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The three events, viz. schizophrenia, dissociative anaesthesia and Near-Death Experience, despite their seemingly unrelated manifestation to each other, have nevertheless similar functional basis. All three events are linked to the glutamate sensitive N-methyl-D-aspartate (NMDA) receptor complex, which serves as their common functional denominator. Arguments and speculations are presented in favor of the view that, the three events might be considered as functional models of each other. Antagonism to the recognition NMDA-site of the receptor induces dissociative anaesthesia and precipitates Near-Death Experience. Agonist reinforcement at the modulatory glycine-site of the receptor counteracts negative symptoms of schizophrenia. Both types of challenges towards the receptor are compatible with a glutamate deficiency concept which underlies the meeting of the three events at the NMDA receptor.

Traditional acupuncture has been used for treating a variety of health conditions. In contrast, Western physicians limited acupuncture to the alleviation of pain. Concomitant with a recent view that not all kinds of pain are within the reach of acupuncture-induced relief, it has been suggested that some conditions other than pain can be effectively treated by this method. Increased release of the neuropeptide beta-endorphin was proposed to explain the antinociceptive function of acupuncture. Even if correct beta-endorphin cannot account for the effect of acupuncture in other conditions. Endorphins might be interacting with cytokines, some of which (e.g. interleukin-10) downregulate the inflammatory component of disorders in which acupuncture may be useful. We present a speculative notion of the view that acupuncture may amplify the interaction between neuropeptides and cytokines. A non-invasive approach, such as immune-committed cells harvested from blood of acupuncture-treated patients, could be used to examine this hypothesis. Inclusion of a placebo group might support the credibility of acupuncture., (Copyright 2002, Elsevier Science Ltd. All rights reserved.)

The levels of the eicosanoids leukotriene B4, prostaglandin E2, prostacycline and thromboxane B2, the cytokines interleukin-1 beta, interleukin-6 and tumour necrosis factor-alpha and soluble intercellular adhesion molecule 1 were measured in ascites and plasma samples of patients with liver cirrhosis (53), peritoneal cancer (26) and spontaneous bacterial peritonitis (10) to assess their value as a possible diagnostic and prognostic parameter in the course of the disease. Soluble intercellular adhesion molecule 1, of the eicosanoids prostaglandin E2 and leukotriene B4, and the protein concentration in ascites were all significantly elevated in ascites of patients with peritoneal cancer in comparison to ascites of patients with liver cirrhosis. In ascites of patients with spontaneous bacterial infection interleukin-6 concentration was significantly elevated and the protein concentration was significantly lower in comparison to the other two groups. None of these parameters, however, seems to be of practical use as a diagnostic parameter, as there is an overlap between all the levels of these mediators in ascites of liver cirrhosis, peritoneal cancer and spontaneous bacterial peritonitis group. Soluble intercellular adhesion molecule 1 levels were much higher in plasma than in ascites, in contrast to interleukin-6 levels which were much higher in ascites than in plasma. Soluble intercellular adhesion molecule 1 in ascites correlated with soluble intercellular adhesion molecule 1 in plasma (r = 0.6926, P = 0.0001). Soluble intercellular adhesion molecule 1, interleukin-6 and the number of polymorphonuclear cells in peritoneal fluid correlated during episodes of infection in patients with a peritonitis. For this reason soluble intercellular adhesion molecule 1 and interleukin-6 could be of prognostic value for patients with peritonitis.

The effect of short- and long-term feeding with L-carnitine, L-acetyl carnitine and L-propionyl carnitine on the production of eicosanoids from in vitro stimulated carrageenan-induced rat peritoneal macrophages was investigated. Both young (4 weeks) and old (18 months) rats were used. A lower number of cells was isolated from the peritonea of treated than control young rats after 4 d feeding, but after 60 d no differences were observed. A similar reduction in cell number was found when old animals were given L-acetyl carnitine or L-propionyl carnitine (acutely) or L-acetyl carnitine or L-carnitine (chronically). Plasma carnitine levels were higher in young rats given carnitine both chronically and acutely. Carnitine derivatives were without effect. In contrast, levels of total carnitine in the plasma of old rats given L-carnitine and L-acetyl carnitine for 4 d and 60 d were higher than in controls. There was no correlation between total plasma carnitine level and effects on prostaglandin, thromboxane and leukotriene B4 (LTB4) production. In young rats the most important changes were observed in relation to the production of prostacyclin (PGI2), measured as 6 keto-prostaglandin F1 alpha. Prostacyclin production was higher in the groups given carnitine or its derivatives. The net result of the changes in PGI2 was that the 6 keto-prostaglandin F1 alpha: thromboxane B2 and the 6-keto-prostaglandin F1 alpha: LTB4 ratios tended to be higher in cells from young animals following short-term feeding with L-carnitine.(ABSTRACT TRUNCATED AT 250 WORDS)

To examine the interactions between the main pro-inflammatory cytokines and eicosanoids produced by human inflammatory cells, human peritoneal macrophages (hp-M phi) were isolated from ascitic fluid of patients with portal hypertension. Interactions between interleukin-1 beta (IL-1 beta), IL-6, tumor necrosis factor alpha (TNF-alpha), leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) were studied by addition or inhibition of several cytokines and eicosanoids: human recombinant IL-1 beta (hrIL-1 beta) addition, LTB4 addition and 5-lipoxygenase inhibition (6-hydroxy-2-(4-sulfamoylbenzylamino)-4,5,7-trimethylbenzothiaz ole hydrochloride (E6080)), PGE2 addition and cyclooxygenase inhibition (indomethacin). In hp-M phi hrIL-1 beta stimulated the LTB4 production, while the PGE2 production was inhibited. HrIL-1 beta had no significant effect on IL-6 production in hp-M phi. LTB4 did not regulate IL-1 beta and IL-6 production. Increasing PGE2 down regulated the TNF-alpha production, but did not effect the IL-1 beta and IL-6 production.

