Prostaglandin receptor site: evidence for an essential role in the action of luteinizing hormone.

A dose-response relation was established between prostaglandins and formation of adenosine 3',5'-monophosphate in the mouse ovary. The prostaglandin antagonist, 7-oxa-13-prostynoic acid, blocked the stimulatory effect of prostaglandin E(1), prostaglandin E(2), and luteinizing hormone on adenosine 3',5'-monophosphate formation in a competitive manner. Kinetic studies made it possible to suggest that there is a single luteinizing-hormone-related prostaglandin receptor in mouse ovaries, and that activation of this prostaglandin receptor is an essential requirement in the action of luteinizing hormone to stimulate adenosine 3',5'-monophosphate formation and steroidogenesis.

Effect of RNA and protein synthesis inhibitors on the release of inflammatory mediators by macrophages responding to phorbol myristate acetate.

The interaction of phorbol myristate acetate with resident populations of mouse peritoneal macrophages causes an increased release of arachidonic acid followed by increased synthesis and secretion of prostaglandin E2 and 6-keto-prostaglandin F1 alpha. In addition, phorbol myristate acetate causes the selective release of lysosomal acid hydrolases from resident and elicited macrophages. These effects of phorbol myristate acetate on macrophages do not cause lactate dehydrogenase to leak into the culture media. The phorbol myristate acetate-induced release of arachidonic acid and increased synthesis and secretion of prostaglandins by macrophages can be inhibited by RNA and protein synthesis inhibitors, whereas the release of lysosomal hydrolases is unaffected. 0.1 microgram/ml actinomycin D blocked the increased prostaglandin production due to this inflammatory agent by more than 80%, and 3 microgram/ml cycloheximide blocked prostaglandin production by 78%. Similar results with these metabolic inhibitors were found with another stimulator of prostaglandin production, zymosan. However, these inhibitors do not interfere with lysosomal hydrolase releases caused by zymosan or phorbol myristate acetate. It appears that one of the results of the interaction of macrophages with inflammatory stimuli is the synthesis of a rapidly turning-over protein which regulates the production of prostaglandins. It is also clear that the secretion of prostaglandins and lysosomal hydrolases are independently regulated.

Physiological and pharmacological regulation of prostaglandin and leukotriene production by macrophages.

The synthesis and secretion of prostaglandins and leukotrienes by mouse peritoneal macrophages is under several regulatory controls. Arachidonic acid must first be released from phospholipid stores by the action of phospholipases. Macrophages have the capacity to deacylate arachidonic acid directly from the SN2 position of phospholipids via the action of a phospholipase A2. In addition, these cells contain a phospholipase C capable of removing inositol-phosphate from phosphatidylinositol generating diacylglycerol. Another enzyme, diacylglycerol lipase is present to then generate arachidonic acid. The free arachidonic acid then enters the cyclooxygenase pathway to generate prostaglandins, the lipoxygenase pathway to generate leukotrienes or both pathways. The nature of the inflammatory stimulus added to these cells determines which of the above pathways become operative. Zymosan and the Ca++ ionophore, A23187 stimulate the synthesis of both prostaglandins and leukotrienes whereas phorbol myristate acetate and lipopolysaccharide induce only the synthesis of prostaglandins. In addition, the synthesis of these two products by macrophages can be regulated by certain antiinflammatory compounds. Indomethacin, aspirin, ibuprofen and benoxaprofen are only inhibitors of the prostaglandin pathway, whereas BW755C, 5,8,11-ETYA, NDGA and sulindac sulfide (high doses) are inhibitors of the synthesis of both prostaglandins and leukotrienes. Dapsone, an effective drug for leprosy, also inhibits the synthesis of both of these products.

rTNF alpha facilitates human polymorphonuclear leukocyte adherence to fibrinogen matrices with mobilization of specific and tertiary but not azurophilic granule markers.

rTNF alpha facilitates highly reproducible adherence of polymorphonuclear leukocyte (PMN) to fibrinogen-coated surfaces in a concentration- and time-dependent manner. The adhesion was maximal with 1.0 nM rTNF alpha within 40-50 min at 37 degrees C. A monoclonal antibody (1B4) directed toward the beta 2-chain of the integrin receptor for fibrinogen (CD11b, CD18) completely inhibited the rTNF alpha induced adhesion. TNF alpha caused a time-dependent secretion of the granule markers gelatinase and lactoferrin but no liberation of myeloperoxidase and minimal production of A alpha(1-21), a specific cleavage product of fibrinogen generated by elastase, as markers for the azurophilic granule. PMN adhered to fibrinogen in the presence of rTNF alpha could be further stimulated with cytochalasin B and N-formyl-methionyl-leucyl-phenylalanine (FMLP) to release azurophilic granule markers as measured by increasing MPO activity and A alpha(1-21) production over time. Thus the rTNF alpha-facilitated adherence of PMN to a fibrinogen matrix provides a system for partial activation of PMN resulting in release of markers of specific and tertiary but not azurophilic granules. Moreover, these conditions should provide an opportunity to define more clearly the signal transduction processes involved in azurophilic granule release.

