High serum levels of TNF-α after its administration for isolation perfusion of the limb

In a phase II study, 18 patients with locally spreading melanoma or sarcoma of lower limb were treated by isolation perfusion (ILP) with hyperthermia and local infusion of high dose of recombinant human tumor necrosis factor α (rHuTNF-α) (4 mg). Bioactive TNF-α and interleukin 6 (IL-6) serum levels were measured serially, In the limb, TNF-α rapidly reached a plateau at 2 μ/ml, while IL-6 appeared later and progressively increased until the end of ILP. In the systemic circulation TNF-α rose up to a median concentration of 31 ng/ml after 1 hour, then decreased and became negligible after 6 hours. IL-6 peaked only after 5 hours after start of ILP (median: 36.7 ng/ml). In patients with substantial leakage towards systemic circulation, both cytokines peaked higher and earlier as compared with patients with minimal leakage. No correlation was found between cytokine levels and severity of side effects which in all cases were reversible. We conclude that high dose TNF-α infusion in ILP results in extremely high levels of bioactive TNF-α in the systemic circulation without irreversible side effect, and provokes a delayed blood release of large amounts of IL-6; there was a correlation between leakage from the limb during procedure and the magnitude of systemic cytokines levels.     

Compartmentalization of TNF and IL-6 in meningitis and septic shock

We examined the compartmentalization of bioactive tumour necrosis factor (TNF) and interleukin 6 (IL-6) to the subarachnoid space and systemic circulation in patients with meningococcal meningitis and septic shock/bacteraemia. In patients with meningitis, median levels of TNF in 31 paired samples of cerebrospinal fluid (CSF) and serum were respectively 783 pg/ml and below detection limit (p < 0.001) and median levels of IL-6 were 150 ng/ml and 0.3 ng/ml (p < 0.0001). In patients with septic shock without meningitis, median levels in paired samples of CSF and serum were respectively below detection limit and 65 pg/ml (not significant, (ns)) (TNF, eleven patients) and 1.3 ng/ml-3 ng/ml (ns) (IL-6, nine patients). The data show that TNF and IL-6 are localized to the subarachnoid space in patients with meningitis although the blood-brain barrier is penetrable to serum proteins. On the other hand, patients with septic shock tend to have cytokines in both serum and CSF.     

Circulating tumour necrosis factor-alpha bioactivity in rheumatoid arthritis patients treated with infliximab: link to clinical response

Our objective was to clarify the heterogeneity in response to infliximab treatment in rheumatoid arthritis (RA); to this end, a bioassay was designed to explore the contribution of circulating tumour necrosis factor (TNF)-alpha bioactivity and its possible link to response. The bioassay is based on the induction of IL-6 and osteoprotegerin (OPG) production by synoviocytes in response to TNF-alpha. RA synoviocytes were cultured with TNF-alpha (5 ng/ml) and 42 RA plasma samples collected just before starting therapy. Levels of IL-6 and OPG were measured in supernatants. In 20 of the patients, plasma samples collected before and 4 hours after the first and the ninth infusions were tested in the same way. Plasma concentrations of TNF-alpha and p55 and p75 soluble receptors were measured using ELISA. TNF-alpha induced IL-6 and OPG production by synoviocytes, which was further increased with patient plasma dilutions and inhibited by infliximab. With plasma samples obtained before the first infusion, the IL-6-induced production was greater in patients with a good clinical response than in the poor responders (44.4 +/- 23.3 ng/ml versus 27.4 +/- 20.9 ng/ml; P = 0.05). This high circulating TNF-alpha bioactivity was strongly inhibited with the first infliximab infusion. The difference between IL-6 levels induced with plasma samples obtained before and 4 hours after the first infusion was greater in patients with a good clinical response (40.0 +/- 23.7 ng/ml versus 3.4 +/- 10.0 ng/ml; P = 0.001). Similar findings were obtained for OPG production (7.0 +/- 6.2 ng/ml versus 0.0 +/- 3.0 ng/ml; P < 0.05). Levels of circulating TNF-alpha bioactivity were predictive of clinical response to TNF-alpha inhibition, confirming a key role for TNF-alpha in these RA patients.     