Production and release of arachidonic acid (AA) compounds (eicosanoids: prostaglandins-cyclooxygenase and leukotrienes-lipoxygenase) and monokines (TNF-alpha, IL-1 and others) play an essential role in the expression of antitumour activity of macrophages (MO). We investigated the possibility of inducing the antitumour activity of peritoneal murine and human MO by regulating their production of eicosanoids and monokines. The antitumour activity of MO was inversely correlated to production of PGE2 and directly correlated to production of leukotrienes (LTC4 and LTD4). Thus, indomethacin rendered murine MO cytostatic against tumour cells and enhanced the antitumour activity of human peritoneal macrophages from renal patients on CAPD (continuous ambulatory peritoneal dialysis), and leukotriene inhibitors (NDGA-nordihydroguaiaretic acid and AA861) prevented antitumour cytostatic activity of MO. Human peritoneal MO collected during periods of inflammation (infectious peritonitis) were more active against tumour cells, especially when cultured in the presence of LPS, and their activity was correlated to increase with the release of TNF and of IL-1 beta. Human peritoneal MO from inflammation-free patients reacted against a human tumour cell line if cultured with LPS and TPA (phorbol-myristate acetate) and were therapeutically effective against the same palpable s.c. tumours implanted in nude mice.

Ascites is a readily available source of human macrophages (M phi), which can be used to study M phi functions in vitro. We characterized the mediators of inflammation produced by human peritoneal M phi (hp-M phi) obtained from patients with portal hypertension and ascites. The production of the cytokines interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) was found to be lipopolysaccharide (LPS) concentration dependent (0-10 micrograms/ml) with a maximal production at 10 micrograms/ml and also dependent on the time of exposure to the stimulus (0-36 h). IL-1 beta, IL-6 and TNF-alpha production after LPS administration reached a plateau at 24 h. In vitro stimulation for 24 h with LPS does not influence the eicosanoid production from endogenous arachidonate. 13 min of exposure of the cells to the calcium ionophore A23187 gives a significant increase in eicosanoid production from both exogenous and endogenous arachidonate. The main eicosanoids produced are the 5-lipoxgenase products LTB4 and 5-hydroxyeicosatetraenoic acid (HETE). The increase in production of the other eicosanoids is not significant. The eicosanoid production depends on the stimulus concentration. The optimal A23187 concentration is 1 microM. Oxygen radical production was measured in the M phi by a flowcytometric method. The fluorescence intensity of phorbol 12-myristate 13-acetate stimulated and dihydro-rhodamine 123 loaded hp-M phi increases significantly after 15 min. We conclude that LPS stimulation of hp-M phi from liver disease results in similar production of IL-1 beta, IL-6 and TNF-alpha, but that the profile of the eicosanoid production of these M phi stimulated with LPS and A23187 differs from M phi of other origin and species.

Antigen challenged alveolar macrophages (ac-AM) showed much higher basal prostaglandin E2 (PGE2) release (4,4-fold) and cAMP content (2,4-fold) than naive alveolar macrophages (AM). In naive AM 1 fM platelet activating factor (PAF) enhanced PGE2 release from 115 to 157 ng/5 x 10(6) cells but was inactive at 1 nM or 1 microM. In ac-AC 1 fM PAF enhanced PGE2 release from 510 to 670 ng/5 x 10(6) cells and inhibited leukotriene B4 (LTB4) release (from 6.0 to 4.8 ng/5 x 10(6) cells). At a 10(6)-fold higher concentration PAF inhibited PGE2 release (from 510 to 400 ng/5 x 10(6) cells) and stimulated LTB4 release (from 6.0 to 8.2 ng/5 x 10(6) cells). PAF-induced increase or decrease in PGE2 release was paralleled by changes in cellular cAMP (+35 and -17%, respectively). The specific PAF-antagonist BN 52021 completely reversed all PAF-induced effects while indomethacin inhibited only PAF-induced increase in PGE2 release and cAMP leaving LTB4 release unaffected. Similarly, the lipoxygenase inhibitor AA-861 inhibited PAF-induced rise in LTB4 release leaving the enhancement in PGE2 release and cAMP content unaffected. Present data show that PAF dose-dependently affects eicosanoid production and cAMP level in alveolar macrophages.

The effect of the phosphodiesterase inhibitor isobutyl-methylxanthine (IBMX), salbutamol and sodium nitroprusside was evaluated regarding PGE2 and LTB4 release and cAMP and cGMP level in human alveolar macrophages obtained from controls and COPD patients. Basal levels per five million control- respectively COPD alveolar macrophages: cAMP 1.2 and 1.0 pmole; cGMP 8.4 and 9.1 fmole; PGE2 120 and 63 pg and LTB4 19.2 and 14.8 pg. In both populations IBMX increased cAMP level by 55-93% and salbutamol+IBMX by 285-252%. Except for the 61% rise in LTB4 release by salbutamol+IBMX the drugs hardly affected PGE2 and LTB4 release from control macrophages. In COPD alveolar macrophages, however, IBMX and IBMX+salbutamol largely reduced PGE2 release (63 vs 11 pg per 10(6) cells) but less efficiently increased LTB4. In both macrophage populations sodium nitroprusside (SNP) substantially increased (3-4 fold) cGMP level but did not affect eicosanoid production. Present results indicate that drugs which enhance cAMP level decrease PGE2 release from COPD macrophages and stimulate the release of LTB4 a chemotactic mediator involved in bronchial inflammatory reactions.

