IL-10 and toll-like receptor-4 polymorphisms and the in vivo and ex vivo response to endotoxin

To determine to what extent lipopolysaccharide-induced IL-10 production capacity is determined by polymorphisms in toll-like receptor-4 (TLR4) and the IL-10 promoter region, we measured in vivo IL-10 and TNF-alpha production in patients undergoing elective cardiopulmonary bypass surgery, a major surgical trauma associated with ischemia-reperfusion injury that triggers an endotoxemia and profound inflammatory response in most patients. Ex vivo the IL-10 and TNF-alpha production was measured in a whole blood stimulation assay, using 3 LPS concentrations. Positive correlations were found between TNF-alpha and IL-10 production ex vivo, upon stimulation with each of the LPS concentrations. Also, the estimated TNF-alpha and IL-10 EC50, and TNF-alpha(max) and IL-10max were positively correlated (r = 0.203; p = 0.023 and r = 0.287; p = 0.001, respectively), indicating that these parameters describing LPS sensitivity and maximal production capacity, respectively, can be estimated by measuring either TNF-alpha or IL-10. Interleukin-10 concentrations in patients experiencing endotoxemia in vivo negatively correlated with the IL-10 levels produced upon stimulation with 1000 ng/mL LPS as well as the estimated IL-10max ex vivo. In vivo, a positive correlation between the TNF-alpha concentration at time-point 2 and the IL-10 concentration at time-point 3 was found, consistent with an important contribution of the magnitude of TNF-alpha release upon the subsequent IL-10 production. Carriers of the IL-10 promoter -1330G, -1082A, -819T, -592A (GATA) haplotype had lower IL-10 production ex vivo upon stimulation with 10 and 100 ng/mL LPS and higher EC50 values (the estimated LPS concentration at which 50% of the maximal IL-10 response is reached) as compared to carriers of the other haplotypes combined, indicating decreased LPS sensitivity ex vivo. These individuals did not differ from the others in interleukin-10 production capacity upon stimulation with a high LPS concentration (i.e., 1000 ng/mL) and the estimated IL-10(max) values, were similar, indicating unimpaired maximal IL-10 production capacity ex vivo. Carriers of the IL-10 promoter AGCC haplotype had lower EC50 values as compared to carriers of the other haplotypes combined, indicating increased LPS sensitivity ex vivo. In accordance with this finding, carriers of the AGCC haplotype had higher circulating IL-10 levels in vivo. The common TLR4 polymorphisms (Asp299Gly and Thr399Ile) were associated with slightly higher IL-10 production capacity ex vivo and in vivo, however, this was not statistically significant. Our results indicate that polymorphisms in the proximal IL-10 promoter region are associated with in vivo and ex vivo LPS sensitivity. The contribution to the inter-individual variation, however, is limited since the variation between individuals in LPS sensitivity and IL-10 production capacity can only partly be attributed to these IL-10 promoter polymorphisms.     

Intrinsic capacity of monocytes to produce cytokines ex vivo in patients with acute lymphoblastic leukaemia

Monocytic cytokine profiles of fifteen children with acute lymphoblastic leukaemia (ALL) were included to determine whether malignancy per se contributes to impaired cytokine profiles in vivo and ex vivo. The ex vivo tumour necrosis factor-alpha (TNF-alpha) and interleukin 1beta (IL-1beta) production was positively correlated with the monocyte number and with the number of intracellular TNF-alpha or IL-1beta positive cells in lipopolysaccharide (LPS)-stimulated MNC cultures. The mean ex vivo TNF-alpha and IL-1beta production per 1x10(4)monocytes in these cultures was not significantly different in children at diagnosis of ALL, at remission or in controls. High IL-10 plasma levels at diagnosis of ALL had no effect on the ex vivo TNF-alpha and IL-1beta production of monocytes in LPS stimulated MNC cultures. These results show that monocytes of ALL patients have a normal intrinsic capacity to produce cytokines ex vivo. However, the decreased monocyte number is responsible for the lower TNF-alpha and IL-1beta concentrations ex vivo upon LPS stimulation.     