Interleukin-6, TNF-alpha and interleukin-1 beta levels in blood and tissue in severely burned rats

Previous studies have demonstrated the early appearance of inflammatory cytokines in the systemic circulation after thermal injury both in humans and animals. The aim of this study was to evaluate the time course of several cytokines, IL-6, TNF-alpha and IL-1beta in serum, lung, liver and brain of severely burned rats during the first week after thermal injury. Cytokine measurements were performed by enzyme-linked immunosorbent assay (ELISA). The comparison between the sham-burned animals and animals with third-degree burns on 20% or 40% of their total body surface area allowed for the study of the inflammatory process relative to the size of the injury. Serum IL-6 levels, which were undetectable in sham-treated animals, peaked during the first hours after injury and were proportionate to the size of the area burned. After a few days, IL-6 increased once more, but only in the most severely burned rats. In lung, liver and brain, low but measurable basal levels of TNF-alpha and IL-1 were detected in sham-burned animals. Strikingly, IL-1beta levels remained significantly elevated in the lung after injury in animals having 20% and 40% burned skin area. Unexpectedly, both TNF-alpha and IL-1beta production decreased gradually in liver and brain after burn injury. Also, the inflammatory response after a burn injury appeared to be biphasic. The first period corresponded to the early release of IL-6 into the circulation, proportional to the severity of the injury. After a few days, a second period was marked by the extension of the inflammatory processes from the injured area to the rest of the body, particularly to lung, which could be considered as at potential risk of involvement in severely burned patients.     

Up-regulation of mucin secretion in HT29-MTX cells by the pro-inflammatory cytokines tumor necrosis factor-alpha and interleukin-6

The pro-inflammatory cytokines IL-6 and TNF-alpha have been implicated in the pathogenesis of otitis media with effusion (OME). A disease where goblet cells proliferate in a modified respiratory epithelium, leading to the accumulation of a mucin-rich effusion in the middle ear cleft. The MUC5AC and MUC5B mucin gene products have been identified as components of these effusions. To determine the effect of IL-6 and TNF-alpha on MUC5AC and MUC5B secretion we have used HT29-MTX goblet cells, which secrete both types of mucins. MUC5AC and MUC5B mucin secretion was measured by an enzyme-linked immunosorbent assay (ELISA) using a specific monoclonal antibody NCL-HGM-45M1 and polyclonal antiserum TEPA, respectively. Time response (0-72 hours) and dose response (1.5-150 ng/ml) studies were carried out. IL-6 and TNF-alpha stimulated MUC5AC and MUC5B mucin secretion in a time dependent manner, both in pre-confluent and post-confluent cells. IL-6 (15 ng/ml and 20 ng/ml) produced a low and prolonged stimulation of mucin secretion that persisted for 72 hours, with peak response at 24 hours after induction. The IL-6-mediated mucin secretion at 24 hours was concentration-dependent, with a maximal effect at 15 ng/ml. TNF-alpha (20 ng/ml) induced rapid stimulation of mucin secretion within the first 24 hours, with peak response at 7 hours after induction. IL-6 and TNF-alpha exposure significantly increased MUC5AC secretion, but not MUC5B secretion. Maximal levels of cytokine-induced mucin secretion were detected in pre-confluent cells that showed one and a half- and two-fold increases in MUC5AC secretion after IL-6 and TNF-alpha stimulation, respectively, in comparison with post-confluent cells. The results presented here suggest that IL-6 and TNF-alpha generate a differential up-regulation of mucin secretion and thus contribute to the expression of mucin genes in inflammatory responses.     

Hyperthermic isolated limb perfusion increases circulating levels of inflammatory cytokines

Hyperthermic isolated limb perfusion is an established method of treatment for regionally advanced melanoma. Recent studies suggest that exogenously administered cytokines potentiate tumor response in patients with in-transit melanoma. We hypothesized that isolated limb perfusion induces an immunogenic response characterized by increased circulating levels of cytokines in the pump circuit, potentially contributing to the antitumor effect. We obtained blood samples from the perfusion circuit and systemic circulation at various intervals from patients undergoing isolated chemotherapeutic perfusion for melanoma. Samples were analyzed for serum cytokine profiles by enzyme-linked immunosorbent assay. When compared with baseline values, significant increases in serum levels of interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor (TNF) occurred within the perfusion circuit during isolated limb perfusion (P<0.05). In addition, there was a corresponding increase in IL-8 within the systemic circulation at the 60-min interval (P<0.05), suggesting some degree of leakage from the isolated circuit due to the extremely high levels of IL-8 in the perfusion circuit. A transient but insignificant decrease in circulating levels of neutrophils was also observed during the perfusion process, which may be attributed to margination. Increased levels of cytokines IL-6, IL-8, and TNF occurred within the isolated circuit during hyperthermic limb perfusion and may contribute to tumor response seen in patients treated with isolated limb perfusion.Presented at the Annual Meeting of the Society of Surgical Oncology, 17–20 March 1994 Houston, Texas     

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.     