Prostaglandin and leukotriene synthesis in mouse ears inflamed by arachidonic acid.

Topical application of arachidonic acid on mouse ears induces the synthesis of prostaglandin E2 and leukotrienes C4 and D4. The increased tissue levels of these products are quantitated by radioimmunoassay. The identity of the leukotrienes was confirmed by immunoreactivity of reverse-phase high-performance liquid chromatography fractions corresponding to authentic standards. Synthesis of the arachidonic acid metabolites precedes or is coincident with increased vascular permeability resulting in an edematous response, as measured by accumulation of [125I]albumin in the ear after i.v. injection or by tissue wet weight. When applied topically, anti-inflammatory drugs such as BW755C (3-amino-1-(m-[trifluoromethyl]phenyl)2-pyrazoline, indomethacin, and nordihydroguaiaretic acid inhibit edema and modulate the appearance of the arachidonic acid products. The data suggest the coinvolvement of prostaglandin E2 and leukotrienes C4 and D4 as mediators of inflammation in this in vivo model.

The role of macrophage secretory products in chronic inflammatory processes.

Mononuclear phagocytes participate in various stages of chronic inflammatory responses and associated diseases. Such participation is mediated by (a) direct interaction with pericellular interstitial tissue components as well as with other cell types present at sites of inflammation and (b) by secretion of soluble mediators. Several of these mediators are synthesized and secreted in increased amounts after macrophages interact with inflammatory stimuli. In this paper we pay particular attention to neutral proteinases and prostaglandins. It is shown that these 2 classes of mediators are released in significant amounts under different conditions. Prostaglandins are synthesized most readily by resident populations of mouse peritoneal macrophages responding to various model inflammatory stimuli. Mouse peritoneal macrophage populations elicited in vivo by inflammatory stimuli are less responsive in this respect. In contrast neutral proteinase secretion does not occur in resident cell populations but is observed on a continuous basis in elicited populations. Such secretion can be increased further by addition of phagocytic stimuli and initiated in resident populations by model inflammatory stimuli such as phorbol myritate acetate. Other secretory products of macrophages with possible relevance to inflammation are discussed briefly. Finally some of the effects of antiinflammatory glucocorticoids, cyclooxygenase inhibitors and dapsone on the secretory activity of macrophages are briefly summarized.

Effects of cyclooxygenase and lipoxygenase inhibitors on inflammation associated with oxazolone-induced delayed hypersensitivity.

Oxazolone-induced delayed hypersensitivity in mice produced swelling with concomitant increased tissue levels of leukotrienes and prostaglandins. Pharmacological agents were coapplied topically with oxazolone at the time of challenge in an attempt to modulate the immune-based inflammation. Dexamethasone inhibited both swelling and increases in eicosanoid levels. Indomethacin reduced prostaglandin levels but failed to inhibit swelling or reduce leukotriene levels. L-651,896 (2,3-dihydro-6-[3-(2-hydroxymethyl)phenyl-2-propenyl]-5-benzofuranol), a 5-lipoxygenase inhibitor, reduced leukotriene levels but did not reduce swelling or prostaglandin levels. A combination of indomethacin and L-651,896 reduced eicosanoid levels but did not reduce swelling. These data suggested that the reduction in tissue levels of 5-lipoxygenase or cyclooxygenase oxygenation products of arachidonic acid either singularly or together did not result in the concomitant reduction of the inflammation associated with oxazolone-induced delayed hypersensitivity.

Pharmacological modulation of eicosanoid levels and hyperalgesia in yeast-induced inflammation.

Injection of brewer's yeast into the rat paw results in edema and a subsequent hyperalgesia. The edema was accompanied by an increase in 5-lipoxygenase products, and the hyperalgesia coincided with the formation of both cyclooxygenase and 5-lipoxygenase products. When administered perorally, indomethacin inhibited cyclooxygenase product formation, phenidone inhibited 5-lipoxygenase product formation, and 3-amino-1-(m-[trifluoromethyl]-phenyl)-2-pyrazoline (BW 755C) inhibited formation of products of both pathways. These compounds were also effective analgesic agents. The correlation of these effects with the suppression of hyperalgesia suggests the participation of products from both cyclooxygenase and 5-lipoxygenase pathways in the mediation of hyperalgesia.