Immune status evaluation of patients with chronic heart failure

Chronic heart failure (CHF) may be considered a state of immune activation and persistent inflammation expressed by increased circulating levels of pro- and anti-inflammatory cytokines. The purpose of the study was to investigate the immune status in patients with CHF compared to normal individuals. We measured serum cytokine levels as well as cytokine production after ex vivo LPS stimulation of whole blood taken from 14 patients with CHF and 14 healthy volunteers. We used 500 pg/ml of LPS for an incubation period of 4h to stimulate 100 microL of whole blood. Patients with CHF had significantly higher levels of TNF-RI, and TNF-RII in serum compared to normal individuals. TNF-alpha, IL-6, and IL-10 did not differ significantly. After LPS stimulation, patients with CHF had significantly higher levels of TNF-alpha and IL-10, and significantly lower IL-6 levels compared to normal individuals. TNF-alpha receptors did not differ significantly. Patients with CHF may be found in a pro- as well as an anti-inflammatory state. They also do not develop endotoxin tolerance in an ex vivo laboratory model using whole blood stimulated with LPS. They may have increased TNF-alpha and IL-10 production after LPS stimulation of whole blood, which may contribute to a worsening of heart function, more severe disease presentation and a worse outcome during infections.     

Reduced glucocorticoid sensitivity of monocyte interleukin-6 release in male employees with high plasma levels of tumor necrosis factor-alpha

Cytokine production by monocytes plays a key role in atherosclerosis. In vitro, preincubation of whole blood with tumor necrosis factor (TNF)-alpha regulates interleukin (IL)-6 release from monocytes stimulated with lipopolysaccharide (LPS). We investigated whether plasma levels of TNF-alpha would also relate to LPS-stimulated monocyte IL-6 production and the inhibitory effect of a glucocorticoid on this process. 224 middle-aged men were assigned to three groups according to tertiles of plasma levels of TNF-alpha. Subjects in the highest tertile (high TNF-alpha, n = 75) were compared to those in the lowest (low TNF-alpha, n = 74) and medium tertile (medium TNF-alpha, n = 75), respectively. In vitro monocyte IL-6 release following lipopolysaccharide (LPS)-stimulation was assessed with and without coincubation with incremental doses of dexamethasone. Monocyte glucocorticoid sensitivity was defined as the dexamethasone concentration inhibiting IL-6 release by 50%. Subjects with high TNF-alpha showed more IL-6 release after LPS-stimulation than those with low TNF-alpha (p =.011). Monocyte glucocorticoid sensitivity was lower in subjects with high levels of TNF-alpha than in subjects with low (p =.014) and with medium (p =.044) levels of TNF-alpha. Results held significance when a set of classic cardiovascular risk factors was controlled for. Our findings suggest that elevated plasma levels of TNF-alpha might enhance LPS-stimulated IL-6 release from circulating monocytes. Such a mechanism might contribute to exaggerated monocyte cytokine release in vivo to any LPS-like danger signal such as related to an infection or cellular stress thereby promoting atherosclerosis.     

Low- versus high-baseline epinephrine output shapes opposite innate cytokine profiles: presence of Lewis- and Fischer-like neurohormonal immune phenotypes in humans?

Immunogenetic mechanisms operating within the immune system are known to influence cytokine profiles and disease susceptibility. Yet the role of the individual's neurohormonal background in these processes remains undefined. Hormonal imbalances are documented in immune-related diseases, but it is unclear whether this represents a secondary phenomenon or a primary "defect" related to specific neurohormonal immune phenotype(s). We report that in a large subpopulation of healthy humans the baseline epinephrine output (but not cortisol and sex steroid hormones) correlated inversely with proinflammatory and positively with anti-inflammatory cytokine production. Thus, low vs high epinephrine excretors had a 2- to 5-fold higher TNF-alpha and IL-12 production but 2-fold lower IL-10 production induced by LPS ex vivo. In alternative settings, we found low baseline levels and profoundly blunted stress-induced epinephrine responses but high TNF-alpha levels in Lewis vs Fischer inbred rats. Additionally, isoproterenol, a beta adrenoreceptor agonist suppressed LPS-induced TNF-alpha production, with more pronounced effect in Lewis than in Fischer rats. In human monocytes, epinephrine and the beta(2) adrenoreceptor agonist fenoterol potently inhibited LPS-induced TNF-alpha and IL-12, but stimulated IL-10 production. The order of potency for hormones able to inhibit IL-12 production ex vivo was: epinephrine > norepinephrine > or = 1,25-(OH)(2) vitamin D(3) > hydrocortisone. This indicates that baseline epinephrine conditions cytokine responsiveness and through this mechanism intrinsic hypo- or hyperactive adrenal medullas in some individuals may shape opposite cytokine profiles. Since Lewis and Fischer rats have opposite susceptibility to experimental immunological diseases, this suggests that the parallel human phenotypes could be linked to differing responsiveness and susceptibility to infections and immune/inflammatory-related conditions.     

