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.

[Host inflammatory and anti-inflammatory response during sepsis]. / Réponse inflammatoire et anti-inflammatoire de l'hôte au cours du sepsis.

Sepsis still remains the major complication for patients admitted in intensive care units (ICU), and is responsible for numerous deaths. ICU patients admitted after sepsis, hemorrhagic shock, severe trauma, severe burns or major surgery show a systemic inflammatory response syndrome (SIRS). This syndrome is characterized by an exacerbation of inflammation, with increased levels of pro- (IL-1ß, TNFα, IL-6, IL-8) as well as anti-inflammatory (IL-10, IL-1Ra, TGFß) cytokines into their bloodstream. During sepsis, the bacteria release microbial motifs such as peptidoglycan, lipopolysaccharide (LPS) and DNA that initiate the inflammatory response, and are involved in the onset of multiple organ failure. The same microbial motifs can also be found in patients with a SIRS of non-infectious origin, following the translocation of bacteria from their digestive tract. This translocation is certainly contributing to the difficulty of discriminating between septic and SIRS patients using biological markers. Furthermore, the host response is accompanied by an alteration of the ex vivo response of circulating leukocytes, particularly monocytes. This hyporesponsiveness to LPS is associated with a decreased activation of the transcription factor NF-κB (required for the expression of pro-inflammatory cytokines) and an increased expression of negative regulators of the NF-κB pathway. However, the leukocyte hyporesponsiveness is not a global phenomenon, it depends on the type of patient, on the receptor-activator pair, on the timing, and on the cytokine.

Acute inflammation causes epithelial invasion and mucosal destruction in experimental shigellosis.

The gram-negative pathogen Shigella flexneri causes bacillary dysentery, an invasive disease of the human colonic mucosa. A major characteristic of the infectious process is the occurrence of an acute inflammatory reaction of mucosal tissues which is generally consequence of primary invasion and destruction of colonic epithelial cells by the pathogen. Confirming in vitro demonstration that S. flexneri is unable to invade the apical pole of colonic cells and that polymorphonuclear (PMN) cells may assist them in reaching the basal side of epithelial cells where they can invade, we have provided here in vivo evidence that S. flexneri enters the epithelial barrier essentially through the dome of lymphoid follicles at the early stage of infection and that subsequent invasion and destruction of the epithelium is primarily due to immigration of leukocytes, particularly PMN that destroy cohesion of the epithelial barrier. These conclusions are based on experiments carried out in infected rabbit ligated intestinal loops, with some animals treated by an anti-CD18 monoclonal antibody that blocked immigration of leukocytes into infected tissues.

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.

Monoclonal immunoglobulin A antibody directed against serotype-specific epitope of Shigella flexneri lipopolysaccharide protects against murine experimental shigellosis.

To determine the role of humoral mucosal immune response in protection against shigellosis, we have obtained a monoclonal dimeric immunoglobulin A (IgA) antibody specific for Shigella flexneri serotype 5a lipopolysaccharide (mIgA) and used a murine pulmonary infection model that mimics the lesions occurring in natural intestinal infection. Adult BALB/c mice challenged with 10(7) S. flexneri organisms developed a rapid inflammatory response characterized by polymorphonuclear cell infiltration around and within the bronchi and strong systemic interleukin 6 response. Implantation of hybridoma cells in the back of mice, resulting in the development of a myeloma tumor producing mIgA in the serum and subsequently secretory mIgA in local secretions, or direct intranasal administration of these antibodies, protected the animals against subsequent intranasal challenge with S. flexneri serotype 5a. Absence of histopathological lesion and significant decrease in bacterial load of the lungs and of systemic interleukin 6 response were the three major criteria of protection. This protection was shown to be serotype-specific and dependent on local concentration of mIgA. These data demonstrate that mucosal antibodies directed against a single polysaccharidic surface epitope of Shigella can protect against the disease.

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.

Role of interleukin-1 in the pathogenesis of experimental shigellosis.

The effect of human recombinant interleukin-1 receptor antagonist on intestinal inflammation, tissue destruction, and bacterial invasion during experimental shigellosis caused by Shigella flexneri was studied in the rabbit-ligated loop infection model. Intravenous infusion of the inhibitor at a dose of 2 mg/kg per h, was initiated 30 min before intestinal loops were ligated and infected, and continued during the 8-h period of infection. The animals treated with IL-1 receptor antagonist showed a striking decrease in inflammation, destruction, and bacterial invasion of their tissues, both at the level of the villous intestine and Peyer's patches. This is conclusive evidence that interleukin-1 plays a critical role in the pathogenesis of shigellosis. This proinflammatory cytokine is here proposed as a major trigger of the inflammatory reaction which is characteristic of this invasive disease of the intestine, due to the particular interaction existing between S. flexneri and macrophages.

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.

The nonspecific nature of endotoxin tolerance.

Re-exposure of organisms or cells to endotoxin after a previous challenge is not accompanied by the profound metabolic changes that are induced by the first encounter with endotoxin. Endotoxin tolerance is not specific to the action of lipopolysaccharide, and crossreactivity with other exogenous stimuli occurs. Various cytokines can mimic the effects of endotoxin in vivo and/or in vitro.

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.

Long-term-impaired expression of nuclear factor-kappa B and I kappa B alpha in peripheral blood mononuclear cells of trauma patients.

