

The capacity to down-regulate the production of IL-8 by LPS-activated human polymorphonuclear cells (PMN) has been demonstrated for IL-4, IL-10, and TGFbeta. We compared their relative capacities and further extended this property to IL-13. We report a great heterogeneity among individuals related to the responsiveness of PMN to the IL-4 and IL-13 inhibitory effects while their response to the IL-10 effect was homogenous. The inhibitory activities were observed at the transcriptional level. IL-8 induction by TNFalpha was, unlike its induction by LPS, resistant to the inhibitory effects of IL-10, IL-4, IL-13 and TGFbeta. Furthermore, IL-10 and IL-4 inhibitory activity were less effective when TNFalpha was acting synergistically with LPS to induce IL-8 production by PMN. LPS-induced cell-associated IL-8, detected in the PMN cultures, could be marginally inhibited by IL-4 and IL-10. Altogether, our data demonstrate that IL-13 is able to inhibit LPS-induced IL-8 production by human PMN, although IL-10 remains the most active anti-inflammatory cytokine. Despite the capacity of IL-4, IL-10, and IL-13 to limit the production of TNFalpha-induced IL-8 in a whole blood assay, none was able to inhibit this production when studying isolated human polymorphonuclear cells.

Recombinant C5a enhances interleukin 1 and tumor necrosis factor release by lipopolysaccharide-stimulated monocytes and macrophages.

Lipopolysaccharide (LPS) is a potent inducer of interleukin 1 (IL 1) synthesis and release, and of tumor necrosis factor (TNF) secretion. Many signals can enhance the LPS-induced production of these cytokines. We have previously observed that addition of low amounts of normal human serum to the culture medium enhances IL 1 production. Among serum factors, anaphylatoxins C3a and C5a and/or their desArg derivatives have been shown to enhance LPS-induced IL 1 and TNF production. However, the capacity of natural anaphylatoxins to induce by themselves the production of cytokines remains a controversial issue. We have investigated the capacity of human recombinant C5a (hrC5a) to induce IL 1 and TNF production. Despite its lack of direct triggering, hrC5a was able to act synergistically with LPS, leading to higher IL 1 and TNF release by human monocytes and mouse peritoneal macrophages. As assessed by the comitogenic assay, hrC5a increased IL 1 release, whereas cell-associated IL 1 activity was not significantly modified. Measurement by enzyme-linked immunosorbent assay of human IL 1 beta led to similar conclusions, whereas measurement of IL 1 alpha by radioimmunoassay indicated, in addition, an increase in intracellular IL 1 alpha.