Arachidonic acid metabolism of rat peritoneal macrophages after passive sensitization and allergen challenge.

The aim of our work was to evaluate the effect of passive sensitization of rat peritoneal macrophages (treatment of cells by an anti-ovalbumin IgE-rich serum) on arachidonic acid (AA) metabolism and the impact of specific antigen (ovalbumin) on this process. Compared to a control treatment without serum, the atopic serum and a serum without IgE, used on [3H]AA-labeled macrophages, increased cyclooxygenase and lipoxygenase eicosanoid production. Sera, used prior to [3H]AA incorporation, induced a decrease of 3H-labeled membrane phospholipids and an increase of lipoxygenase metabolites in the [3H]AA incorporation medium. To establish if the serum-induced catabolism of AA differed according to whether it was externally added to the culture medium or incorporated into membrane phospholipids, we studied the eicosanoid secretion of [3H]AA-prelabeled macrophages, treated by the serum and incubated with [14C]AA. It was confirmed that phospholipid-incorporated AA was catabolised following the cyclooxygenase and lipoxygenase pathways and external AA preferentially following the lipoxygenase pathway. The allergen increased the eicosanoid formation of passively sensitized macrophages but not that of cells treated by the serum without IgE. Our data suggest that changes occurring in passive sensitization, on AA mobilization from membrane phospholipids and on AA catabolism, induced by the serum, are independent of IgE and must be taken into consideration on interpreting the allergen effect.

Possible implication of arachidonic acid metabolism in the decrease of chemiluminescence production after exposure of murine peritoneal macrophages to diethylnitrosamine and tumour promoter, 12-O-tetradecanoylphorbol-13-acetate.

The effect of the carcinogen diethylnitrosamine (DEN) on prostaglandins (PGs), leukotrienes (LTs) and reactive oxygen intermediates production by murine peritoneal macrophages was assessed. In vitro exposure to DEN (0.8, 1.6 and 8 mM) resulted in a dose-dependent stimulation of the PGs and LTs generation by macrophages. DEN-exposed peritoneal macrophages demonstrated enhanced production of arachidonic acid (AA) metabolites following stimulation with 12-O-tetradecanoylphorbol 13-acetate (TPA) as compared to macrophages stimulated with TPA alone. Studies of [3H]AA release from glycerolipids of prelabelled macrophages and of the distribution of AA metabolites between intra and extracellular compartments indicated that DEN induced de novo synthesis of AA metabolites. The stimulation of AA metabolism by DEN was decreased by H-7 and staurosporine, protein kinase C (PKC) inhibitors, and so could be dependent on PKC activation. The generation of PGs by macrophages after DEN exposure was also inhibited by indomethacin (cyclo-oxygenase inhibitor). DEN at high concentrations (1.6-16 mM) inhibited chemiluminescence production by peritoneal macrophages in a dose-dependent manner, triggered by tumour promoter TPA; lower concentrations (0.8 and 1.2 mM) increased this reactive oxygen intermediates dependent chemiluminescence production induced by TPA. The role of AA metabolism in the alteration of chemiluminescence production by murine peritoneal macrophages treated in vitro with DEN and triggered by TPA has been evaluated by using AA metabolism inhibitors. The stimulation of chemiluminescence by TPA was inhibited by the addition of phospholipase A2 (PLA2) inhibitor, 4-p-BPB; this metabolic inhibitor did not affect the decrease of chemiluminescence production induced by DEN. The cyclo-oxygenase (CO) inhibitor, indomethacin, reversed the inhibition of TPA-induced chemiluminescence caused by DEN. These results suggest that AA and/or a lipoxygenase product can potentiate the reactive oxygen intermediates production by macrophages stimulated by TPA. The CO pathway could be involved in the inhibition by DEN of the reactive oxygen intermediates generating enzyme system. It is suggested that this inhibition could be related to AA metabolites issued from the CO pathway or to DEN oxygenated metabolites issued from the co-oxidation of the DEN by the PGs endoperoxide synthase. These results also raise the problem of macrophage dysfunction by chemical carcinogens and the implication of the CO pathway in this process.

The lavage fluid from a patient with alveolar proteinosis inhibits the in vitro chemiluminescence response and arachidonic acid metabolism of normal guinea pig alveolar macrophages.