Human peritoneal macrophages were collected from dialysis bags of renal patients on Continuous Ambulatory Peritoneal Dialysis (CAPD), during an inflammation-free period. The macrophage suspension was cultured in presence of bacterial lipopolysaccharide (LPS) and phorbol myristate acetate (TPA). The cultured macrophages were tested for therapeutic effectiveness against a human tumor-cell line, RC43, implanted subcutaneously in NMRI nude mice. The macrophages were injected around the tumor starting from the 14th day after inoculation, when the tumor growth was already detectable (mean tumor size 7 mm). Three injections of macrophages on days 14, 18 and 21 induced hemorrhagic patches at the tumor site and almost complete regression of the tumor. One injection of macrophages cultured either in presence of LPS+TPA or of LPS+TPA+PGE2 resulted in marked slow-down of the tumor growth. Injection of either TNF-alpha (4000 U/mouse) or PGE2 (150 ng/mouse) given at the site of the palpable small tumor had no effect. Macrophages cultured in medium or in medium supplied with either TPA, LPS or TPA+LPS, were not effective in nude mice bearing large (16 to 19 mm) tumors. The results obtained suggest that activated human macrophages might be therapeutically effective at certain stages of human cancer.

This review describes the potential role of macrophages in defense against cancer cells and the regulatory involvement of inflammatory mediators in this role. Interactions between macrophage-derived cytokines (tumor necrosis factor alpha, interleukin-1, IL-6) and their interrelationships with eicosanoids (mainly the cyclooxygenase product prostaglandin E2 and some lipoxygenase metabolites) represent a network that controls the expression of antitumor activity of macrophages either in a cell-to-cell contact system between the effector and the target tumor cell or as cell-free soluble products. Attention is given to the influence of tumor burden on production of cytokines and eicosanoids by macrophages and to the production of these mediators by tumor cells. Emphasis is placed on the roles of TNF-alpha and PGE2 in links between inflammatory and antitumor functions of macrophages. Finally, the perspectives and still existing problems in clinical implications of macrophage-derived cytokines are discussed in terms of a conceivable macrophage-directed immunotherapy of cancer.

Human peritoneal macrophages collected from renal patients on continuous ambulatory peritoneal dialysis (CAPD) during inflammation-free periods were induced to express antitumor activity in vitro when cultured in the presence of bacterial lipopolysaccharide (LPS) and even more activity when they were kept in the presence of LPS + IND (indomethacin). The antitumor activity was expressed against a human tumor-cell line, RC43, either in a cell-to-cell contact set-up between the macrophages and the RC43 target cells or when the tumor cells were exposed to supernatants of the cultured macrophages. The antitumor activity of macrophages was correlated to a marked increase in production of tumor necrosis factor-alpha (TNF alpha), not correlated to an increase in nitrite production and inversely correlated to the production of PGE2. The RC43 tumor cells were susceptible to recombinant human TNF alpha, recombinant human IL-1 beta, sodium nitrite and the leukotriene LTB4. The results obtained suggest that activated human macrophages might represent a useful tool for cancer immunotherapy.

In this study we evaluated the role of epithelial versus subepithelial peptidases in the responses of isolated peripheral and central human airways to VIP. Human airways were obtained at thoracotomies (n = 8) and studied in organ baths. Intact or epithelium-denuded strips of central and peripheral airways were incubated with or without a cocktail of peptidase inhibitors containing phosphoramidon (2.5 micrograms/ml), leupeptin, aprotinin, captopril, soybean trypsin inhibitor (all 20 micrograms/ml), and bestatin (2.8 micrograms/ml). After precontraction with histamine (5 x 10(-6) M), cumulative concentration-response curves to VIP (10(-10) to 10(-7) M) were obtained. Both intact central and peripheral airways showed only minor relaxations to VIP irrespective of the precontraction level. Removal of the epithelium and addition of peptidase inhibitors additively increased the sensitivity (> 20-fold) and maximal response to VIP in both central and peripheral airways. We conclude that (1) VIP relaxes both central and peripheral human airways but only in the absence of epithelium and/or the presence of peptidase inhibitors, and (2) both epithelial and subepithelial peptidases are important in the inactivation of VIP in human airways.

The effect of diclofenac sodium was investigated on haemodynamics, haematologic and blood glucose values as well as the release of eicosanoids, tumor necrosis factor (TNF) and platelet activating factor (PAF) in anaesthetized pigs receiving 5 micrograms.kg-1 Escherichia coli lipopolysaccharide (LPS) over 60 min into the superior mesenteric artery. The animals were observed for an additional period of 2 h after the termination of LPS infusion. 15 of the 31 animals infused with LPS and not treated with diclofenac sodium died within 30 min after the commencement of LPS infusion (non-survivors), while the other 16 survived the experimental period of 3-h, though in a shock state (survivors). No alterations were observed in plasma concentrations of PAF or eicosanoids (TXB2, 6-keto PGF1 alpha and LTB4), but a marked increase was detected in TNF release in the non-survivors. A significant, though transient, increase in concentrations of PAF, TNF and eicosanoids studied characterized the survivors. Another group of 7 LPS-infused pigs was treated with diclofenac sodium (2 mg, kg-1, i.v. bolus 60 min before the start of LPS infusion, followed by a continuous infusion of 1 mg kg-1 h-1) 1 mg/kg-1/h-1. This treatment prevented death and shock despite the high concentrations of TNF and PAF. Concentrations of both cyclooxygenase and 5-lipoxygenase enzymes products were reduced. These data indicated that the beneficial effect of diclofenac sodium in LPS induced shock may be related to the reduced production of eicosanoids.