Lethality during continuous anthrax lethal toxin infusion is associated with circulatory shock but not inflammatory cytokine or nitric oxide release in rats

Although circulatory shock related to lethal toxin (LeTx) may play a primary role in lethality due to Bacillus anthracis infection, its mechanisms are unclear. We investigated whether LeTx-induced shock is associated with inflammatory cytokine and nitric oxide (NO) release. Sprague-Dawley rats with central venous and arterial catheters received 24-h infusions of LeTx (lethal factor 100 microg/kg; protective antigen 200 microg/kg) that produced death beginning at 9 h and a 7-day mortality rate of 53%. By 9 h, mean arterial blood pressure, heart rate, pH, and base excess were decreased and lactate and hemoglobin levels were increased in LeTx nonsurvivors compared with LeTx survivors and controls (diluent only) (P < or = 0.05 for each comparing the 3 groups). Despite these changes, arterial oxygen and circulating leukocytes and platelets were not decreased and TNF-alpha, IL-beta, IL-6, and IL-10 levels were not increased comparing either LeTx nonsurvivors or survivors to controls. Nitrate/nitrite levels and tissue histology also did not differ comparing LeTx animals and controls. In additional experiments, although 24-h infusions of LeTx and Escherichia coli LPS produced similar mortality rates (54 and 56%, respectively) and times to death (13.2 +/- 0.8 vs. 11.0 +/- 1.7 h, respectively) compared with controls, only LPS reduced circulating leukocytes, platelets, and IL-2 levels and increased TNF-alpha, IL-1 alpha and -1 beta, IL-6, IL-10, interferon-gamma, granulocyte macrophage-colony stimulating factor, RANTES, migratory inhibitory protein-1 alpha, -2, and -3, and monocyte chemotactic protein-1, as well as nitrate/nitrite levels (all P < or = 0.05 for the effects of LPS). Thus, in contrast to LPS, excessive inflammatory cytokine and NO release does not appear to contribute to the circulatory shock and lethality occurring with LeTx in this at model. Although therapies to modulate these host mediators may be applicable fo shock caused by LPS or other bacterial toxins, they may not with LeTx.     

Flow cytometrical determination of regulatory cytokines (IL-10, IL-12) and circulating dendritic cell cytokines in allergic asthmatic children

Interleukin (IL)-10 and IL-12 have been suggested to be key regulators in the pathogenesis of allergic asthma. Several of the secretion products of dendritic cells (DC), such as IL-12, IL-10, IL-1beta and TNF-alpha, are considered to play a role in allergic asthma. This study compares the production of IL-10 and IL-12 in allergic asthmatic children (n = 17) and controls (n = 14) by measuring their extracellular secretion in whole blood samples after stimulation, using a microsphere-based immunoassay. Additionally, we assessed intracellular production of IL-1beta, TNF-alpha, IL-12 and IL-10 by circulating DC in stimulated whole blood samples of asthmatic and healthy children. The concentration of IL-10 in the supernatants of LPS-stimulated whole blood was significantly lower in allergic asthmatic children as compared to healthy children (463 (207-768) vs 881 (364-2626) pg/mL; p = 0.005). When a combined LPS and IFN-gamma stimulation was used, IL-10 production decreased significantly as compared to LPS alone, especially in healthy children. Consequently, no difference in IL-10 production after LPS/IFN-gamma stimulation was found between healthy and allergic children. In contrast to isolated LPS stimulation, stimulation with LPS/IFN-gamma induced higher IL-12 production; allergic asthmatic children showed a significantly lower IL-12 secretion after LPS/IFN-gamma stimulation as compared to healthy children (20 (5-247) vs 208 (7-775) pg/mL; p=0.03). Moreover, the number of IL-12 producing CD11c-positive DC (DC1) tended to be lower in asthmatic children compared to healthy children (0.05 (0.00-0.45) vs 0.27 (0.00-0.83) 10(6)/L) and correlated with the extracellular release of IL-12 in asthmatic children (r = 0.65; p = 0.016). The number of IL-1beta and TNF-alpha producing CD11c-positive DC (DC1) was comparable between healthy and asthmatic children. We hypothesize that the decreased production of IL-10 and IL-12 is responsible for Th2 polarized responses in allergic asthmatic children.     