Evidence for IFN-gamma as a mediator of the lethality of endotoxin and tumor necrosis factor-alpha.

Current evidence indicates that endogenously produced peptide cytokines, most notably TNF-alpha and IL-1, mediate the lethality of experimental endotoxemia. Because circulating serum levels of IFN-gamma can be detected soon after TNF-alpha and IL-1 in response to endotoxin, we investigated the role of IFN-gamma in endotoxin and TNF-alpha lethality. Specific neutralizing antibodies to murine TNF-alpha (anti-TNF-alpha Ab) or murine IFN gamma (anti-IFN-gamma Ab) produced in our laboratory protected mice against the lethality of Escherichia coli endotoxin (LPS) administered 6 h later. Serum IFN-gamma levels 2 h after i.v. LPS were lower in mice treated with anti-TNF-alpha Ab compared to mice that received nonimmune IgG (median less than 2.5 vs 3.0 U/ml, P2 less than 0.05). In contrast, serum TNF-alpha levels 1 h after i.v. LPS peaked more than fourfold higher in mice treated with anti-IFN-gamma Ab compared to controls (median greater than 6400 vs 1405 pg/ml, p2 less than 0.05). Doses of TNF-alpha (300 micrograms/kg) and IFN-gamma (50,000 U) which were well tolerated when given individually were synergistically lethal in combination (0% lethality vs 100% lethality, P2 less than 0.001), and were associated with higher serum levels of IL-6 than with either cytokine alone. Anti-IFN-gamma Ab provided complete protection against exogenous human rTNF-alpha at the LD100 dose (1400 micrograms/kg, p2 less than 0.001), and in fact prevented lethality at doses four- to fivefold greater than the LD100 human rTNF-alpha (up to 6000 micrograms/kg). We conclude that IFN-gamma is synergistic with TNF-alpha, is essential for the lethality of LPS and TNF-alpha, and may have modulating effects on the negative control of serum levels of TNF-alpha after LPS in mice.     

Opiate regulation of IL-1beta and TNF-alpha in cultured human articular chondrocytes

In order to investigate if beta-endorphins anti-inflammatory effect in cartilage-damaging states is mediated via tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta), we examined its influence on these two cytokines in vitro. Human articular chondrocytes were obtained from patients undergoing total knee arthroplasty and stimulated with beta-endorphin (60-6000 ng/ml). Protein levels of TNF-alpha and IL-1 beta were measured by ELISA in supernatants from articular chondrocyte cultures. beta-Endorphin significantly increased the levels of IL-1 beta for all concentrations used after 15 min incubation, and when stimulated with 600 and 6000 ng/ml after 24 h incubation. The opioid-induced increase in IL-1 beta was blocked by naltrexone in the group tested. TNF-alpha expression was also significantly stimulated by 60 and 600 ng/ml beta-endorphin after 15 min, an effect blocked by naltrexone in the group tested. These findings indicate that the mechanism of beta-endorphins anti-inflammatory influence in cartilage-damaging states is not apparently mediated via these two cytokines modulation.     