Human peritoneal macrophages (hp-M phi) are a source of inflammatory mediators. After stimulation in vitro for 24 h with LPS there was a significant increase in cytokine production (IL-1, IL-6 and TNF alpha), but not in the production of eicosanoids from endogenous arachidonate. Leukotrienes are the predominant eicosanoids formed after stimulation with calcium ionophore for 15 min, while prostaglandin formation is insignificant. The fluorescence intensity of TPA-stimulated and DHR123 loaded hp-M phi (a measure of the respiratory burst) increases significantly in a short period of time. Hp-M phi will be useful as a model for testing the effects of anti-inflammatory drugs on eicosanoid and cytokine production and respiratory burst activity in vitro.

The current study was undertaken to compare two methods for the efficiency of measuring tumor necrosis factor (TNF-alpha) in biological fluids, which is species undependent, reliable, sensitive, simple and not expensive. We have compared the MTT tetrazolium cytotoxic assay [1,2] and the 3H-thymidine (3H-TdR) incorporation cytostatic assay for measuring the anti-tumor activity of human recombinant TNF-alpha, of human colonic tissue and of supernatants of in vitro stimulated human and rat peritoneal macrophages. Two target cell-lines, namely murine myelomonocytic leukaemia WEHI-164- and L-929-transformed murine fibroblast cell-lines, were used in the MTT assay. The L-929 line was also used in the 3H-TdR assay. WEHI-164 was more sensitive than the L-929 cell-line in the MTT cytotoxic assay. Furthermore, the MTT assay was more sensitive to TNF-alpha than the 3H-TdR assay. Both methods can be used for the detection of anti-tumor activity in biological fluids but the MTT cytotoxic method has the advantage of being more sensitive and more simple.

THE acyl carrier coenzyme A (CoA) is involved in fatty acid metabolism. The carnitine/CoA ratio is of particular importance in regulating the transport of long-chain fatty acids into mitochondria for oxidation. Also CoA has a role in the formation and breakdown of products from both the cyclooxygenase and lipoxygenase pathways of the precursor arachidonic acid. In the present study the effect of 4 days feeding of 300 mg/kg/day of L-carnitine, acetyl Lcarnitine and propionyl L-carnitine on the basal and calcium ionophore (A23187) stimulated release of arachidonic acid metabolites from rat carrageenin elicited peritoneal cells was investigated. There were two series of experiments carried out. In the first, the harvested peritoneal cell population consisted of less than 90% macrophages and additional polymorphonuclear (PMN) leucocytes. The basal release of prostaglandin E(2) (PGE(2)), 6-ketoprostaglandin F(1alpha) (6-keto-PGF(1alpha)) and leukotriene B(4) (LTB(4)) was stimulated by all treatments. The A23187 stimulated release of 6-keto-PGF(1alpha) and LTB(4) was increased by all three compounds. The 6-keto-PGF(1alpha):TxB(2) and 6-keto-PGF(1alpha):LTB(4) ratios were increased by carnitine treatment. These results suggested that carnitine could modify the macrophage component of an inflammatory site in vivo. In the second series of experiments the harvested cell population was highly purified (>95% macrophages) and none of the compounds fed to the rats caused a change of either eicosanoid or TNFalpha formation. Moreover the 6-keto-PGF(1alpha):TxB(2) and 6-keto-PGF(1alpha):LTB(4) ratios were not enhanced by any of the compounds tested. It is conceivable that in the first series the increased ratios 6-keto-PGF(1alpha):TxB(2) and 6-keto-PGF(1alpha):LTB(4) reflected the effect of carnitine or its congeners on PMN leucocytes rather than on macrophages.

Continuous lipopolysaccharide (LPS) infusion in pigs induced death in approximately half of the animals and a prolonged state of shock (up to 3 hours of experimental observation period, i.e., two hours after discontinuation of LPS infusion) in the surviving animals. Lethal-induced shock was marked by huge release of Tumor Necrosis Factor (TNF) into the blood, whereas eicosanoid and Platelet Activating Factor (PAF) levels remained unchanged. In pigs surviving LPS-infusion but still remaining in a state of shock, transient increases in PAF and thromboxane levels were observed, whereas prostacyclin and leukotrienes values remained above normal levels up to the end of the observation period. It is concluded that different types of mediators play a role in LPS-induced lethal shock as compared to non-lethal prolonged state of shock.