Tumour tissue microenvironment can inhibit dendritic cell maturation in colorectal cancer

Inflammatory mediators in the tumour microenvironment promote tumour growth, vascular development and enable evasion of anti-tumour immune responses, by disabling infiltrating dendritic cells. However, the constituents of the tumour microenvironment that directly influence dendritic cell maturation and function are not well characterised. Our aim was to identify tumour-associated inflammatory mediators which influence the function of dendritic cells. Tumour conditioned media obtained from cultured colorectal tumour explant tissue contained high levels of the chemokines CCL2, CXCL1, CXCL5 in addition to VEGF. Pre-treatment of monocyte derived dendritic cells with this tumour conditioned media inhibited the up-regulation of CD86, CD83, CD54 and HLA-DR in response to LPS, enhancing IL-10 while reducing IL-12p70 secretion. We examined if specific individual components of the tumour conditioned media (CCL2, CXCL1, CXCL5) could modulate dendritic cell maturation or cytokine secretion in response to LPS. VEGF was also assessed as it has a suppressive effect on dendritic cell maturation. Pre-treatment of immature dendritic cells with VEGF inhibited LPS induced upregulation of CD80 and CD54, while CXCL1 inhibited HLA-DR. Interestingly, treatment of dendritic cells with CCL2, CXCL1, CXCL5 or VEGF significantly suppressed their ability to secrete IL-12p70 in response to LPS. In addition, dendritic cells treated with a combination of CXCL1 and VEGF secreted less IL-12p70 in response to LPS compared to pre-treatment with either cytokine alone. In conclusion, tumour conditioned media strongly influences dendritic cell maturation and function.     

Hormonal and metabolic responses to intracarotid and intrajugular infusion of beta-endorphin in normal dogs

The hormonal and metabolic responses of beta-endorphin infused cephalad into the carotid artery, or via the jugular vein, were examined in 10 normal dogs. The intracarotid administration of beta-endorphin resulted in significant increases in plasma glucagon, adrenocorticotropin, and cortisol levels. Hepatic glucose production increased only transiently and there was no significant change in glucose disappearance or plasma glucose concentrations. Infusion of beta-endorphin in the jugular vein gave rise to significant increases in glucagon and cortisol levels and to a transient increase in plasma epinephrine. Although no significant changes in glucose kinetics could be demonstrated, there was a slight transient decrease in plasma glucose concentrations. In conclusion, both intracarotid and intrajugular infusions of beta-endorphin stimulated glucagon secretion independent of circulating catecholamines, and increased cortisol release, probably through activation of the pituitary-adrenocortical axis.     

TNF-alpha promoter, Nod2 and toll-like receptor-4 polymorphisms and the in vivo and ex vivo response to endotoxin