Formation of polymorphonuclear leukocyte elastase: alpha 1 proteinase inhibitor complex and A alpha (1-21) fibrinopeptide in human blood stimulated with the calcium ionophore A23187. A model to characterize inhibitors of polymorphonuclear leukocyte elastase.

Incubation of human blood with the secretagogue A23187 resulted in the formation of increased plasma concentrations of polymorphonuclear leukocyte (PMN) elastase: alpha 1 proteinase inhibitor (PMNE:alpha 1 PI) complex as well as A alpha(1-21) fibrinopeptide [A alpha(1-21)]. The formation of these species was both time and A23187 concentration dependent. Using a sandwich ELISA and a radioimmunoassay, we determined the comparative potencies of several compounds to inhibit the formation of PMNE: alpha 1 PI complexes and A alpha(1-21), respectively. L-658,758, a substituted cephalosporin, essentially irreversible elastase inhibitor, inhibited the formation of PMNE: alpha 1 PI and A alpha(1-21) with IC50 values of 38 and 15 microM, respectively. L-683,845, a monocyclic beta-lactam, was much more potent against isolated PMNE than L-658,758. However in this system it was approximately equivalent to L-658,758 with an IC50 of 15 microM against both species. ICI-200,880, a competitive slow-binding elastase inhibitor, was significantly less potent to inhibit A alpha(1-21), having an IC50 of 75 microM, while Declaben, a reversible noncompetitive inhibitor, was inactive at concentrations as great as 200 microM. We propose that evaluating inhibitors in the complex milieu of blood will provide a useful method to predict their therapeutic potential in vivo.

Inhibition of the release of prostaglandins, leukotrienes and lysosomal acid hydrolases from macrophages by selective inhibitors of lecithin biosynthesis.

The release of the inflammatory mediators, prostaglandins (PGs), leukotrienes (LT) and lysosomal acid hydrolases (LAH), by macrophages is stimulated by endocytic stimuli such as zymosan. This process can be interfered with by specific inhibitors of phosphatidylcholine (PC) biosynthesis. The diphenylsulfone dapsone and three analogs selectively inhibited [14C]choline incorporation into PC but had varied effects on inhibition of mediator release by macrophages. Dapsone inhibited the release of PGs, LT and LAH, whereas the three closely related structural analogs inhibited LAH release only, with little or no effect on PG production.

Pharmacological effects of non-steroidal antiinflammatory agents on prostaglandin and leukotriene synthesis in mouse peritoneal macrophages.

Resident mouse peritoneal macrophages, exposed to zymosan, synthesized and released products of both the cyclooxygenase and lipoxygenase pathways. The effects of various non-steroidal antiinflammatory agents were evaluated for their abilities to inhibit zymosan-stimulated prostaglandin E2 (PGE2) and leukotriene C4 (LTC4) synthesis. The order of potencies to inhibit PGE2 synthesis and release was: indomethacin greater than or equal to sulindac sulfide greater than ibuprofen greater than or equal to aspirin greater than 3-amino-1-[3-(trifluoromethyl)-phenyl]-2-pyrazoline (BW755C) greater than benoxaprofen greater than or equal to nordihydroguaiaretic acid (NDGA) greater than 5,8,11-eicosatriynoic acid (ETYA). BW755C and ETYA also inhibited zymosan-stimulated LTC4 production. None of the compounds tested showed selective inhibition of lipoxygenase products.

Biologically active derivatives of fatty acids: prostaglandins, thromboxanes, and endoperoxides.

The causal role assigned to the E and F prostaglandins in inflammatory processes, implied by the antiinflammatory action of prostaglandin synthetase inhibitors, is not consistent with the findings reported here that a compound (MK-447) capable of increasing levels of these prostaglandins is antiinflammatory in classical animal models of acute inflammation. That both MK-447 and prostaglandin synthetase inhibitors depress the enzymatic formation of PGG2 from arachidonic acid suggests that this endoperoxide plays a pivotal role in acute inflammation. However, in view of the intermediate nature of PGG2, it seems likely that such a pivotal role for this substance is a function of its ability to be converted to other inflammatory mediators. Possible candidates for a causal role are thromboxane A2 (TXA2) prostacyclin (PGI2), both of which derive from PGG2. However, direct evidence is presented to show that an oxygen equivalent released in the enzymatic conversion of PGG2 to PGH2 is a prime factor in inflammation.