In vitro secretion of the prostanoids PGE2 and PGI2 and of the cytokine IL-1 beta by peritoneal macrophages obtained from CAPD patients during episodes of peritonitis and infection free periods, was determined, after culturing with or without 5 micrograms/ml of LPS. The release of PGE2 and PGI2 as measured by its stable metabolite 6-keto-PGF alpha was determined in 10 episodes of peritonitis and 10 infection free periods. IL-1 beta release was determined in 14 episodes of peritonitis and 20 infection free periods. PGI2 release from macrophages declined sharply during peritonitis both in the absence and presence of LPS in the culture medium (p less than 0.005). A tendency to decreased PGE2 release was found during peritonitis, when macrophages were cultured in the absence of LPS. In the presence of LPS, the same amounts of PGE2 were released during peritonitis and during an infection free period. On the other hand, peritoneal macrophages released significantly more IL-1 beta during peritonitis as compared to an infection free period, provided that the cells were in vitro stimulated with LPS. In view of the interregulatory effects between prostanoids and macrophage cytokines in their production, these findings may indicate that the impaired release of PGI2 during peritonitis has allowed the macrophages to secrete more IL-1 beta after in vitro stimulation with LPS. This implies that PGI2 and PGE2 may play a distinct role in the regulation of cytokine secretion by these cells.

Strips or rings of airway tissue are often used to study contractile responses of human airways in vitro. These preparations have the disadvantage that it is impossible to deliver stimuli selectively to the mucosal or serosal surface. Hence, they allow only for a limited evaluation of the modulatory role of the airway epithelium. We developed an in vitro model that allows independent stimulation from either the serosal or the mucosal side of human peripheral airways. Segments of human peripheral airways were perfused with a Krebs solution at a constant pressure, and responsiveness was measured as a change in flow rate. Pressure/flow relationships indicated laminar flow over a wide pressure range, and a working pressure of 6 cm H2O was chosen because this is a physiological transpulmonary pressure. When stepwise stretching the airway to 180% of its length, we noted an increase in baseline flow and a decrease in flow reduction after methacholine 10(-5) M. At 140% of the length, accurate and reproducible measurements of the sensitivity (EC50) to methacholine were obtained, and airway closure did not occur. A one-way analysis of variance (ANOVA) revealed that the between-patients differences accounted for 91% of the total variability for -log EC50. We conclude that this in vitro model offers interesting possibilities for evaluating the modulatory effects of the human airway epithelium. In addition, the model provides the opportunity to study human small-airway mechanical properties and secretory functions.

We have investigated the effects of inflammatory mediators and beta-adrenoceptor agonists on the adenylyl cyclase responsiveness in alveolar macrophages from control subjects, patients suffering from chronic obstructive pulmonary disease (COPD) and asthmatics. Basal cyclic AMP (cAMP) levels in alveolar macrophages from COPD patients were significantly elevated (plus 42%) as compared to controls. In addition, the adenylyl cyclase responsiveness to prostaglandin E2, histamine and the beta-adrenoceptor agonist salbutamol was significantly impaired in alveolar macrophages from COPD patients and asthmatics. The lipid mediator platelet activating factor showed no effect on cAMP production in all three alveolar macrophage populations. Furthermore, the cAMP-enhancing effects of isoprenaline, salbutamol and histamine appeared to be mediated via beta 2-adrenoceptors and histamine H2-receptor subtypes respectively. Taken together, these data suggest an intrinsic desensitization phenomenon in alveolar macrophages from COPD patients and asthmatics.

We have reported previously that macrophages obtained from renal patients on continuous ambulatory peritoneal dialysis (CAPD) during an episode of infectious peritonitis display a decrease in intracellular cAMP levels and in spontaneous in vitro release of PGE2 and PGI2. Such macrophages also release large quantities of IL-1 beta and TNF alpha when stimulated in vitro by LPS. In view of the interregulatory effects between PGE2 and macrophage cytokines (IL-1 beta and TNF alpha) in their production, we examined in the present work to what extent the LPS-induced release of either IL-1 beta or TNF alpha in vitro from CAPD-originated peritoneal macrophages is affected by graded doses of exogenous PGE2 (range 0-1000 ng/ml) and by the cyclooxygenase inhibitor indomethacin (INDO) (10(-6) M). IL-1 beta and TNF alpha were determined using an enzyme-linked immunoabsorbent assay and an immunoradiometric assay, respectively. We found that PGE2 invariably induced a dose-dependent decrease in TNF alpha release. In peritoneal macrophages collected during an infection-free period, TNF alpha release decreased from 3225 pg/ml (controls) to 353 pg/ml at 1000 ng/ml of PGE2, and in peritoneal macrophages collected during an episode of infectious peritonitis, it decreased from 4100 pg/ml (controls) to 545 pg/ml at 100 ng/ml of PGE2. However, PGE2 failed to influence the secretion of IL-1 beta. INDO induced an approx. two-fold increase in TNF alpha release, but had no effect on IL-1 beta release. These findings indicate that exogenous and endogenous PGE2 controls the release of TNF alpha rather than IL-1 beta from LPS-stimulated peritoneal macrophages.

Density-defined macrophages isolated from fluids of patients with liver cirrhosis mainly generated the 5-lipoxygenase products leukotriene B4 (LTB4, 16%) and 5-hydroxy-eicosatetraenoic acid (5-HETE, 24%) and the cyclooxygenase products 12-hydroxyheptadecatrienoic acid (HHT, 22%) and thromboxane B2 (TXB2, 18%). The synthesis of eicosanoids was linear with the maturity of the macrophage subpopulations, suggesting that eicosanoid production is increased in in-vivo activated macrophages.