SYSTEMIC RELEASE OF SOLUBLE TNF RECEPTORS AFTER HIGH-DOSE TNF IN ISOLATED LIMB PERFUSION

Isolated limb perfusion (ILP) with high dose tumour necrosis factor (TNF), interferon γ and melphalan (TIM) is an efficient treatment for patients with regionally advanced melanoma and sarcoma. In 44 patients, we determined the kinetics of soluble TNF receptors (sTNF-RI and RII) plasma concentrations, and correlated them with systemic TNF and interleukin 6 (IL-6) levels and shock. Seven patients treated conventionally by ILP without cytokine served as controls.Elevated levels of both sTNF-Rs were observed within 30 min after beginning of the TIM-ILP. A first peak of sTNF-Rs levels was observed 3 h after ILP and was followed by a rapid decrease reaching a nadir at 12–14 h post ILP. This first peak was followed by a second, long-lasting elevation of both sTNF-Rs levels persisting for 4 to 5 days after TIM-ILP. Patients treated by ILP without TNF/interferon γ (IFN-γ) had no detectable increase in either sTNF-Rs or in circulating TNF, demonstrating that the release of TNF-Rs was dependent upon the administration of TNF/IFN-γ. High plasma levels of TNF and IL-6 were observed in patients that had more than 5% leakage during the TIM-ILP, but no significant correlation between TNF levels and the peak values of both sTNF-Rs was observed. The levels of TNF and IL-6 were, however, significantly related to each other. TNF systemic levels, but not sTNF-Rs concentrations, correlated significantly with the severity of the shock observed after TIM-ILP. Patients in which sTNF-RII concentration was in excess over circulating TNF, had no shock or grade I shock only, suggesting that sTNF-RII may play a protective, although limited, role in inhibiting activity of circulating TNF.     

Ghrelin as an acute-phase reactant during postoperative stress response.

Ghrelin is a growth hormone-releasing peptide, discovered in 1999 by Kojima et al. Its potential role in inflammation and stress response is not yet clear. The purpose of this study was to characterize perioperative levels of circulating ghrelin in relation to different surgical procedures. The authors compared plasma ghrelin changes with cortisol, cytokines, and acute-phase proteins. The prospective study was performed on 22 patients with resection for colon cancer (group 1). Group 2, functioning as a comparative group, consisted of 22 patients with elective laparotomic cholecystectomy. Plasma concentrations of ghrelin, cortisol, tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta, IL-6, IL-8, soluble IL-2 receptor, C reactive protein, and alpha1-antitrypsin were estimated repeatedly during a 72-hour postoperative period. Data revealed significant elevation of plasma ghrelin 24 hours after resection of coli (median 508.0 ng/l, interquartile range 398.2-633.7 ng/l) in relation to both preoperative levels (317.6 ng/l, 253.4-355.1 ng/l, p<0.01) and group 2 maximal postoperative levels (386.2 ng/l, 324-432 ng/l, p<0.05). Ghrelin levels returned to initial status 36-48 hours after surgery with subsequent decline to subnormal levels. The regression coefficient was the highest for ghrelin and TNF-alpha 24 hours after laparotomy (r=0.64, p<0.05) and for ghrelin and IL-6 24 hours after surgery (r=0.56, p<0.05). Maximal postoperative levels of all tested parameters except for cortisol and IL-1beta differed significantly between both patient groups at p<0.05. After large abdominal surgery, ghrelin shows itself as an acute-phase reactant. The significant correlation between ghrelin and inflammatory cytokines supposes their regulatory role in this period. Our comparison of more- and less-invasive surgical procedures with similar nutritional restrictions argues for a dominant role of inflammatory factors in postoperative ghrelin elevation.     

IL-6/IFN-beta-2 as a circulating hormone. Induction by cytokine administration in humans

IL-6/IFN-beta 2 is a family of phosphoglycoproteins ranging in size from 19 to 30 kDa which elicits a broad range of physiologic and immune responses. Several cytokines, including TNF, have been shown to stimulate IL-6 production in cell culture. In this report, we describe the rapid induction of circulating biologically active IL-6 by the systemic administration of rTNF to patients with cancer. Low levels of IL-6 activity could be detected in the sera of patients as early as 5 min after rTNF infusion. IL-6 levels peaked approximately 2 to 3 h after rTNF bolus administration and were undetectable in most cases within 8 h. IL-6 was detected in two separate bioassays--the hybridoma B9 proliferation and the hepatocyte-stimulating factor assay. Maximum detectable levels of IL-6 ranged from 160 to 310 hybridoma growth factor units and 11-82 ng/ml in the hepatocyte-stimulating factor assay. IL-6 induction decreased after serial, daily doses of rTNF. Serial serum samples of patients receiving IL-2 or IFN-alpha were also assayed for IL-6 production. IL-2-treated but not IFN-alpha-treated patients generated low levels of IL-6 (range less than 20 to 95 hybridoma growth factor units/ml). Interestingly, in patients treated with IL-2, serum levels of TNF were detectable and peak TNF activity preceded measurable IL-6 levels. Serum levels of acute phase plasma proteins and of corticosteroid rose in response to rTNF administration. C-reactive protein increased (2.5 to 4.0-fold) within 8 h of rTNF administration and cortisol levels rose (10- to 20-fold) within 4 h after rTNF injection. We conclude that rTNF administration in man leads to the induction of circulating IL-6 which, due to its broad range of activities, may be an important physiologic signal regulating the immune response.     