Effect of dialyser composition and reuse on neutrophil count and elastase alpha-1 proteinase inhibitor complex formation.

To assess the inter-relationship of leucopenia and PMN elastase release we undertook a prospective crossover study of 6 patients dialysed with new and reused cuprophane, cellulose acetate and polysulfone membranes. Serial blood samples were analysed for PMN count, and elastase-alpha 1-proteinase inhibitor complex (E alpha 1PI) concentrations. After 15 min dialysis with new membranes median PMN counts fell by 72.2%, 25.3% and 22.1% with cuprophane, cellulose and polysulfone, respectively. With reuse the decreases were reduced to 6.4%, 8% and 13.6%. All membranes produced a gradual increase of E alpha 1PI. Median E alpha 1PI accumulation rates (ng ml-1 min-1) with new membranes were 175, 169 and 187 for cuprophane, cellulose acetate and polysulfone, respectively. With reuse of cuphrophane and cellulose acetate these rates fell to 99 and 109 (p less than 0.05 and p less than 0.05, respectively), however, with polysulfone it remained unchanged at 180 ng ml-1 min-1. This study highlights differences between two aspects of the neutrophil response to haemodialysis, and demonstrates that extrapolation from individual parameters to conclusions concerning biocompatibility may be inappropriate.

Effect of tumor necrosis factor on granule release and LTB4 production in adherent human polymorphonuclear leukocytes.

Tumor necrosis factor (rTNF) has previously been shown to induce PMN chemotaxis, stimulate PMN adhesion to vascular endothelium and stimulate hydrogen peroxide secretion from PMNs adhered to biological surfaces. We investigated the activity of both rTNF alpha and rTNF beta on adherent and suspension cultures of human PMNs. rTNF alpha selectively stimulated the release of the specific granule in a dose dependent manner. Exocytosis of the specific granule was measured with an enzyme-immunoassay for lactoferrin and a radioassay for vitamin B12-binding protein. Adherent PMNs released up to 60% of the total lactoferrin content of the cells with no increase in myeloperoxidase (MPO) secretion when stimulated with 0.1-10 nM rTNF alpha. The PMNs in suspension cultures also selectively released the specific granule, although total release was reduced suggesting that adherence of PMNs increased their ability to respond to physiological stimuli. When PMNs in suspension cultures or adherent cells were stimulated with rTNF alpha, no LTB4 production was detectable, yet the cells retained the ability to synthesize LTB4 when stimulated with calcium ionophore A23187. Neither rTNF alpha or rTNF beta stimulated the release of the azurophilic granule, measured by the secretion of MPO and neutrophil elastase activity. These results suggest that a function of rTNF alpha and rTNF beta on PMNs is the release of the contents within the specific granule.

The role of arachidonic acid metabolism in the activities of interleukin 1 and 2.

Several investigations have suggested that products of arachidonic acid metabolism have modulatory effects on the development of cellular immunity. In this report we have studied the role of arachidonic acid metabolism in the specific effects of interleukin 1 (IL 1) induction of interleukin 2 (IL 2), and also IL 2 stimulation of proliferation and interferon-gamma (IFN-gamma) production. Utilizing cell lines that are specifically responsive to IL 1 or IL 2, it was found that both interleukins stimulate lipoxygenation of arachidonic acid in their respective target cell. The ability of each interleukin to induce monohydroxyeicosatetraenoic acid (HETE) correlated with the induction of secondary lymphokine secretion. Utilizing selective and partially selective pharmacologic inhibitors of arachidonic acid metabolism, the data suggest that the participation of lipoxygenase activity is required for both IL 1 induction of IL 2 production and IL 2 regulation of proliferation and IFN-gamma secretion. The same requirement for lipoxygenase activity was seen when phorbol myristate acetate (PMA) was used as a secretory stimulant, suggesting a similar mode of action for stimulation-secretory activity between PMA and interleukins. Studies performed with an endogenous inhibitor of 5-lipoxygenase (15-HETE) demonstrated the requirement of this enzyme system for IL 2-dependent proliferation and IFN-gamma production. Although leukotrienes could replace IL 2 for IFN-gamma secretion, they had no effect on IL 2 growth promotion. The results suggest that both IL 1 and IL 2, and PMA, may share the lipoxygenase pathway of arachidonic acid metabolism which is a component of the intracellular signal transduction process that regulates secretory activity and/or cellular proliferation.