The role of platelet activating factor (PAF) in endotoxic shock was investigated in anaesthetized pigs receiving 5 micrograms/kg E. coli endotoxin (LPS) into the superior mesenteric artery over a 60 min period. Concentrations of PAF and tumor necrosis factor (TNF) were measured in blood obtained from the superior mesenteric vein and aorta before, during and 60 min after the LPS infusion. The effect of 4 mg/kg of BN 52021, a PAF receptor antagonist, given as a bolus injection 5 min prior to LPS infusion and/or PAF administration into the superior mesenteric vein was studied on systemic and regional hemodynamic variables. Eight of the 17 animals infused with LPS died within 30 min after start of LPS, while the other 9 survived the experimental period of 3 h, though in a shock state. In survivors, PAF concentration in both superior mesenteric vein and aorta increased twenty-fold at 30 min of endotoxaemia, but rapidly returned back towards normal values. No changes in PAF release, but a marked rise in TNF production were measured in non-survivors. Exogenous administration of PAF (0.01 micrograms/kg) produced similar hemodynamic effects as observed in survivors. BN 52021 markedly reduced the effects of PAF on arterial blood pressure for over 1 h. Treatment with BN 52021 (4 mg/kg), injected 5 min prior to LPS infusion, failed to exert any effect on the surviving rate. However, in survivors all circulatory and laboratory parameters studied were improved after treatment with BN 52021. PAF release observed during LPS infusion in survivors may play a role in the development of shock; however, its role in the rapid death seems to be negligible. Present results clearly demonstrate that endotoxin shock is not crucially dependent on one class of mediators.

1. The effects of indomethacin were investigated on haemodynamics, haematological and blood glucose values, and the release of tumour necrosis factor (TNF), platelet activating factor (PAF) and eicosanoids in anaesthetized pigs receiving 5 micrograms kg-1 E. coli lipopolysaccharide (LPS) over 60 min into the superior mesenteric artery. The animals were observed for an additional period of 2 h after the termination of LPS infusion. 2. Eight of the 17 animals infused with LPS and not treated with indomethacin died within 30 min after the beginning of LPS infusion (non-survivors), while the other 9 survived the experimental period of 3 h though in a state of shock (survivors). 3. No alterations were observed in plasma concentrations of PAF and eicosanoids (thromboxane B2 (TXB2), 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha) and leukotriene B4 (LTB4] in non-survivors. However, a marked increase was detected in TNF release. A significant, though transient, increase in concentrations of PAF, TNF and eicosanoids occurred in the survivors. The peak in the concentrations of PAF and TXB2 preceded the maximum in TNF values in survivors. 4. Another group of 7 LPS-infused pigs was treated with indomethacin (2 mg kg-1, i.v. bolus 60 min before the start of LPS infusion, followed by a continuous infusion of 3 mg kg-1 h-1). This treatment prevented death and shock despite the high concentrations of circulating TNF and PAF. Concentrations of cyclo-oxygenase enzyme products were reduced, whereas LTB4 release was not affected. The effect of indomethacin on haemodynamic changes occurred earlier than on cyclo-oxygenase products.5. In another group of 6 pigs indomethacin (2mg kg- 1, i.v.) was given 20-25 min after the start of LPS infusion at which time mean arterial blood pressure (MABP) had decreased below 40mmHg indicating imminent death. This indomethacin treatment immediately reversed the hypotension, restored the organ perfusion, delayed the haemoconcentration and thrombocytopenia and prevented death. However, TNF and PAF concentrations remained elevated. Concentrations of cyclo-oxygenase products studied were reduced by the end of the observation period, whereas LTB4 production was unaffected.6. The decrease in MABP induced by exogenous PAF was temporarily prevented by indomethacin.7. These data indicate that the beneficial effect of indomethacin in LPS-induced septic shock is related to cyclo-oxygenase inhibition as well as to a direct vasoconstrictor property of the drug.

Using membrane fractions (MF) from guinea pig alveolar macrophages (AM), we investigated the effects of sensitization and antigen challenge on the stepwise activation of adenylyl cyclase considering receptor binding, G-protein coupling and direct stimulation of the enzyme. Receptor binding studies, using [125I]ICYP as the beta-adrenoceptor specific ligand, show that neither receptor number (Bmax) nor receptor affinity constants (Kd values) were affected by sensitization or antigen challenge. Using forskolin as a direct stimulant of AC, alterations in the enzymatic activity of AC could be excluded. Pretreatment of the different MF with cholera toxin (CT, a toxin which eliminates GTPase activity) and subsequent stimulation of AC with GTP, shows an increased responsiveness in MF from sensitized and antigen challenged AM. In addition, pretreatment of MF from naive AM with increasing doses of CT results in a maximal AC response at the higher concentrations used (50-100 micrograms/mL), an effect not observed in MF from sensitized and antigen challenged AM. In these MF, the AC response still increases after pretreatment with such doses of CT. These data suggest that the enhanced AC responsiveness in AM, induced by sensitization and antigen challenge, results from alterations in alpha s-subunits.

Platelet-activating factor (PAF) induces a dose-dependent biphasic response in adenylyl cyclase activity in antigen-challenged alveolar macrophages (AM), but not in naive AM. Intracellular cyclic AMP levels are enhanced by very low concentrations of PAF (10(-13)-10(-10) M) and decreased by higher PAF concentrations (10(-8)-10(-5) M). The PAF response of adenylyl cyclase could be completely blocked by pretreatment with the PAF receptor antagonist BN 52021. The adenylyl cyclase stimulatory and inhibitory phases are reversed by indometacin (inhibiting cyclo-oxygenase) and AA 861 (inhibiting lipoxygenase). These results show that the PAF-induced response of adenylyl cyclase activity in antigen-challenged AM is achieved by its modulation of intracellular arachidonic acid metabolism.