Proinflammatory cytokines interact synergistically with epidermal growth factor to stimulate PGE2 production in amnion-derived cells.

Recent evidence has implicated cytokines and growth factors in the initiation of parturition in women. In the present study, the amnion-derived cell line WISH was used to determine whether proinflammatory cytokines (interleukins 1 beta, 6, and 8, tumor necrosis factor-alpha, and granulocyte/macrophage colony stimulating factor) could amplify epidermal growth factor-induced prostaglandin E2 production. WISH cells were preincubated with cytokines (0.0001-10 ng/ml) for 60 min and then challenged with EGF (10 ng/ml) for 4 hrs after which PGE2 production was measured by radioimmunoassay. EGF, IL-1 beta and TNF-alpha alone caused a dose-dependent increase in PGE2 production, while IL-6, IL-8 and GM-CSF were ineffective over the dose range tested. When cells were preincubated with IL-1 beta or TNF-alpha, there was a dose-dependent potentiation of EGF-induced PGE2 production that was greater than the sum of EGF alone and IL-1 beta or TNF-alpha alone. In each case, the minimum dose of IL-1 beta or TNF-alpha which amplified EGF-induced PGE2 production was 0.1 ng/ml (p less than 0.05, Student's t-test). These data show that low concentrations of IL-1 beta or TNF-alpha may serve to amplify EGF-mediated PGE2 biosynthesis in amnion-derived cells and suggest that cytokines may modulate EGF function in responsive cells.     

HIV infection of placental macrophages: effect on the secretion of HIV stimulatory cytokines.

Vertical transmission of HIV-1 can occur at three different stages: during gestation, delivery and breast feeding. To determine the role of cytokines in vertical transmission of HIV during gestation, we studied the secretion of IL-1beta, TNF-alpha and IL-6 from in vitro infected and Mock-infected placental macrophages (Hofbauer cells) in comparison to blood monocyte derived macrophages (MDM). Hofbauer cells stimulated with lipopolysaccharide (LPS) secreted lower levels of HIV stimulatory cytokines (6-8 ng/ml) in the supernatants than MDM (26 ng/ml, p<0.005). Cytokine levels in MDM decreased upon HIV infection to 7 ng/ml. IL-6 was the major cytokine produced after LPS stimulation by the two cell populations (p<0.005), being MDM the major cytokine producer. In vitro infection studies with a M-tropic virus (HIV-BaL) indicated that MDM were 10x more susceptible to HIV than placental macrophages (p=0.001). Our results indicate that although macrophages from term placenta secrete lower amount of HIV stimulatory cytokines than MDM, there was no correlation between the levels of cytokines and HIV production by both cells.     

The transcriptional control of TGF-beta in human osteoblast-like cells is distinct from that of IL-1 beta.

Transforming growth factor beta (TGF-beta) and interleukin 1 (IL-1) are among the most potent osteotropic cytokines. The expression of mRNA for both TGF-beta and IL-1 beta was studied in human osteoblast-like cells in vitro. These cells constitutively expressed TGF-beta but not IL-1 beta mRNA. Treatment of the cells with the systemic hormones 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] (10(-8) M) and parathyroid hormone (10(-7) M) induced an increase in TGF-beta mRNA but failed to stimulate the production of IL-1-beta mRNA. Retinoic acid (10(-8) M) had no effect on either mRNA species. The cytokines IL-1 alpha (200 pg/ml), tumour necrosis factor alpha (TNF-alpha) (17 ng/ml) and bacterial lipopolysaccharide (LPS) (500 ng/ml) stimulated the production of IL-1 beta mRNA after 6-8 hours. This was followed by an increase in protein production after 24 hours. In contrast, the production of TGF-beta mRNA remained constant after treatment with these agents. Treatment of the cells with hydrocortisone (10(-8) M) resulted in the suppression of both TGF-beta and IL-1 beta mRNA. However, when the stimulating agent 1,25-(OH)2D3 was added in conjunction with hydrocortisone the mRNA expression of TGF-beta mRNA returned to 70% of the stimulated level. In contrast, the addition of the stimulatory agent IL-1 alpha to hydrocortisone-treated cells resulted in no increase in IL-1 beta mRNA. In-situ hybridization demonstrated both TGF-beta and IL-1 beta mRNA at the cellular level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of short-term intralipid infusion on the immune response during low-dose endotoxemia in humans