Humans exhibit substantial inter-individual differences in TNF-alpha production upon endotoxin stimulation. To determine to what extent the lipopolysaccharide-induced TNF-alpha production capacity in vivo and ex vivo is determined by polymorphisms in toll-like receptor-4 (TLR4), the TNF-alpha promoter region and Nod2, we screened for two TLR4 polymorphisms, a Nod2 polymorphism and the TNF-alpha promoter polymorphisms. We measured the perioperative endotoxemia and TNF-alpha production and the TNF-alpha production capacity of each patient in a whole-blood stimulation assay using blood drawn before anesthesia, using various LPS concentrations, in patients undergoing elective cardiac surgery. This operation represents a major surgical trauma associated with ischemia-reperfusion injury and triggers an endotoxemia and profound inflammatory response. In vivo TNF-alpha production was positively correlated with the level of endotoxemia after aortic declamping; thus TNF-alpha levels were higher in patients having endotoxemia compared to patients without endotoxemia. This correlation was observed in patients with any of the genotypes studied, and did not differ between the various genotypes. In vivo TNF-alpha levels correlated best with those ex vivo after stimulation with 1000 ng/mL LPS, and the estimated maximal TNF-alpha release capacity. Subjects with the wild-type TLR4 gene had similar levels of TNF-alpha upon LPS stimulation ex vivo as compared with patients carrying Asp299Gly and/or the Thr399Ile TLR4 polymorphism. Our results indicate that polymorphisms in the TLR4 receptor, Nod2 and TNF-alpha promoter region are not strongly associated with in vivo and ex vivo TNF-alpha production capacity upon endotoxin stimulation. This suggests that in this model of natural LPS release, the variation between individuals in TNF-alpha release can only modestly be determined by genetic background (TNF-alpha promoter, Nod2 and TLR4) of the individual.     

Exaggerated human monocyte IL-10 concomitant to minimal TNF-alpha induction by heat-shock protein 27 (Hsp27) suggests Hsp27 is primarily an antiinflammatory stimulus

Unlike more well-studied large heat shock proteins (hsp) that induce both T cell antiinflammatory (IL-10, IL-4) and macrophage proinflammatory (TNF-alpha, IL-15, IL-12) cytokines, hsp27, a small hsp, has been primarily identified as a substrate of mitogen-activated protein kinase-activated protein kinase-2 involved in the p38 signaling pathway and activated during monocyte IL-10 production. Hsp27 can also act as an endogenous protein circulating in the serum of breast cancer patients and a protein whose induction correlates to protection from LPS shock. However, the cytokine-stimulating properties of hsp27 have been unexplored. In this study, exogenous hsp27 is demonstrated for the first time as a potent activator of human monocyte IL-10 production, but only a modest inducer of TNF-alpha. Although exogenous hsp27 stimulation activated all three monocyte mitogen-activated protein kinase pathways (extracellular signal-related kinase (ERK) 1/2, c-Jun N-terminal kinase, and p38), only p38 activation was sustained and required for hsp27 induction of monocyte IL-10, while both ERK 1/2 and p38 activation were required for induction of TNF-alpha when using the p38 inhibitor SB203580 or the ERK inhibitor PD98059. Hsp27's transient activation of the c-Jun N-terminal kinase pathway, which can down-regulate IL-10, may contribute to its potent IL-10 induction. Hsp27's ERK 1/2 activation was also less sustained than activation by stimuli like LPS, possibly contributing to its modest TNF-alpha induction. The failure of either PD98059 or anti-TNF-alpha Ab to substantially inhibit IL-10 induction implied that hsp27 induces IL-10 via activation of p38 signaling independently of TNF-alpha activation and may be predominantly an antiinflammatory monokine stimulus.     

Continuous treatment with recombinant Mycobacterium tuberculosis CFP-10-ESAT-6 protein activated human monocyte while deactivated LPS-stimulated macrophage

Influence of the recombinant culture filtered protein 10 (CFP-10) and early-secreted antigenic target 6kDa protein (ESAT-6) (r-CFP-10-ESAT-6, rCE) of Mycobacterium tuberculosis (Mtb) on human monocyte and macrophage activation was investigated using human monocyte, monocyte like THP-1 cell line and monocyte derived macrophage (MDM). rCE solely enhanced TNF-alpha release from human monocytes and THP-1 cells in a dose- and time-dependent manner. rCE enhanced expression of CD80 and CD40, it also synergized with IFN-gamma in induction of TNF-alpha production and HLA-DR expression. Pharmacological agents that selectively inhibit mitogen activated protein kinase activation markedly suppressed rCE-induced TNF- alpha release. However, continuous presence of rCE (>72h) during monocyte to macrophage differentiation inhibited macrophage response to LPS stimulation. Collectively, these data suggest that rCE might have differential influence on monocyte and macrophage activation, which might be correlated with Mtb immune evasion.     