1. We studied the effect of hyperosmolarity on human isolated airways because a better understanding of the effect of hyperosmolarity on the human airway wall may improve insight into the pathophysiology of hyperosmolarity-induced bronchoconstriction in asthma. 2. In cartilaginous bronchial rings dissected from fresh human lung tissue, hyperosmolar krebs-Henseleit buffer (450 mosM, extra sodium chloride added) evoked a biphasic response: a rapid relaxation phase (peak after 5.0 +/- 0.3 min) followed by a slow contraction phase (peak after 25.4 +/- 0.8 min). 3. With the histamine (H1) receptor antagonist mepyramine, the contraction phase was reduced to 41.2% of the control value (P less than 0.001), with atropine to 50.0% (P less than 0.01), with the local anaesthetic lignocaine to 48.7% (P less than 0.05) and with mepyramine together with atropine to 19.2% (P less than 0.001). 4. With the inhibitor of neutral metalloendopeptidase, phosphoramidon, the contraction phase increased to 128.0% of the control value (P less than 0.05) and after removal of the epithelium to 131.8% (P less than 0.05). 5. Indomethacin, the leukotriene C4/D4 (LTC4/D4) antagonist FPL 55712 or the blocker of nerve conduction, tetrodotoxin, had no effect on the contractile phase. 6. The relaxation phase was not altered by any of these drugs nor by epithelial denudation. The relaxation phase was also unchanged in the presence of alpha-chymotrypsin, which degrades muscle relaxing peptides such as vasoactive intestinal peptide. 7. Hyperosmolar buffer slightly increased the sensitivity and maximal response to methacholine as well as the cholinergic twitch to electric field stimulation. 8. We conclude that hyperosmolarity releases acetylcholine, histamine and neuropeptides in the human airway wall in sufficient quantities to contract airway smooth muscle. This release itself or its effect on airway muscle is modulated by the airway epithelium. The mechanism of the relaxation phase may be an unknown smooth muscle relaxing substance or a direct effect on the airway muscle, related to ion fluxes.

The release of eicosanoids during endotoxin shock was investigated in anesthetized pigs receiving 5 micrograms/kg Escherichia coli lipopolysaccharide (LPS) over 60 min into the superior mesenteric artery. TXB2, 6-keto PGF1 alpha and LTB4 concentrations in blood obtained from the superior mesenteric vein (SMV), right ventricle (RV) and aorta, during LPS infusion and an additional period of 2 h, were assessed along with hemodynamic variables, blood gases and pH and laboratory parameters. Half of the animals died within 30 min after termination of LPS infusion (non-survivors, n = 8), while the other half survived the experimental period of 3 h, though in a shock state (survivors, n = 9). The non-surviving pigs demonstrated progressively reduced cardiac output, hypotension and hypoperfusion in all organs. The surviving pigs demonstrated also a reduced cardiac output, which however was compensated by an elevated systemic vascular resistance resulting in a maintenance of arterial blood pressure. After exhausting this compensation the flow to non-vital organs increased and consequently arterial blood pressure was reduced resulting in hypoperfusion. In survivors a marked, though, transient increase was measured in concentrations of TXB2 and 6-keto PGF1 alpha level. A significant increase was measured in plasma concentration of LTB4 in SMV without any elevation in RV and aorta. LTB4 production started when prostanoid release had decreased. In contrast to survivors, no changes could be observed in eicosanoid release for non-survivors. A correlation was observed between systemic vascular resistance and TXB2 to 6-keto PGF1 alpha ratio.(ABSTRACT TRUNCATED AT 250 WORDS)

In asthma a temporal association exists between the late allergic reaction (LAR), the influx of granulocytes into the airway wall, and an increase in bronchial responsiveness. We therefore tested the hypothesis that activated human granulocytes constrict isolated human airways and increase their sensitivity to cholinergic stimuli. Bronchial rings were dissected from 23 lung tissue specimens collected at thoracotomy and studied isotonically in organ baths. Airways were incubated with 1, 2, 5, 10, or 20 x 10(6) granulocytes from normal or atopic donors. Activation of the cells with serum-treated zymosan (STZ, 0.2 mg/ml), which itself did not alter baseline airway caliber, resulted in a bronchoconstriction proportional to the number of zymosan-activated granulocytes (ZAG) present (rs = 0.79, p less than 0.001). This contraction was reduced by about 70% with the leukotriene C4/D4 receptor antagonist FPL 55712 (11.5 microM; p less than 0.001) or with the lipoxygenase inhibitor nordihydroguaiaretic acid (10 microM; p less than 0.001). The scavengers of activated oxygen molecules superoxide dismutase (300 U/ml) and bovine catalase (5,000 U/ml), the cyclooxygenase inhibitor indomethacin (10 microM), or the histamine (H1) receptor antagonist mepyramine (2.8 microM) had no effect. Granulocyte suspensions from atopic donors contained more eosinophils (p less than 0.001), and the magnitude of the contraction to 10 x 10(6) ZAG was related to the proportion of eosinophils (rs = 0.66, p less than 0.01). The sensitivity of the airways to methacholine was unchanged in the presence of 1, 2, or 5 x 10(6) ZAG and decreased with 10 or 20 x 10(6) ZAG (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