Novel anti-inflammatory effects of insulin have recently been described, and insulin therapy to maintain euglycemia suppresses the plasma levels of free fatty acids (FFA) and increases the survival of critically ill patients. We aimed to explore the effect of short-term high levels of plasma FFA on the inflammatory response to a low dose of endotoxin. Fourteen healthy male volunteers underwent the following two trials in a randomized crossover design: 1) continuous infusion of 20% Intralipid [0.7 ml.kg(-1).h(-1) (1.54 g/kg)] for 11 h, and 2) infusion of isotonic saline for 11 h (control). In each trial, heparin was given to activate lipoprotein lipase, and an intravenous bolus of endotoxin (0.1 ng/kg) was given after 6 h of Intralipid/saline infusion. Blood samples and muscle and fat biopsies were obtained before the Intralipid/saline infusion and before as well as after infusion of an endotoxin bolus. Plasma levels of FFA, triglycerides, and glycerol were markedly increased during the Intralipid infusion. Endotoxin exposure induced an increase in plasma levels of TNF-alpha, IL-6, and neutrophils and further stimulated gene expression of TNF-alpha and IL-6 in both skeletal muscle and adipose tissue. The systemic inflammatory response to endotoxin was significantly pronounced during Intralipid infusion. Short-term hyperlipidemia enhances the inflammatory response to endotoxin, and skeletal muscle and adipose tissue are capable of producing essential inflammatory mediators after endotoxin stimulation.     

Proinflammatory cytokines alter/reduce esophageal circular muscle contraction in experimental cat esophagitis

Cholinergic mechanisms are largely responsible for esophageal contraction in response to swallowing or to in vitro electrical field stimulation (EFS). After induction of experimental esophagitis by repeated acid perfusion, the responses to swallowing and to EFS were significantly reduced but contraction in response to ACh was not affected, suggesting that cholinergic mechanisms are damaged by acid perfusion but that myogenic mechanisms are not. Measurements of ACh release in response to EFS confirmed that release of ACh was reduced in esophagitis compared with normal controls. To examine factors contributing to this neuropathy, normal esophageal strips were incubated for 1-2 h with the proinflammatory cytokines IL-1beta (100 U/ml), IL-6 (1 ng/ml), or TNF-alpha (1 ng/ml). IL-1beta and IL-6 levels, measured by Western blot analysis, increased in esophagitis compared with normal circular muscle. IL-1beta and IL-6 reduced contraction in response to EFS (2-10 Hz, 0.2 ms) but did not affect ACh-induced contraction, suggesting that these cytokines inhibit ACh release without affecting myogenic contractile mechanisms. EFS-induced ACh release was significantly reduced in normal esophageal strips by incubation in IL-1beta or IL-6, suggesting that they may contribute to the contractility changes. TNF-alpha at 1 ng/ml, however, did not affect the response to ACh or to electrical stimulation but inhibited both at higher concentrations. TNF-alpha levels were low in normal muscle and did not increase with esophagitis. The data suggest that the proinflammatory cytokines IL-1beta and IL-6 contribute to reduced esophageal contraction by inhibiting release of ACh from myenteric neurons.     