Changes in plasma IL-1beta, TNF-alpha and IL-6 after total hip replacement surgery in general or regional anaesthesia

Different anaesthetic methods influence the neuro-immuno-endocrine biologic responses to surgery and may thus possibly interfere with the postoperative course and development of complications. The neuroendocrine system is closely related to the cytokine network. In this study, the effects of general anaesthesia (n=6) and regional spinal/epidural anaesthesia (n=6) on the cytokine response (IL-1beta, TNFalpha, IL-6) to uncemented total hip replacement surgery were evaluated. The postoperative clinical course was uneventful in every case. In both groups, only very low values of plasma IL-beta were measured perioperatively, whereas plasma IL-6 increased postoperatively with peak values 4 h after surgery. The changes in plasma TNF-alpha were not significant. No significant differences in plasma TNF-alpha or IL-6 were found between patients operated in general or in regional anaesthesia. This suggests minor influence of plasma cytokines on the possible beneficial effects of regional anaesthesia on the clinical course after surgery in low risk patients. There were slightly higher TNF-alpha and IL-6 levels after the operation and significantly lower cortisol levels during the operation in the regional anaesthesia group compared to the general anaesthesia group, giving rise to a significant inverse correlation between peak values of IL-6 and peak values of cortisol. This supports the theory that after surgery the inhibitory effect of cortisol on monocyte cytokine production overrides adrenergic stimulation.     

Interleukin-6 and interleukin-15 are selectively regulated by lipopolysaccharide and interferon-gamma in primary pig adipocytes

3T3-L1 adipocytes express the lipopolysaccharide (LPS) receptor and respond to direct stimulation with the antigen by increasing the expression of inflammatory mediators. Activation of this receptor by its ligand in the macrophage causes the activation and translocation of nuclear factor-kappaB (NF-kappaB) to the nucleus where it regulates the expression of proinflammatory cytokines and other target genes. We investigated whether LPS could stimulate NF-kappaB translocation in primary pig adipocytes and regulate the expression and secretion of TNF-alpha and IL-6. LPS clearly induced the nuclear translocation of NF-kappaB and also upregulated (P < 0.05) the mRNA expression and secretion of IL-6 into the culture medium. An induction of TNF-alpha expression by LPS was not detected, but with extended incubation (8 h), there was a modest increase (P < 0.09) in the media concentration of this cytokine. Inhibition of either ERK1/2, PKC, or the inhibitory G protein (Gi) with U-0126, bisindolylmaleimide HCl, and pertussis toxin, respectively, blocked (P < 0.05) the increase in IL-6 expression caused by LPS. Because LPS administration in vivo increases circulating concentrations of IFN-gamma, and because this cytokine also regulates multiple immune modulators in the adipocyte, we also determined whether IFN-gamma regulates cytokine expression in primary adipocytes. Although the expression of IL-6 and TNF-alpha was unresponsive to IFN-gamma, the expression of IL-15 was markedly upregulated (P < 0.01). Furthermore, the induction of IL-15 expression by IFN-gamma was blocked by inhibition of PKC. These data indicate that NF-kappaB is responsive to LPS in the adipocyte and also identify key mediators of LPS-induced IL-6 expression. In addition, we provide novel evidence that IFN-gamma targets the adipocyte to induce IL-15 expression, thus indicating a possible role for the adipocyte in the regulation of T-cell function and muscle metabolism during the innate immune response.     

TNF-alpha drives human CD14+ monocytes to differentiate into CD70+ dendritic cells evoking Th1 and Th17 responses

Many mechanisms involving TNF-alpha, Th1 responses, and Th17 responses are implicated in chronic inflammatory autoimmune disease. Recently, the clinical impact of anti-TNF therapy on disease progression has resulted in re-evaluation of the central role of this cytokine and engendered novel concept of TNF-dependent immunity. However, the overall relationship of TNF-alpha to pathogenesis is unclear. Here, we demonstrate a TNF-dependent differentiation pathway of dendritic cells (DC) evoking Th1 and Th17 responses. CD14(+) monocytes cultured in the presence of TNF-alpha and GM-CSF converted to CD14(+) CD1a(low) adherent cells with little capacity to stimulate T cells. On stimulation by LPS, however, they produced high levels of TNF-alpha, matrix metalloproteinase (MMP)-9, and IL-23 and differentiated either into mature DC or activated macrophages (M phi). The mature DC (CD83(+) CD70(+) HLA-DR (high) CD14(low)) expressed high levels of mRNA for IL-6, IL-15, and IL-23, induced naive CD4 T cells to produce IFN-gamma and TNF-alpha, and stimulated resting CD4 T cells to secret IL-17. Intriguingly, TNF-alpha added to the monocyte culture medium determined the magnitude of LPS-induced maturation and the functions of the derived DC. In contrast, the M phi (CD14(high)CD70(+)CD83(-)HLA-DR(-)) produced large amounts of MMP-9 and TNF-alpha without exogenous TNF stimulation. These results suggest that the TNF priming of monocytes controls Th1 and Th17 responses induced by mature DC, but not inflammation induced by activated M phi. Therefore, additional stimulation of monocytes with TNF-alpha may facilitate TNF-dependent adaptive immunity together with GM-CSF-stimulated M phi-mediated innate immunity.     

