Changes in resident rat peritoneal macrophage eicosanoid release, induced by altering the buffer K+/Na+ ratio, are Ca2+ dependent.

Altering the buffer K+/Na+ ratio ([K+]o/[Na+]o) resulted in a biphasic change in the basal release of prostaglandin E2, 6-ketoprostaglandin F1 alpha (the stable breakdown product of prostaglandin I2) and thromboxane B2 (the stable metabolite of thromboxane A2), from resident rat peritoneal macrophages. Changing the [K+]o (at the expense of [Na+]o) from 15 mM to 0 mM or 75 mM (combined concentration of [K+]o and [Na+]o was maintained at 150 mM) resulted in a stimulation of 6-ketoprostaglandin F1 alpha and thromboxane B2 release. Prostaglandin E2 synthesis was also stimulated when the [K+]o was decreased from 15 mM to 0 mM. When the [K+]o was increased to 45 mM, prostaglandin E2 formation was inhibited but returned to values observed at 15 mM K+ when the [K+]o was further increased to 75 mM. Prostaglandin E2 synthesis at 75 mM K+ was still only 40% of that measured in the absence of K+, however. When cells were incubated in a Ca2+-free medium (+EDTA) eicosanoid release was drastically reduced and the changes in arachidonic acid metabolite release observed on changing the buffer [K+]o/[Na+]o were abolished. Total release of radiolabel ([14C]arachidonic acid and its radiolabelled metabolites) from macrophages prelabelled with [14C]arachidonic acid followed the same pattern as basal eicosanoid release, suggesting that changing [K+]o influenced phospholipase A2 activity, and hence, substrate availability. At all [K+]o values, from 0 mM to 75 mM, cicletanide reduced the release of radioactivity from macrophages prelabelled with [14C]arachidonic acid (by about 15%). In the presence of [K+]o, cicletanide had a stimulatory effect on the metabolism of the free fatty acid which masked the decrease in eicosanoid release expected due to inhibition of arachidonic acid release from phospholipid. In the presence of 5 mM K+, cicletanide inhibited the basal release but enhanced the arachidonic acid-stimulated synthesis of eicosanoids from resident macrophages in a dose-related fashion, confirming the dual action of the drug, i.e., the inhibitory effect on arachidonic acid release and the stimulation of arachidonic acid metabolism. The possible in vivo significance of these results is discussed.

Involvement of inflammatory mediators in macrophage antitumor activity.

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.

Interactions between inflammatory mediators in expression of antitumor cytostatic activity of macrophages.

Antitumor properties and participation in inflammatory events are important characteristics of activated macrophages. We show here that both antitumor cytostatic function of macrophages and participation of these cells at inflammatory sites are controlled by two main groups of mediators: cytokines (IL-1, TNF alpha) and eicosanoids (prostanoids and leukotrienes). These two groups of mediators represent a complex system of mutual interactions in regulation of their production and activities. Multiple sets of experiments with murine macrophages are discussed in favor of the views that PGE2 and lipoxygenase products oppose each other's actions, and that the regulating role of PGE2 in the secretions of cytokines are of pivotal importance in antitumor cytostasis of macrophages in vitro. Such observations can be extended to a situation ex vivo, showing that human macrophages harvested from inflammatory sites have markedly augmented cytostatic expression. It thus appears that the antitumor cytostatic function of macrophages is related to the production of inflammatory mediators by these cells. Accordingly, it might be that occurrence of inflammation in tumor-bearing individuals plays a role in the promotion of antitumor activity of macrophages.

Activity of human peritoneal macrophages against a human tumor: role of tumor necrosis factor-alpha, PGE2 and nitrite, in vitro studies.

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.

Prostaglandin E2 inhibits the release of tumor necrosis factor-alpha, rather than interleukin 1 beta, from human macrophages.

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.

Interactions between cytokines and eicosanoids: a study using human peritoneal macrophages.

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.

Serum levels of tumor necrosis factor determine the fatal or non-fatal course of endotoxic shock.

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.

Low concentrations of prostaglandin E2 inhibit the prostacyclin-induced elevation of cyclic adenosine 3',5'-monophosphate in elicited populations of rat peritoneal macrophages.