Nuclear factor (NF)-kappa B expression and dimer characteristics were studied in peripheral blood mononuclear cells (PBMCs) of major-trauma patients and healthy controls. Analysis of PBMCs on days 1, 3, 5, and 10 after trauma revealed that expression of both p65p50 heterodimers and p50p50 homodimers was significantly reduced compared with that in controls. In vitro lipopolysaccharide (LPS) stimulation of PBMCs induced NF-kappa B translocation. However, throughout the survey, p65p50 activation remained significantly lower in trauma patients than in controls. After LPS stimulation in vitro, the p65p50/p50p50 ratio was significantly lower in PBMCs from trauma patients than from healthy controls. The ex vivo expression of I kappa B alpha was higher in PBMCs of controls than of trauma patients. LPS did not induce I kappa B expression in PBMCs from trauma patients, but strong induction was obtained with staphylococci, suggesting that this defect is not universal and depends on the nature of the activating signal. Although no direct correlation was found between levels of interleukin-10 or transforming growth factor-beta and NF-kappa B, these immunosuppressive cytokines were significantly elevated in trauma patients by 10 days after admission. The long-term low-basal and LPS-induced nuclear translocation of NF-kappa B recalled long-term immunoparalysis observed in patients with severe inflammatory stress such as trauma.

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.

Polymyxin-B inhibition of LPS-induced interleukin-1 secretion by human monocytes is dependent upon the LPS origin.

Polymyxin-B (PMB) is an antibiotic known to inhibit various biological activities induced by lipopolysaccharides (LPS). We have investigated the ability of PMB to inhibit LPS-induced interleukin-1 (IL-1) secretion by human monocytes in vitro. Interleukin-1 was assayed by the conventional comitogenic assay using mice thymocytes. Our data demonstrate that PMB (1-2 micrograms/assay)-mediated inhibition of LPS-induced IL-1 secretion depends on the origin of the LPS. Interleukin-1 secretions induced by Escherichia coli and Acinetobacter calcoaceticus LPSs, when used at 1 microgram/assay were completely inhibited by PMB, whereas those induced by Neisseria gonorrheae, Neisseria meningitidis, Bordetella pertussis, and Salmonella enteritidis LPSs were unaffected. Neisseria meningitidis, the most potent IL-1 inducer tested could be inhibited by PMB only at concns below 5 ng/assay; when the assay was performed in the presence of serum (0.2%) PMB could not completely inhibit Neisseria meningitidis LPS-induced IL-1 secretion at LPS doses as low as 100 pg/assay. Polymyxin B itself, at doses greater than 50 micrograms/assay, stimulated IL-1 secretion and acted synergistically with LPS to induce IL-1 secretion when used at 10 micrograms/assay. Potential relevance of Lipid A-mediated IL-1 secretion and the use of PMB to detect endotoxin contamination is discussed.

Interleukin-1 induction by lipopolysaccharides: structural requirements of the 3-deoxy-D-manno-2-octulosonic acid (KDO).

We previously showed the importance of the 3-deoxy-D-manno-2-octulosonic acid (KDO) residue in endotoxins (lipopolysaccharides, LPS) for the induction of the synthesis and release of interleukin-1 (IL-1) by human monocytes. We further investigated the effect of some structural variations within the KDO molecule on IL-1 production induced by LPS. Deamination of Bordetella pertussis LPS, followed by mild anhydrous acidic methanolysis released a hexasaccharide (fragment B'), which had a terminal methyl ketoside KDO residue with a methyl-esterified carboxyl group. This fragment was unable to induce IL-1 production by human monocytes. Fragment B' could be converted into an active hexasaccharide by de-esterification (fragment B-OMe), but not by reduction of the methyl ester group. The KDO residues in the LPS of some bacterial species have been shown to be phosphorylated and we observed that these LPS were weak IL-1 inducers. Phosphorylated KDO present in Vibrio cholerae and B. pertussis LPS respond poorly in the thiobarbiturate assay (specific for KDO). However, if these LPS were dephosphorylated with aqueous hydrofluoric acid (HF) their KDO response in this assay was increased 5.4- to 2.6-fold, respectively. In parallel, the HF-treated LPS were more potent IL-1 inducers than untreated endotoxins. These data confirm that the KDO residue(s) present in all endotoxins play(s) a major role in the signal(s) leading to IL-1 production by human monocytes, and show that IL-1 induction by LPS (1) requires a free carboxyl group in the KDO and (2) is correlated with the degree of substitution of the KDO.

Interleukin 1 secretion by human monocytes stimulated by the isolated polysaccharide region of the Bordetella pertussis endotoxin.

The isolated polysaccharide chain, PS-1, of the Bordetella pertussis endotoxin was examined by isoelectric focusing, SDS-polyacrylamide gel electrophoresis and gel filtration for heterogeneity and for possible contamination by the parent endotoxin. This polysaccharide, previously found to be a very potent, macrophage-dependent, polyclonal B-cell activator and to mediate the specific binding of the endotoxin to macrophages, stimulated the interleukin 1 (IL 1) secretion by human monocytes; its potency was similar to that measured for the endotoxin. It was concluded that endotoxin-induced IL 1 production may be initiated by the interaction of the polysaccharide chain of the B. pertussis endotoxin and a specific structure present on macrophages.

[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.

Purification of rabbit B spleen cells by removal of adherent and of T cells.

B lymphocytes from the rabbit spleen were freed of T cells by removal of cells which formed rosettes with papain-treated rabbit erythrocytes. Additional purification could be achieved if fractionation by rosette removal was preceded by removal with a magnet of cells which adhered to or ingested poly L-lysine coated iron core particles. Cell yield and purification were assessed by complement mediated cytotoxic kill of B and T cells with antibody directed against RABELA and RTLA, respectively. Other criteria depended on determination of the number of Fc receptor bearing cells and of thymidine uptake by cells which were stimulated with concanavalin A, PHA or with antibody directed against the allotypic specificity of receptor Ig light chains. Purified preparations of B cells were obtained in a yield of about 20% of the B cells in the original spleen and contained less than 10% of cells which were not B cells. This method allows purification which does not interfere with the membrane of the isolated cells.