Effect of catecholamines on intracellular cytokine synthesis in human monocytes.

Proinflammatory cytokines produced by monocytes, like Interleukin-6 (IL-6), Interleukin-8 (IL-8), and tumor necrosis factor (TNF-alpha) are known for their pivotal role in the initiation of the inflammatory response following cardiopulmonary bypass (CPB). Catecholamines like epinephrine (Epi) and norepinephrine (Nor) are often necessary to stabilize the cardiac function in the early postoperative period and may influence the cytokine expression in monocytes. In this study we investigated the effects of Epi and Nor on IL-6, IL-8 and TNF-alpha expression in human monocytes stimulated with lipopolysaccharide (LPS) in whole blood, analyzed intracellularly by flow cytometry. Kinetics of intracellular proinflammatory cytokine production and LPS ED(50) were obtained. To simulate different stages of inflammation in vivo, varying concentrations of LPS (0.2 ng/ml, 1 ng/ml and 10 ng/ml) were used for stimulation. After a stimulation with LPS TNF-alpha was the first produced cytokine, followed by IL-8 and IL-6. All cytokines peaked from 3 h to 6 h. Epi and Nor had comparable effects on the expression of IL-6, IL-8 and TNF-a in monocytes. Both inhibited IL-6 and TNF-alpha expression in a concentration dependent manner whereas IL-8 expression remained unchanged. We conclude that monocytes are targets for Epi and Nor concerning their cytokine expression. The inhibiting effects of Nor and Epi were almost identical for all cytokines. Cytokine expression was affected most at low LPS concentrations.     

Evaluation of tumor necrosis factor alpha production in ex vivo short term cultured whole blood from women with polycystic ovary syndrome

In mammals, the pleiotropic biological functions of tumor necrosis factor alpha (TNF-alpha) may include important effects on human reproductive physiology. Thus, chronic anovulation, oligo or amenorrhea, infertility, hyperandrogenism, obesity, insulin resistance and increased TNFalpha serum levels have been observed in women affected by polycystic ovary syndrome (PCOS). Whole blood short - term cell cultures (WBSC) are simple systems where the capacity to produce TNF-alpha by circulating leukocytes, mainly of the macrophage/monocyte lineage, can be accurately quantified. Given the relevance of monocytes/macrophages in the production of TNF-alpha, in this study, in a control-case approach, WBSC from women with PCOS were analyzed in their basal and lipolysaccharide (LPS)- stimulated capacity to produce the cytokine. These measurements did not correlate with the increased serum levels of the cytokine and the normal levels of cortisol, found in PCOS women. Increased serum TNF-alpha levels in PCOS women correlated positively with body mass index and negatively with insulin sensitivity. In spite of the increased serum TNF-alpha levels in PCOS women, basal and LPS stimulated production of the cytokine, by the ex vivo WBSC from these patients, were within normal values.     