Elevation of cyclic adenosine 3',5'-monophosphate (cyclic AMP) in elicited populations of rat peritoneal macrophages was used as a parameter to examine the influence of prostaglandin E2 (PGE2) on the effects of prostacyclin (PGI2) and (+/-)-5E-13,14-didehydro-carbo-prostacyclin (DDH-carbo-PGI2) in vitro. PGE2, within the range of 1.4 X 10(-9) to 1.2 X 10(-8)M, caused a concentration-dependent inhibition of the rise in cyclic AMP induced by 2.8 X 10(-6) M PGI2 or DDH-carbo-PGI2. With higher concentrations of PGE2 the inhibition was either non-existent or masked by the effect of PGE2 per se on cyclic AMP levels. The present findings suggest that the earlier observed low responsiveness of granuloma macrophages to PGI2, in terms of rise in cyclic AMP, is possibly due to permanent exposure of these cells to environmental endogenous PGE2.

Time-dependent stimulatory and inhibitory effects of prostaglandin E1 on exudative and tissue components of granulomatous inflammation in rats.

1 The effects of prostaglandin (PGE(1)), following local administration during different phases of developing sponge-induced granulomata, were studied in normal and essential fatty acid deficient (EFAD) rats.2 In normal rats, a single dose of 1 mug PGE(1) on implantation (day 1) increased exudate production without altering total leucocyte counts after 6 h and stimulated granulomatous tissue formation after 8 days.3 Repeated daily administration of the same dose of PGE(1) on days 1 to 3 had no effect, while administration on days 4 to 7 (i.e. when tissue growth is already in progress) inhibited granuloma formation.4 In EFAD rats, which are known to produce only very small amounts of endogenous prostaglandins, acute (6 h) exudate formation was unaffected by 0.05 mug PGE(1). However, early stimulatory and later inhibitory effects of 0.05 mug PGE(1) per day were obtained on the granulomatous tissue, similar to those obtained with the 20 fold higher dose in normal rats.5 The early stimulatory action of PGE(1) on granulomatous tissue formation was enhanced, in normal rats, by concomitant administration of 10 mug theophylline. This latter compound did not influence the later inhibitory effect of PGE(1).6 These results indicate that PGE(1) exerts either pro- or anti-inflammatory actions on the proliferative (tissue) component of the inflammatory process, depending on the time of administration. While the stimulatory effect following early administration may have been secondary to an initial cyclic adenosine 3',5'-monophosphate-mediated, vascular response, such a mechanism is unlikely to have been responsible for the later anti-inflammatory action of PGE(1).7 The implications of these results are discussed in relation to the postulated negative-feedback role of endogenous PGE in chronic inflammation.

Prostaglandin E2 inhibits and indomethacin and aspirin enhance, A23187-stimulated leukotriene B4 synthesis by rat peritoneal macrophages.

1. The calcium ionophore, A23187, stimulated leukotriene B4 (LTB4), thromboxane B2 (TxB2) and prostaglandin E2 (PGE2) synthesis by 4 day carrageenin-elicited rat peritoneal macrophages. 2. At concentrations of 2 x 10(-7)-2 x 10(-5) M indomethacin and aspirin enhanced A23187-stimulated LTB4 synthesis and inhibited PGE2 and TXB2 formation. 3. PGE2 inhibited A23187-stimulated LTB4 and TXB2 formation as well as the augmentation of LTB4 release caused by aspirin and indomethacin. However, PGE2 was ineffective when the cells were challenged with arachidonic acid (AA). 4. Dibutyryl adenosine 3':5'-cyclic monophosphate (db-cyclic AMP) partially inhibited A23187-stimulated LTB4 production. 5. Our results suggest that PGE2 inhibits macrophage LTB4 synthesis by limiting the availability of AA. Indomethacin and aspirin, possibly by removing the regulatory effect of PGE2, promote the synthesis of the pro-inflammatory LTB4.

Sequential release of tumour necrosis factor, platelet activating factor and eicosanoids during endotoxin shock in anaesthetized pigs: protective effects of indomethacin.

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.

Effects of hyperosmolarity on human isolated central airways.

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.

