Mary Ellen Dahlgren

Macrophages synthesise and release prostaglandins in response to inflammatory stimuli

WHEN macrophages encounter inflammatory stimuli either in vivo or in vitro, they respond by releasing a number of products which may account for the central role that this cell has in chronic inflammatory diseases1. These products include hydrolytic enzymes active at neutral2 or acidic pH (ref. 1), components of both the classical3 and alternate pathway4 of complement, factors modulating responses of lymphocytes to antigens and mitogens5, and factor(s) influencing the proliferation6 and synthesis of collagen7 by fibroblasts. We now show that macrophages whose phospholipid components were labelled with 3H-arachidonic acid also synthesise and release 3H-prostaglandins (PGs) in response to inflammatory stimuli. These observations are consistent with the findings that human macrophages on intrauterine devices8 and guinea pig macrophages responding to lymphokines9 release PGs.

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.

Phorbol myristate acetate induces the secretion of an elastase by populations of resident and elicited mouse peritoneal macrophages

Cultured thioglycollate-elicited mouse peritoneal macrophages secrete an enzyme which hydrolyzes [3H]elastin prepared by NaB3H4 reduction of bovine ligamentum nuchae elastin. Over a 24 h culture period, 3.5 . 10(6) thioglycollate-elicited cells secrete sufficient enzyme to solubilize 200--350 micrograms [3H]elastin in 18h. Secretion at this rate continues for at least 6 days in culture. Secretion of the enzyme is stimulated 3-fold by exposure of the cultured cells to 10(-7) M phorbol myristate acetate, whereas the parent alcohol 4 alpha-phorbol is inactive in this respect. Enzyme activity is linearly related to the amount of conditioned medium assayed and is linear over incubation times up to 30 h. Unlabelled elastin competitively inhibits the solubilization of [3H]elastin. The solubilization rate is doubled if the substrate is pretreated with sodium dodecyl sulfate, but the rate of solubilization of this pretreated substrate increases with time. Resident peritoneal macrophages secrete barely detectable amounts of elastase, but phorbol myristate acetate (10(-7) M) stimulates its secretion in amounts comparable to those secreted by phorbol myristate acetate-stimulated thioglycollate-elicited cells. Dexamethasone (10(-9) M) inhibits phorbol myristate acetate-induced secretion by 50%, but 10(-6) M indomethacin is without effect. The secreted enzyme has the characteristics of a metalloproteinase.

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.

Inhibition of human leukocyte elastase. 6. Inhibition by 6-substituted penicillin esters.

Abstract Penicillin amides substituted at C-6 with either an α- or β-trifluoroacetamido or an α-alkoxy functionality are reported as human leukocyte elastase (HLE) inhibitors. The structure activity relations for these derivatives are discussed and compared to the corresponding known cephalosporin structures in terms of chemical stability, HLE inhibition, and efficacy in an intratracheal (IT) lung hemorrhage assay.

Inhibition of human leukocyte elastase. 5. Inhibition by 6-alkyl substituted penem benzyl esters.

Abstract Penem benzyl esters substituted at the 6-position with small alkyl groups and at the 3-position with a variety of carbon and heteroatom groups were prepared as inhibitors of human leukocyte elastase (HLE). The structure activity relations found for these positions are discussed in relation to know cephalosporin inhibitors and some important stereochemical implications are reported for inhibition of HLE by these β-lactam structures.

Normal Phase High‐Performance Liquid Chromatography of Pneumocandins: In Situ Modification of Silica with l‐Proline To Separate Structural Analogues

Lipopeptides such as pneumocandin B0 are often produced by fermentation processes. Many compounds with similar structures (structural analogues), and hence similar physiochemical properties, are coproduced in the fermentation. We employed high performance liquid chromatography using silica gel as the stationary phase and a ternary ethyl acetate/MeOH/water mobile phase to separate pneumocandin B0 from these structural analogues. Despite extensive efforts to optimize this system, two key structural analogues, pneumocandin E0 and pneumocandin B5, continued to be poorly resolved from the main product peak (pneumocandin B0). As a result, feed load was restricted and productivity was limited. In situ modification of the silica gel stationary phase with l‐proline or other amino acids significantly enhances the resolution of the two key structural analogues from the compound of interest, enabling a two‐fold increase in productivity. Results of a systematic study showed that the amine group in l‐proline and other amino acids plays a key role in the modification of the surface of the silica gel to mediate the selectivity enhancement.