Methylenedioxymethamphetamine (MDMA; Ecstasy) suppresses IL-1beta and TNF-alpha secretion following an in vivo lipopolysaccharide challenge

In this study we examined the effects of methylenedioxymethamphetamine (MDMA) administration on responsiveness to an in vivo immune challenge with lipopolysaccharide (LPS; 100 microg/kg; i.p.). LPS produced an increase in circulating IL-1beta and TNF-alpha in control animals. MDMA (20 mg/kg; i.p.) significantly impaired LPS-induced IL-1beta and TNF-alpha secretion. The suppressive effect of MDMA on IL-1beta secretion was transient and returned to control levels within 3 hours of administration. In contrast, the MDMA-induced suppression of TNF-alpha secretion was evident for up to 12 hours following administration. In a second study we examined the effect of co-administration of MDMA (5, 10 and 20 mg/kg; i.p.) on LPS-induced IL-1beta and TNF-alpha secretion, and demonstrated that all three doses potently suppressed LPS-induced TNF-alpha secretion, but only MDMA 10 and 20 mg/kg suppressed LPS-induced IL-1beta secretion. In addition, serum MDMA concentrations displayed a dose-dependent increase, with the concentrations achieved following administration of 5 and 10 mg/kg being in the range reported in human MDMA abusers. In order to examine the possibility that the suppressive effect of MDMA on IL-1beta and TNF-alpha could be due to a direct effect of the drug on immune cells, the effect of in vitro exposure to MDMA on IL-1beta and TNF-alpha production in LPS-stimulated diluted whole blood was evaluated. However IL-1beta or TNF-alpha production were not altered by in vitro exposure to MDMA. In conclusion, these data demonstrate that acute MDMA administration impairs IL-1beta and TNF-alpha secretion following an in vivo LPS challenge, and that TNF-alpha is more sensitive to the suppressive effects of MDMA than is IL-1beta. However the suppressive effect of MDMA on IL-1beta and TNF-alpha could not be attributed to a direct effect on immune cells. The relevance of these findings to MDMA-induced immunomodulation is discussed.     

Are lipid mediators implicated in the production of pro- and anti-inflammatory cytokines during cardiopulmonary bypass graft with extracorporeal circulation?

In this study the authors assessed the sequential release of lipid mediators (TXB2, PGE2, 6-keto-PGF1alpha, LTB4, LTC4, PAF), pro-inflammatory cytokines (IL-6, IL-8, TNF-alpha) and anti-inflammatory cytokines (IL-4, IL-10) in 17 patients undergoing coronary artery bypass graft (CABG) with extracorporeal circulation (ECC). Time course of appearance of inflammatory mediators revealed the early and transient increase in lipid mediator plasma concentrations (6-keto-PGF1alpha, LTB4, LTC4, PAF) whereas cytokines (IL-6, IL-8, IL-10) were involved only in late pre- and post-operative periods. No variation of TXB2, PGE2, IL-4 and TNF-alpha levels were found. No correlation was documented between the levels of lipid mediators and pro- or anti-inflammatory cytokines suggesting that lipidic compounds are not implicated in the genesis of cytokines which appear much later involved. Despite the common use of high doses of aprotinin (a non-specific enzyme inhibitor) in hope to abrogate the inflammatory response to cardiopulmonary bypass procedure, this study reports the persistent release of several inflammatory compounds that might be involved in the post-CABG multiple organ failure syndromes.     

Endotoxemia and release of tumor necrosis factor and interleukin 1 alpha in acute heatstroke

To determine whether endotoxemia and release of tumor necrosis factor (TNF-alpha) and/or interleukin 1 alpha (IL-1 alpha) are involved in the pathogenesis of heatstroke, 17 adult patients with a mean rectal temperature of 42.1 +/- 0.2 degrees C were studied. Blood samples were taken on admission and after cooling was completed. TNF-alpha and IL-1 alpha levels were measured by enzyme-linked immunosorbent assay, and lipopolysaccharide (LPS) content was measured by the chromogenic substrate modification of the Limulus amebocyte lysate. TNF-alpha, IL-1 alpha, and LPS were elevated in all patients [199 +/- 25 (SE) pg/ml, 480.5 +/- 68.3 pg/ml, and 8.60 +/- 1.19 ng/ml, respectively, compared with normal control values of 31.4 +/- 8.4 pg/ml, 53.7 +/- 5.32 pg/ml, and less than 9 pg/ml]. There was no significant correlation between temperature and the circulating concentration of TNF-alpha, IL-1 alpha, and LPS. Postcooling TNF-alpha, IL-1 alpha, and LPS concentrations were significantly decreased but still above normal control values. The findings suggest that these mediators may have a role in the pathogenesis of heatstroke that could change the strategy of management.