1-Hydroxy-3-amino-pyrrolidone-2(HA-966): a new GABA-like compound, with potential use in extrapyramidal diseases.

1. The drug HA-966 (1-hydroxy-3-amino-pyrrolidone-2), which chemically resembles the cyclic form of GABA, has been studied for neuro-pharmacological properties and for effects on the catecholamine content of the corpus striatum.2. The acute effects on spontaneous behaviour of rodents included flaccid catalepsy and reversible tranquillization in doses which were 5% or less of the lethal dose. Long lasting depression of the CNS, followed by complete recovery, was produced in the cat and the dog. In the monkey HA-966 caused periodical sleeping episodes.3. The exploratory behaviour and the amphetamine-induced motor activity in mice were blocked by HA-966. The toxicity of amphetamine in aggregated mice was only moderately reduced, suggesting that HA-966 differs from neuroleptics.4. Tremors induced by chemical agents (nicotine, zinc and tremorine) were markedly inhibited by HA-966. The muscarinic effects of tremorine were not reduced by HA-966, indicating a selective central antitremor effect.5. HA-966 elevated the threshold to strychnine convulsions and abolished the ipsilateral flexor reflex, while not having motor endplate blocking properties. It is suggested that HA-966 depresses central internuncial neurones.6. In rats and rabbits HA-966 produced synchronous EEG and inhibited the sensory arousal in doses not causing sedation. In the monkey the drug caused a periodical dissociation between ;sleep-EEG' and behaviour.7. In rat brain, HA-966 selectively elevated the dopamine content in the corpus striatum, while no changes in noradrenaline and 5-hydroxytryptamine contents could be demonstrated. The effect was still present when dopa synthesis was inhibited with alpha-methyl-p-tyrosine.8. Several effects of intravenously administered HA-966 became manifest after an appreciable delay and in hepatectomized mice the effects were much reduced. It is postulated that HA-966 is converted to a pharmacologically active metabolite.9. The results are discussed in the light of current views on drug therapy in extrapyramidal conditions and a GABA-related hypothesis as to the mode of action of HA-966 is presented.

Cyclic-AMP level and eicosanoid release from alveolar macrophages are differentially affected by high and low dose of platelet activating factor.

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.

Sensitization enhances the adenylyl cyclase responsiveness in alveolar macrophages. Changes induced at post-receptor level.

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.

Effects of changes in osmolarity on isolated human airways.

The effects of hypo- and hyperosmolarity on the function of isolated human airways were studied. Changes in osmolarity induced an increasing bronchoconstriction that was proportional to the magnitude of the change in osmolarity. Hypertonicity-induced airway narrowing resulted when buffer was made hypertonic with sodium chloride or mannitol but not with urea. The airways showed no tachyphylaxis to repetitive exposure to hypo- and hypertonic buffer of 200 and 600 mosM, respectively. The bronchoconstriction was not secondary to stimulation of H1 or leukotriene C4/D4 receptors or the release of prostaglandins in the preparation. The bronchoconstriction in hypotonic buffer was totally dependent on extracellular calcium, whereas in hypertonic buffer the bronchoconstriction seemed partially dependent on intracellular calcium release. Isoprenaline prevented the bronchoconstriction in hyper- or hypotonic buffer of 450 and 250 mosM but not in buffer of 600 and 150 mosM. It is concluded that hypo- and hypertonic buffers lead to bronchoconstriction via different mechanisms, which relate to influx of extracellular calcium in hyposmolar buffer and probably to release of calcium from intracellular stores in hypertonic buffer. In strongly hypertonic buffer, part of the bronchoconstriction may be due to osmotic shrinkage. The relevance of our data for the mechanism of bronchoconstriction after inhalation of hypo- or hypertonic saline depends on whether changes in osmolarity around the airway smooth muscle occur in asthmatics but not in normal subjects, and this has not yet been established.

Nonneural components in the response of fresh human airways to electric field stimulation.