The role of tumor necrosis factor alpha (TNF alpha) in endotoxin-induced shock was investigated in pigs receiving 5 micrograms kg-1 of Escherichia coli endotoxin (LPS) during 60 min of continuous infusion into the superior mesenteric artery. LPS concentration in aortic plasma, as determined by a chromogenic Limulus amoebocyte lysate (LAL) test, reached a peak of approximately 1000 ng l-1 during LPS infusion, and declined rapidly after discontinuation of the infusion. Serum TNF levels were determined by a bioassay using the L929 murine transformed fibroblast line. Eight of the 17 animals infused with LPS died within 30 min after beginning LPS administration, while the other 9 pigs survived beyond the experimental observation period of 3 h, although they were in a state of shock. No difference in LPS concentration was found between the survivors and the non-survivors. However, the serum TNF levels in non-survivors were significantly higher than in survivors when measured at 30 min after beginning LPS administration. In survivors, the peak increase in serum TNF levels was measured at 60 min after the beginning of LPS injection and returned rapidly to the baseline values. Although the role of TNF inducing rapid death seems to be dominant, the hemodynamic, hematology and blood chemistry disturbances seen during shock continued in survivors long after the return of TNF to baseline levels. These findings indicate that besides TNF other mediators are also involved in the LPS infusion-induced shock.

We studied the effect of cooling on the responses of isolated human airways to the beta-agonist isoproterenol, the alpha/beta-agonist norepinephrine in the presence of the beta-blocker timolol, methacholine, leukotriene C4 (LTC4), and histamine. In addition, the effect of cooling on baseline airway tone and responses to electric field stimulation (EFS) was studied. At 27 degrees C the sensitivity (-logEC50) and maximal response to isoproterenol were unchanged. No measurable response was found to alpha-adrenergic stimulation with norepinephrine + timolol either before or during cooling. At 27 degrees C and 21 degrees C the sensitivity and maximal contraction to methacholine and LTC4 as well as the contraction to a single dose of histamine were reduced. Cooling diminished baseline airway tone. EFS produced a rapid cholinergic contraction followed by a deflection below baseline and a sustained noncholinergic contractile response, which was substantially reduced by the LTC4/D4 receptor antagonist FPL 55712 (11.5 microM) at all three temperatures. Cooling decreased the cholinergic response to EFS and increased the sensitivity to EFS-induced relaxation. In contrast, the sustained noncholinergic contractile response to EFS was not changed, suggesting that cooling facilitates the synthesis of LTC4/D4 that follows EFS and/or inhibits its inactivation. We conclude that in nonasthmatic, isolated human airways slow cooling of the airway wall down to 21 degrees C does not cause bronchoconstriction and does not increase the responsiveness to contractile or relaxing agonists. However, cooling increases the sensitivity to EFS-induced relaxation and might facilitate the accumulation of leukotriene C4/D4 in the airway wall.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of an inflammatory environment on the antitumor cytostatic ability of human macrophages was examined. Peritoneal macrophages of patients on continuous ambulatory peritoneal dialysis (CAPD) were collected, when CAPD was without complication, during an intercurrent infectious inflammation and after recovery. Inhibition of 3H-thymidine uptake served as a measure of cytostasis by macrophages co-cultured with target murine cells MOPC-315 plasmacytoma, WEHI-3B myelomonocytic leukemia and L929 transformed fibroblasts. Macrophages from inflammatory peritoneum expressed a markedly enhanced cytostasis, irrespective of the nature of the tumor cell. Endotoxin (LPS) challenge of inflammatory macrophages failed to further reinforce the cytostasis towards MOPC-315 plasmacytoma, but reinforced the cytostasis towards WEHI-3B leukemia (sensitive to inhibition by IL-1) and towards L929 (sensitive to TNF alpha). Cytostasis by supernatants of human peritoneal macrophages against L929 was markedly inhibited by anti-rHuTNF alpha and against WEHI-3B by anti-rHuIL-1 beta. The results suggest a link between inflammatory function and antitumor cytostasis by macrophages. This link is constituted by mediators involved in the activation process of macrophages.

We have reported previously that human peritoneal macrophages collected from patients on Continuous Ambulatory Peritoneal Dialysis (CAPD) during an episode of peritonitis secrete increased amounts of interleukin-1 (IL-1), as compared to those collected during an infection free period, provided the cells were stimulated in vitro by LPS. We now report that such macrophages release also higher amounts of Tumor Necrosis Factor (TNF), if collected during peritonitis and stimulated subsequently in vitro by LPS. The increase in release of TNF was ascertained by radio-immunoassays as well as by bioassay of cytostatic effect against the highly sensitive TNF target-cell line L929 murine transformed fibroblasts. The present reported results, in addition to previously reported data on release of IL-1, indicate that induction of release of cytokines from human peritoneal macrophages is a dual stepwise process: first priming in vivo in an inflammatory environment and, secondly stimulation in vitro by LPS.

Atrial natriuretic factor (ANF, 10(-7) M) and sodium nitroprusside (SNP, 10(-5)-10(-3) M) stimulated cGMP production in human peritoneal macrophages (HPM). This suggests the existence of two separate forms of guanylate cyclase in HPM, e.g. the receptor-related form by ANF and the soluble form by SNP. In parallel with the rise in cGMP levels, both agents provoked a decrease in cAMP levels. Increasing the concentration of the phosphodiesterase inhibitor IBMX (0.2 mM to 1.0 mM) in the incubation media resulted in a significantly greater rise in cGMP levels which was accompanied by a profound decrease in cAMP levels. ANF did not exert any direct or GTP-related effect on cAMP production, which is in contrast to its action in other tissues. These results suggest that cAMP levels can be modulated through a cGMP signal, most likely at the production level. Results also give substantial evidence for the presence of a ANF receptor site on human peritoneal macrophages.