Barbara J. Bowman

Properties of Interleukin-1 as a complete secretagogue for human neutrophils

Human monocyte-derived Interleukin-1 (IL-1) stimulated a concentration-dependent extracellular release of azurophil (myeloperoxidase) and specific (vitamin B12-binding protein) granule constituents from cytochalasin B-treated human neutrophils. The serine protease inhibitors, L-1-tosylamide-2-phenylethyl-chloromethyl ketone (TPCK) and N-alpha-p-tosyl-L-lysine-chloromethyl ketone (TPCK) as well as an inhibitor of thiol protease activity, p-hydroxymercuribenzoate (PHMB), suppressed granule enzyme release from neutrophils activated with IL-1. Cycloheximide, an inhibitor of protein synthesis, had no effect on IL-1-induced neutrophil degranulation. Neutrophils pretreated with IL-1 were rendered unresponsive to subsequent exposure to this stimulus. IL-1-elicited granule exocytosis appears to be stimulus specific in that N-formyl-methionyl-leucyl-phenylalanine (FMLP), 1-0-hexadecyl/octadecyl-2-0-acetyl-sn-glyceryl-3-phosphorycholine (AGEPC), and 5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (LTB4) were capable of eliciting a secretory response from IL-1-pretreated cells.

The systemic response to inflammation

Abstract Various acute phase reactants (APR) have been studied in rats with different types of inflammation. These APR (serum albumin, serum globulins, plasma fibrinogens, serum alpha-2-glycoprotein [GP], total serum glycoproteins, serum mucoproteins) and neutrophils and lymphocytes reflect the onset, intensity, and duration of many types of experimental inflammation. The APR and cellular changes, like the inflammatory processes eliciting them, are stereotyped. Chemically induced, chronic immunologic, and delayed hypersensitivity-type acute inflammatory reactions cannot be distinguished by the APR. The most sensitive and quantitatively reliable index for the assessment of the “anti-inflammatory” action of the various steroidal and nonsteroidal drugs is the behaviour of the serum alpha-2-glycoproteins. This is also the most sensitive index of the “anti-inflammatory” action of adrenocortical steroids (or their absence, as shown in adrenalectomized rats). The various serum protein changes in rats with inflammation occur in response to greater sequestration rates at the sites of inflammation. Acute and chronic inflammation can be induced with a variety of substances affecting cell membranes (Filipin, digitonin, saponin, nystatin, d -α-tocopherol, vitamin A, and etruscomycin, to name but a few). These inflammatory reactions are inhibited in vivo by both steroidal and nonsteroidal “anti-inflammatory” drugs. We have visualized the process of inflammation as a wave of leaky membranes. The protective effects of nonsteroidal “anti-inflammatory” drugs on heat-induced and hypotonic erthrocyte lysis in vitro partially confirm the idea. The anti-arthritic effects of cytarabine (cytosine arabinoside, an immunosuppressant drug) also have been studied. Cytarabine has no systemic or local “anti-inflammatory” effect in a variety of experimental assay systems. It inhibits completely the onset of adjuvant arthritis and renders animals tolerant to further adjuvant inoculations. The APR cannot be used exclusively to distinguish between the different steroidal, non-steroidal, and immunosuppressant types of “anti-inflammatory” drugs. They are useful, however, in the overall assessment of the “anti-inflammatory” effects of drugs in all types of inflammation. The APR should be incorporated as standard procedures in laboratories “discovering” new “anti-inflammatory” drugs. Emphasis is placed on the APR as necessary requirements for the assessment of “anti-inflammatory” drugs because for example, Filipin-induced hind-paw edema can be inhibited in vivo by the i.p. administration of digitonin, another lytic agent which produces inflammation.

Anti-inflammatory and PG inhibitory effects of phenacetin and acetaminophen.

Although acetaminophen and phenacetin do not inhibit PG synthesis when added directly to isolated rat platelets and when given orally, both drugs inhibit carrageenan-induced hindpaw edema — a widely used inflammatory assay method. It does not appear that the anti-inflammatory effects of these non-steroidal drugs (unlike other well-known non-steroidal drugs) can be explained on the basis of PG-synthetase inhibition.

Effect of 6,9-deepoxy-6,9-(phenylimino)-Δ 6,8-prostaglandin 11 (U-60,257), an inhibitor of leukotriene synthesis, on human neutrophil function

Abstract A23187, a calcium ionophore, stimulated a time-dependent generation of 5(S), 12(R)-dihydroxy-6,8,10,14-eicosatetraenoic acid (leukotriene B4), production of superoxide anion (O2−) and release of granule-associated β-glucuronidase and lysozyme by human neutrophils. Leukotriene B4 also elicited the selective release of granule enzymes from cytochalasin B-treated neutrophils. U-60,257, a recently identified inhibitor of leukotriene (LT) C4 and D4 synthesis, caused a dose-related (1–10 μM) suppression of LTB4 production by A23187-activated neutrophils. Degranulation and O2− generation by neutrophils exposed to A23187 and the chemotactic oligopeptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP), were also inhibited with U-60,257.