Pulmonary alveolar proteinosis is characterized by the accumulation of a lipoproteinaceous material within the alveoli of the lung. It is well established that patients with pulmonary alveolar proteinosis have a high incidence of complicating pulmonary infections possibly resulting from defects of antibacterial functions of alveolar macrophages. Moreover, for unclear reasons, an inflammatory response in the airways is frequently absent. In order to investigate the role of the lipoproteinaceous material in these two patterns, we tested the in vitro effects of a lavage fluid from a human pulmonary alveolar proteinosis on the secretion of reactive oxygen intermediates and arachidonic acid metabolites by normal guinea pig alveolar macrophages. After incubation with the lipoproteinaceous material, the luminol-enhanced chemiluminescence of zymosan-triggered alveolar macrophages was reduced in a dose-dependent fashion. The lipoproteinaceous material similarly reduced the chemiluminescence response in a cell-free xanthine-xanthine oxidase system generating superoxide anions. This latter observation suggests that the lipoproteinaceous material acts as a scavenger for superoxide anions produced by alveolar macrophages. Its purified protein or phospholipid fractions also resulted in a general inhibition of the secretion of arachidonic acid metabolites by alveolar macrophages challenged in vitro with zymosan. Our results suggest that the alveolar filling material of pulmonary alveolar proteinosis may inhibit the action of antibacterial and/or proinflammatory agents produced by alveolar macrophages. We speculate that such effects of the lipoproteinaceous material may play a role in vivo in the high incidence of pulmonary infections and in the absence of discernible interstitial or intraalveolar inflammation seen in pulmonary alveolar proteinosis.

[Effects of cotrimoxazole on some macrophage functions: microbicide, tumoricide, production of free oxygen radicals, prostaglandins and leukotrienes]. / Effets du cotrimoxazole sur certaines fonctions de macrophages: microbicidie, tumoricidie, production de radicaux libres oxygénés, de prostaglandines et de leucotriènes.

In order to demonstrate an immunomodulating effect of cotrimoxazole, we investigated its influence on some macrophage (M phi) functions in culture: P815 tumor cells killing, Toxoplasma gondii killing, production of free oxygen radicals by luminol-dependent chemiluminescence, prostaglandins and leukotrienes secretion evaluated after incorporation of tritiated arachidonic acid. In vitro, cotrimoxazole inhibited in a dose-dependent fashion the chemiluminescence of murine resident peritoneal or guinea pig alveolar M phi. Production of prostaglandin (PG) 6-keto-F1 alpha, PGF2 alpha, and 5-hydroxyeicosatetraenoic acid by resident peritoneal M phi was also inhibited. However, PGD2 synthesis by alveolar M phi was enhanced. A second study was performed on peritoneal M phi, resident or elicited in vivo by one intra-peritoneal injection of an extract from Mycobacterium Tuberculosis membranes and obtained from mice pretreated or not by cotrimoxazole per os. Resident M phi from cotrimoxale-treated animals showed increased production of leucotriene B4 compared to M phi from controls. 6-keto-PGF1 alpha and free oxygen radicals production by elicited M phi was greatly enhanced by cotrimoxazole whereas thromboxane B2 was reduced. Finally cotrimoxazole enhanced intracellular killing of Toxoplasma gondii and cytotoxicity for tumor cells P815 by resident but not by elicited M phi. It is concluded that cotrimoxazole can modulate MO activation and some M phi functions involved in immune homeostasis. This data could help to understand why an antibiotic such as cotrimoxazole, which is known to be frequently used in immunocompromised hosts, is also efficient in Wegener's granulomatosis.

1-naphthyl N-methyl carbamate effect on intra- and extracellular concentrations of arachidonic acid metabolites, and on the chemiluminescence generation by mouse peritoneal macrophages.

1-Naphthyl N-methyl carbamate (carbaryl), potent carbamate insecticide with anticholinesterase activity, was tested for its ability to affect mouse peritoneal macrophages in particular arachidonic acid (AA) metabolism and oxidative burst. Carbaryl inhibited in a dose-related manner the reactive oxygen intermediate dependent chemiluminescence (CL) induced by opsonized zymosan (OZ), 12-O-tetradecanoyl phorbol-13-acetate (TPA) and calcium ionophore (A23187); this carbamate did not affect CL-mediated by AA. The intracellular and extracellular concentrations of prostaglandins (PGs) and 5-hydroxyeicosatetraenoic (5-HETE) generated in macrophages stimulated with OZ has been investigated for various periods. Carbaryl effect displayed two successive phases on AA metabolism stimulation. In a first phase (up to 2-15 min), carbaryl did not alter the rapid AA metabolite synthesis (total amount of intra- and extracellular metabolites) but it increased intracellular concentration of PGE2, PGA2, PGF2 alpha and decreased 5-HETE intracellular concentration. In a second phase (after 2-15 min), carbaryl inhibited AA metabolite synthesis. The release of cyclooxygenase (CO) and lipoxygenase (LO) metabolites decreased, in particular PGF2 alpha and PGD2 which in addition seemed to be submit to a cellular retention; the inhibition of other metabolite release appeared essentially related to the inhibition of their synthesis since the intracellular amount did not augment. The inhibition by carbaryl of the NADPH-oxidase dependent CL induced by OZ may be related to the alteration of the intra- and extracellular concentrations of AA metabolites.