Fresh human bronchi, obtained at thoracotomy and maintained at 37 degrees C, were studied in vitro to investigate their response to electric field stimulation (EFS). We found complex responses that were not only composed of a rapid initial nerve-mediated cholinergic contraction and a non-adrenergic nerve-mediated relaxation, but, in 80% of preparations, also of a tonic contraction with a sustained time course. This sustained phase was not blocked by the nervous conductance blocker tetrodotoxin (TTX) and was therefore not neurally mediated. Controlled transient cooling to 4 degrees C in the organ bath reduced this sustained phase selectively for several hours. The leukotriene (LT) antagonist FPL 55712, dexamethasone, which inhibits phospholipase A2, and the antiasthmatic drug cromolyn all reduced the sustained phase significantly. In 20% of strips, an additional TTX-resistant contraction was seen directly after the cholinergic phase. This contraction could be inhibited by indomethacin. A similar small peak sometimes appeared after selective blocking of either the cholinergic or the sustained phases. Experiments in which the epithelium was removed from the strips suggested that this indomethacin-sensitive response, but not the sustained phase, was dependent on the presence of epithelium. These results show that EFS of fresh human bronchi stimulated cholinergic and nonadrenergic inhibitory nerves and gave rise to a partly epithelium-dependent synthesis of arachidonic acid metabolites, which caused contractile responses that interfered with the neurally mediated responses.

Macrophage cytokines render WEHI-3B tumor cells susceptible to cytostasis by prostaglandins.

The growth of the murine myelomonocytic leukemia tumor, WEHI-3B, has been shown to be inhibited by a two-step treatment: first, incubation for one hour with either interleukin-1 (human recombinant IL-1 alpha or tumor necrosis factor (human recombinant TNF-alpha); second, subsequent exposure to prostaglandins. Preincubation with IL-1 rendered the tumor cells more susceptible to subsequent treatment with either prostaglandin E2 or to the stable synthetic analogue of prostacyclin DC-PGI2. Preincubation with TNF-alpha rendered the tumor cells more susceptible to further treatment with PGE2 but not with DC-PGI2. Preconditioning of the tumour cells with either IL-1 alpha or TNF alpha did not affect cytostasis by subsequent culture of tumor cells in presence of either one of the cytokines. It is concluded that the interactions between macrophage cytokines and prostaglandins in enhancement of antitumor activity might imply first binding or induction of certain modifications in the tumor cells by the cytokines which render the cells more susceptible to exposure to prostaglandins.

The adenylate cyclase of rheumatoid synovial fluid macrophages is more sensitive for dl-5E, 19,14-di dehydro-carbo-prostacyclin, a stable prostacyclin analogue, than for prostaglandin E2.

dl-5E, 19,14-di dehydro-carbo-prostacyclin (DDH-carbo PGI2), a stable prostacyclin (PGI2) derivative, but not prostaglandin (PG) E2, stimulated the adenylate cyclase of synovial fluid macrophages, isolated from rheumatoid patients with an active synovitis, in a dose dependent manner (10-1000 ng/ml). DDH-carbo PGI2 also stimulated synovial macrophage cAMP synthesis when injected into the knee joint. Exogenous arachidonic acid (AA) had little effect on cyclic-AMP (cAMP) formation or PGI2 release (assayed as 6ketoPGF1 alpha). It stimulated, however, the release of PGE2 and, to a lesser extent, thromboxane (Tx) A2 (measured as TxB2).

Specific lipoxygenase inhibition reverses macrophage cytotasis towards P815 tumor cells in vitro induced by the calcium ionophore A23187.

A23187-stimulated cytostatic activity of peritoneal macrophages towards P815 tumor cells served as a model for macrophage activation: a macrophage enriched preparation, separated on the basis of cell size in a discontinuous FCS gradient column, expressed cytostatic activity when stimulated by A23187. This was inhibited dose-dependently, by AA-861 but not by nordihydroguaiaretic acid (NDGA). AA-861 inhibited 5-lipoxygenase specifically, NDGA inhibited both 5-lipoxygenase- and cyclooxygenase activity. The ratio cyclooxygenase/lipoxygenase products increased with AA-861 but not with NDGA. These results show that lipoxygenase products are necessary for expression of cytostatic activity of these arachidonic acid metabolite-producing macrophages and that the ratio cyclooxygenase/lipoxygenase metabolites plays an important role in macrophage activation.