An evaluation of the relationship between arachidonic acid lipoxygenation and human neutrophil degranulation

Abstract Exposure of neutrophils to the calcium ionophore A23187 (5 μ M ) resulted in a time-dependent generation of 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) and the selective release of the granule-associated enzymes, β-glucuronidase and lysozyme. The acetylenic fatty acids, 5,8,11,14-eicosatetraynoic acid, 4,7,10,13-eicosatetraynoic acid, 6,9,12-octadecatriynoic acid, and 10,13-eicosadiynoic acid, which inhibit the lipoxygenase and/or cyclooxygenase pathways of arachidonic acid metabolism, caused a dose-related inhibition of 5-HETE production (50–100 μ M ) by and enzyme release (0.01–100 μ M ) from A23187-stimulated neutrophils. However, these compounds also enhanced 5-HETE synthesis at concentrations (1–10 μ M ) which suppressed enzyme release. Certain phenylhydrazone derivatives, while having no effect on β-glucuronidase or lysozyme release, caused a significant inhibition of A23187-induced 5-HETE generation. Further, the aforementioned acetylenic acids as well as 5,8-tetradecadiynoic acid, which is inactive against 5-lipoxygenase, inhibited N -formyl-methionyl-leucyl-phenylalanine-induced granule exocytosis even though this secretagogue failed to stimulate 5-HETE synthesis. These data suggest that the 5-lipoxygenation of arachidonic acid may not be required for granule enzyme release from human neutrophils.

The pharmacology of 2-(2-fluoro-4-biphenylyl)Propionic acid (Flurbiprofen) A potent non-steroidal anti-inflammatory drug

Flurbiprofen is a potent, orally active, nonsteroidal anti-inflammatory drug in experimental animals. It is less toxic and more potent than indomethacin in certain types of acute and chronic inflammation in the rat. Like indomethacin, flurbiprofen has a prolonged duration of antiinflammatory action. It is effective in all in vitro assay systems used currently for the assessment of other nonsteroidal anti-inflammatory drugs. It is more potent than indomethacin in only one of these in vitro systems, red cell aggregation and sedimentation caused by the addition of dextran. Flurbiprofen may prove to be a useful adjunct for the treatment of various inflammatory diseases in man.

Stimulation of human neutrophil degranulation with 1-0-octadecyl-2-0-acetyl-sn-glyceryl-3-phosphorylcholine: Modulation by inhibitors of arachidonic acid metabolism

Abstract 1-0-octadecyl-2-0-acetyl-sn-glyceryl-3-phosphorylcholine (C 18 -AGEPC) stimulated a concentration (10- 10 -10- 6 M)-dependent release of granule-associated enzymes from human neutrophils. Cells which were not preincubated with cytochalasin B prior to exposure to C 18 -AGEPC did not degranulate. C 18 -AGEPC-elicited enzyme release was significantly reduced, but not abolished, in the absence of extracellular calcium. The lipoxygenase inhibitor, nordihydroguaiaretic acid and the lipoxygenase/cyclo-oxygenase inhibitor, 5,8,11,14-eicosatetraynoic acid, an acetylenic analog of arachidonic acid, caused a concentration-dependent suppression of enzyme discharge from neutrophils exposed to C 18 -AGEPC. However, the cyclo-oxygenase inhibitors, indomethacin, ibuprofen and flurbiprofen had no effect on C 18 -AGEPC-induced enzyme extrusion.

Glucocorticoid and Mineralocorticoid Activities of Δ1-Fluorohydrocortisonei

Summary (1) Comparisons have been made of relative potencies of hydrocortisone, fluorohydrocortisone, Δ1-hydrocortisone, and Δ1-fluorohydrocortisone in causing deposition of liver glycogen and reduction of sodium excretion in the adrenalectomized rat. (2) In glycogen deposition potency, the compounds have activity of the following magnitude: Δ1-fluorohydrocortisone acetate-50; fluorohy-drocortisone acetate-12.6; Δ1-hydrocorti-sone-2.9; hydrocortisone-1. (3) In sodium-retaining potency, the compounds have activity of the following magnitude: Δ1-fluorohydrocortisone acetate-4.6; fluorohydro-cortisone acetate-5.0; Δ1-hydrocortisone acetate-very slight; hydrocortisone acetate -very slight; DOCA-1.

Anomalous biological effects of salicylates and prostaglandins

While some salicylates (salicyclic acid and salicylaldehyde, especially) are as potent as aspirin as acute, orally-active anti-inflammatory drugs in the rat, they are either inactive or far less potent as PG synthesis inhibitors when added directly to isolated platelets or when given orally.Although PGE1 and PGE2 produce anti-ulcerogenic effects when given to rats in the presence of selected nonsteroidal anti-inflammatory drugs, they fail to inhibit the acute anti-inflammatory and anti-nociceptive effects of these drugs. They are anti-inflammatory and anti-nociceptive under certain experimental conditions. PGE1 and PGE2 can also behave as hypothermic agents when given subcutaneously. Related studies, using PG synthesis stimulators in vivo and in vitro (substituted phenylureas), also cause anti-nociception and hypothermia.All of these indirect studies, when taken together, infer that PG synthesis inhibitionper se fails to explain, entirely, the pharmacologic effects of non-steroidal anti-inflammatory drugs. They also suggest that the precise role of certain PGs in toxicopharmacology is far from simple and straightforward.

Pro-Inflammatory Effects of Certain Prostaglandins

Several groups of workers have suggested that certain prostaglandins, especially PGE(1), elicit vascular permeability responses following their local inoculation into the skin of man and experimental animals (Willis, 1969; Arora et al, 1970; Giroud and Willoughby, 1970; Kaley and Weiner, 1971; Crunkhorn and Willis, 1971; Sondergard and Greaves, 1971). Prostaglandins of various types have been isolated from inflammatory lesions of both species (Willis, 1969; Greaves et al, 1971). In consequence, certain prostaglandins are listed among the numerous other chemical mediators of the inflammatory process. Prostaglandins are released by almost all kinds of disturbing influences on the microenvironment of cells (Piper and Vane, 1971). Moreover, it has been shown that the synthesis and release of certain prostaglandins is inhibited by aspirin and indomethacin in the isolated spleen, sensitized lungs and isolated platelets (Smith and Willis, 1971; Vane, 1971; Ferriera et al, 1971).