Chemiluminescence response and arachidonic acid metabolism of macrophages induced by gamma-hexachlorocyclohexane (lindane).

The influence of gamma-hexachlorocyclohexane (gamma-HCCH) on arachidonic acid (AA) metabolism and oxygen metabolite production was investigated on mouse peritoneal macrophages gamma-HCCH stimulated 6KPGF1 alpha, PGE2, LTC4, LTB4 and HETEs production and increased the luminol-dependent chemiluminescence (CL). Lindane acted synergistically with phorbol ester on prostaglandins-leukotrienes (PGs-LTs) and CL production. Similar stimulation of CL and PGs-LTs production was found after challenge by the calcium ionophore A23187. The implication of calcium mobilization in lindane effects was proposed.

Lipid peroxidation in rat liver microsomes: influence of carcinogenic N-nitrosocarbaryl.

The reactivities of carbaryl, N-methyl 1-naphthylcarbamate insecticide and its N-nitrosated derivative carcinogenic, N-nitrosocarbaryl, were investigated on the microsomal hepatic lipid peroxidation and NADPH-dependent reductase activities. The in vivo treatment by N-nitrosocarbaryl produced a reduction in lipoperoxidative degradation induced in vitro by NADPH with regard to the formation of malonaldehyde and conjugated dienes. Carbaryl, its precursor did not affect lipid peroxidation under the same in vivo conditions. Moreover, following administration of the 2 compounds, the activities of NADPH-cytochrome c reductase as well as NADPH-neotetrazolium reductase were significantly decreased by N-nitrosocarbaryl but not influenced by carbaryl. Correspondingly, in vitro studies were performed; different action patterns of the 2 tested xenobiotics were also noted after treatment of rat liver microsomes in vitro by carbaryl and N-nitrosocarbaryl especially in their ability to cope with microsomal oxygen activation. N-Nitrosocarbaryl proved to have a potent inhibitor concentration effect on NADPH-dependent chemiluminescence response in vitro; carbaryl was virtually ineffective on this parameter. No significant difference appeared in the affinity of N-nitrosocarbaryl and carbaryl for the microsomal phospholipids. From the in vitro explorations, it was suggested that carcinogenic N-nitrosocarbaryl may be involved in the inhibition mechanism of microsomal lipid peroxidation through decreases in both NADPH-dependent reductase activities and superoxide generation.

Interaction of carbaryl and N-nitrosocarbaryl with microsomal monooxygenase activities.

The in vitro interactions of carbaryl and carcinogenic N-nitrosocarbaryl with rat liver microsomal monooxygenase activities are compared. The inhibitory effect of the nitroso-compound is demonstrated to be non-competitive on aminopyrine N-demethylase, p-nitroanisole O-demethylase and aniline hydroxylase. The nature of the inhibition induced by the parent amide is found to be competitive on aminopyrine N-demethylase and p-nitroanisole O-demethylase. Correspondingly, in vitro studies of the metabolism of the two compounds were carried out: they both yield formaldehyde. Moreover, N-nitrosocarbaryl is denitrosated through a NO-cytochrome P-450 complex during microsomal metabolism. The toxic effects and biological activities of the two compounds are discussed on the basis of data of metabolic studies and different patterns of enzyme inhibition.

The effect of carbaryl on the arachidonic acid metabolism and superoxide production by mouse resident peritoneal macrophages challenged by zymosan.