Cytokine-mediated modulation of leptin and adiponectin secretion during in vitro adipogenesis: evidence that tumor necrosis factor-alpha- and interleukin-1beta-treated human preadipocytes are potent leptin producers

Over the last decade, compelling evidence has been presented that cytokines affect adipocyte tissue formation and function. In this study we explored the effect of pro-inflammatory (i.e. interleukin (IL)-1beta, IL-6, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha) versus anti-inflammatory cytokines (i.e. IL-4, IL-10, and transforming growth factor (TGF)-beta1) on leptin and adiponectin secretion during in vitro human adipogenesis. Confirmative to previous reports, conversion of precursor preadipocytes into mature adipocytes was completely inhibited upon exposure to TNF-alpha, IL-1beta, IFN-gamma, or TGF-beta1. Hence, all these anti-adipogenic cytokines prevented release of adipocyte-specific adiponectin. IFN-gamma also strongly reduced leptin production (> or =85%). However, TNF-alpha, IL-1beta, and TGF-beta1 stimulated leptin production from preadipocytes in the absence of mature adipocytes (20.6+/-5.4 ng/ml, 100.8+/-18.2 ng/ml, and 5.4+/-0.4 ng/ml, respectively, compared to 6.6+/-0.8 ng/ml in control adipocyte cultures on day 21; n=4). IL-4, IL-6 and IL-10 did not, or only slightly, affect adipocyte differentiation and their hormonal secretion. In conclusion, adiponectin and leptin are both synthesized by adipocytes, whereas leptin is also produced by preadipocytes upon TNF-alpha or IL-1beta stimulation. These data suggest that preadipocytes could contribute more to total circulating leptin levels than has been previously considered, especially in diseased conditions were these pro-inflammatory factors play a prominent role.     

Effects of lipopolysaccharide on neurokinin A content and release in the hypothalamic-pituitary axis

The administration of bacterial lipopolysaccharide (LPS) markedly affects pituitary secretion, and its effects are probably mediated by cytokines produced by immune cells or by the hypothalamo-pituitary axis itself. Since neurokinin A (NKA) plays a role in inflammatory responses and is involved in the control of prolactin secretion, we examined the in vivo effect of LPS on the concentration of NKA in hypothalamus and pituitary (assessed by RIA) and serum prolactin levels in male rats. One hour after the intraperitoneal administration of LPS (250 microg/rat), NKA content was decreased in the posterior pituitary but not in the hypothalamus or anterior pituitary. Three hours after injection, LPS decreased NKA concentration in the hypothalamus and anterior and posterior pituitary. In all the conditions tested, LPS significantly decreased serum prolactin. We also examined the in vitro effects of LPS (10 microg/ml), interleukin-6 (IL-6, 10 ng/ml) and tumor necrosis factor alpha (TNF-alpha, 50 ng/ml) on hypothalamic NKA release. Interleukin-6 increased NKA release without modifying hypothalamic NKA concentration, whereas neither LPS nor TNF-alpha affected them. Our results suggest that IL-6 may be involved in the increase of hypothalamic NKA release induced by LPS. NKA could participate in neuroendocrine responses to endotoxin challenge.     

Outer membrane vesicles from Neisseria meningitidis: effects on cytokine production in human whole blood

The Norwegian group B meningococcal outer membrane vesicle (OMV) vaccine consists of outer membrane proteins (OMPs) as main antigens with significant amounts of lipopolysaccharide (LPS; 5-9% relative to protein). We have studied the ability of this OMV vaccine preparation to induce secretion of pro-inflammatory cytokines, tumour necrosis factor alpha (TNF-alpha), interleukin 1beta (IL-1beta), interleukin 6 (IL-6), interleukin 8 (IL-8) and anti-inflammatory cytokines, interleukin 4 (IL-4), interleukin 10 (IL-10) and interleukin 13 (IL-13) in a human whole blood model. Plasma levels of TNF-alpha, IL-1beta, IL-6 and IL-8 were massively increased; mean peak levels of TNF-alpha 44 696+/-7764, IL-1beta 38 043+/-5411, IL-6 10 057+/-1619 and IL-8 30 449+/-5397 pg/ml were obtained with an OMV-LPS concentration of 1 microg/ml; corresponding levels in control plasmas were below the detection limit of the assay. Mean maximal level of IL-10 (2540+/-144 pg/ml) was obtained at OMV-LPS concentration of 10 microg/ml, after 24 h; while the level in control plasma was below detection limit. OMV-LPS did not induce release of IL-4 and IL-13 in doses from 0.001-10 microg/ml. The present results show that OMVs from meningococci have potent pro-inflammatory properties and are likely to contribute to the observed local and systemic inflammatory effects.