Carbaryl, a broad spectrum insecticide with anticholinesterase activity, was tested for its ability to disturb resident peritoneal macrophages stimulated by opsonized zymosan. The effect of carbaryl on superoxide production and on the release of [1-14C] arachidonic acid and 14C-labelled prostaglandins was dose-dependent. For 2.5 X 10(-6) M of carbaryl, superoxide production and prostaglandin release were not significantly inhibited. At 12.5 X 10(-6) M, the inhibitory effect was apparent for superoxide production (33%) and for the release of 6 KPGF1 alpha (60%), PGE2 (42%), PGF2 alpha (38%), PGD2 (33%). Carbaryl had no effect on the level of free arachidonic acid. Insecticide at 12.5 X 10(-6) M significantly decreased the deacylation of the phosphatidylcholine (20%). Incubation of resident peritoneal macrophages with indomethacin studied conjointly decreased only the prostaglandin release. These results suggest that carbaryl decreases the sequence of events following the binding of a particulate agent to its receptor and leading to the induction of phospholipase activity and the subsequent release of 20:4 and the oxidative burst in the cells. The effect of this pesticide on phospholipid metabolism and its consequences on macrophage stimulation are discussed. Ecto-serine esterase inhibition in the effect mechanism of the pesticide was suggested.

Effect of fasting and streptozotocin in the obese-hyperglycemic (ob/ob) mouse. Apparent lack of a direct relationship between insulin binding and insulin effects.

The decrease of insulin binding to plasma membranes of liver, adipose, and muscle tissues observed in obese-hyperglycemic (ob/ob) mice was reversed towards normal by prolonged fasting or streptozotocin treatment. The extent of this reversal was related to that of the decrease in hyperinsulinemia of the obese mice. In contrast, binding of glucagon to liver plasma membranes was little influenced by fasting or streptozotocin treatment of obese animals. The relationship between insulin binding and metabolic effects of the hormone did not appear to be identical in all tissues. In muscle, insulin binding and insulin effect on glucose uptake and metabolism changed in parallel--i.e., when binding increased, tissue sensitivity to the hormone increased. In the liver, the increase in insulin binding that followed fasting or streptozotocin treatment was not accompanied by any detectable metabolic effect of insulin on hepatic metabolism. A similar situation appeared to prevail in adipose tissue. The varying relationships observed between the state of insulin binding to membranes and the target tissue responsiveness to the hormone probably reflect the multiplicity of the factors operative in these processes and help us to understand why the over-all obese-hyperglycemic syndrome of ob/ob mice cannot be improved simply by decreasing endogenous hyperinsulinemia.

Insulin receptors in the heart muscle. Demonstration of specific binding sites and impairment of insulin binding in the plasma membrane of the obese hyperglycemic mouse.

The presence of insulin receptors in the heart muscle was investigated by measuring the binding of 125I-insulin to specific subcellular fractions of the rat and mouse myocardium. 125I-insulin bound to the plasma membrane fraction with a high degree of specificity and affinity. Insulin analogues competed with 125I-insulin in direct proportion to their biologic potency in vitro. Unlabeled insulin within the range of its concentrations in vivo inhibited 15 to 60 per cent of the 125I-insulin binding. The specific binding sites were finite in number and represented about 90 per cent of the total binding. The insulin-binding capacity of the plasma membrane fraction was twelve- to fifteenfold higher than that of the mitochondrial fraction. As in the liver, the binding was time- and temperature-dependent with a slower but higher binding achieved at a lower temperature. The binding sites appeared to be heterogeneous with respect to affinity. At 5 degrees C., the "higher-affinity" site had a K of about 2 times 10(9) M-1. No more than 10 per cent of the 125I-insulin was degraded by the heart plasma membranes after one hour at 30 degrees C. or twenty-two hours at 5 degrees C. Studies in the obese hyperglycemic (ob/ob) mouse revealed that the insulin binding is impaired in the heart muscle of this animal. Over a wide range of insulin concentrations, the plasma membrane fraction of ob/ob mice bound only 25 to 40 per cent as much insulin as did membranes of the thin littermates, suggesting that, as in the liver, the fat tissue, and the thymic lymphocyte, the number of insulin-binding sites is decreased in the heart of the ob/ob mouse. This defect selectively affected the plasma membrane fraction and could not be explained by differences in membrane purification or insulin-degrading activity. Heart and liver membranes of forty-hour fasted ob/ob mice bound two to three times as much insulin as did membranes of ob/ob mice fed ad libitum. These studies demonstrate and characterize the binding of insulin to heart muscle membranes; they extend to the heart muscle the insulin receptor defect also found in liver membranes and cells, in fat cell membranes, and in thymic lymphocytes of the ob/ob mouse.