Susan S. Iden

Phorbol myristate acetate-induced release of granule enzymes from human neutrophils: inhibition by the calcium antagonist, 8-(N,N-diethylamino)-octyl 3,4,5-trimethoxybenzoate hydrochloride.

Abstract Phorbol myristate acetate (PMA) stimulated the extracellular release of the granule-associated enzyme lysozyme from human neutrophils. The extrusion of lysozyme was not accompanied by the release of β-glucuronidase or the cytosol enzyme lactate dehydrogenase. A time dependent PMA-induced release of lysozyme occurred in the absence of extracellular calcium and when neutrophils were preincubated with EGTA. 8-(N,N-diethylamino)-octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8), an antagonist of intracellular calcium, caused a dose-dependent inhibition of lysozyme release from neutrophils exposed to PMA in a calcium-free medium. This effect of TMB-8 could be reversed by the addition of calcium to the extracellular medium. These studies indicate that TMB-8 represents a valuable pharmacologic tool used to define the dependence of a secretagogue such as PMA on intracellular as opposed to extracellular calcium.

Pharmacological modulation of chemotactic factor-elicited release of granule-associated enzymes from human neutrophils: Effects of prostaglandins, nonsteroid anti-inflammatory agents and corticosteroids

Abstract Human neutrophils demonstrated a selective release of granule-associated β-glucuronidase and lysozyme but not of cytoplasmic lactate dehydrogenase during cell contact with N -formyl-methionyl-leucyl-phenylalanine (FMLP). Enzyme discharge was dependent upon treatment of neutrophils with cytochalasin B prior to exposure to FMLP. Prostaglandins (PG) D 2 , E 2 and I 2 inhibited enzyme release from cytochalasin B-treated neutrophils incubated with FMLP in phosphate buffered saline, pH 7.4, at 37°. Flurbiprofen, ibuprofen, indomethacin, ketoprofen and benoxaprofen reduced the extrusion of β-glucuronidase and lysozyme from FMLP-stimulated neutrophils; acetylsalicylic acid was inactive. Methylprednisolone sodium succinate, hydrocortisone sodium succinate, prednisolone sodium succinate and triamcinolone acetonide hemisuccinate also demonstrated the capacity to inhibit the selective release of granule-associated enzymes from human neutrophils. Aldosterone hemisuccinate and deoxycorticosterone hemisuccinate were inactive. These studies indicate that certain pharmacological and therapeutic agents may function to alleviate various inflammatory conditions by curtailing the extrusion of degradative enzymes from neutrophils.

Characteristics of N-formyl-methionyl-leucyl-phenylalanine as an inducer of lysosomal enzyme release from human neutrophils.

N-Formyl-methionyl-leucyl-phenylalanine (FMLP) stimulated a timeand concentration-dependent release of granule associatedβ-glucuronidase and lysozyme but not cytoplasmic lactate dehydrogenase from human neutrophils. Maximum discharge of lysosomal enzymes occurred two minutes after cell contact with FMLP. The percent of total enzyme activity released is insignificant when cells are not preincubated with cytochalasin B prior to being exposed to FMLP (10−10-10−7 M); although 11.2 ± 1.3 and 12.4 ±1.1% of total activity forβ-glucuronidase and lysozyme, respectively, is secreted from neutrophils with 10−4 M FMLP in the absence of cytochalasin B. FMLP-stimulated release of lysosomal enzymes occurs but is significantly curtailed in the absence of extracellular calcium. Incubation of neutrophils with EGTA in calcium-free medium, however, had no effect on FMLP-elicited lysosomal enzyme extrusion. 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), a purported antagonist of intracellular calcium, demonstrated a dose-dependent inhibition of FMLP-induced release ofβ-glucuronidase and lysozyme from cytochalasin B-treated neutrophils in the absence of extracellular calcium. This effect of TMB-8 could be reversed with the addition of calcium to the extracellular medium. These studies indicate that TMB-8 may be useful in elucidating the role of calcium in the mechanism of lysosomal enzyme release.

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.

Properties of calcium ionophore-induced generation of superoxide anion by human neutrophils.

Exposure of human neutrophils to the calcium ionophore, A23187, eventuates in a time- and concentration-dependent generation of Superoxide anion (O2−) in the presence but not absence of extracellular calcium. The selective requirement for calcium is demonstrated by the observation that magnesium caused a dose-related inhibition of A23187-stimulated O2− generation. Preincubation of neutrophils with cytochalasin B prior to their interaction with A23187 results in a significant enhancement of O2− production. The activity of the O2−-generating system was maximum at 37°C and was significantly curtailed at lower and higher temperatures. A23187-induced O2− generation was inhibited by the sulfhydryl reagents.N-ethyl maleimide (NEM) and iodoacetic acid (IA), and by the metabolic inhibitor 2-deoxy-D-glucose (2-DG) in the absence of glucose and cytochalasin B. Cyanide was inactive. Therefore, A23187 represents a useful pharmacologic probe for investigating the divalent cation and metabolic requirements of the neutrophil O2−-generating system.

Activation of the human neutrophil secretory process with 5(S),12(R)-dihydroxy-6, 14-cis-8,10-trans-eicosatetraenoic acid

Exposure of human neutrophils to 5(S),12(R) dihydroxy-6, 14-cis-8, 10-trans-eicosatetraenoic acid (leukotriene B4, LTB4) resulted in a time- and concentration- (10−9-10−6 M) dependent extracellular release of granule-associated lysozyme and myeloperoxidase (MPO). Enzyme extrusion was negligible if cells were not pretreated with cytochalasin B prior to exposure to LTB4. A time-dependent deactivation of granule exocytosis was observed in neutrophils which were stimulated with LTB4 prior to contact with cytochalasin B. LTB4-induced enzyme release was markedly enhanced in the presence of extracellular calcium. Nevertheless, significant enzyme discharge occurred in the absence of extracellular calcium, and the percent of total activity released was not altered in the presence of EGTA. The calmodulin antagonist, trifluoperazine (TFP), and the intracellular calcium antagonist, 8-(N, N-dietliylamino)-octyl-(3,4,5-trimethoxy)benzoate hydrochloride (TMB-8), caused a dose-related inhibition of enzyme release from LTB4-stimulated neutrophils. Degranulation was suppressed by the glycolytic inhibitor, 2-deoxy-D-glucose (2-DG), and the sulfhydryl reagents iodoacetic acid (IA) andN-ethylmaleimide (NEM). Sodium cyanide was inactive. Two inhibitors of transmethylation, 3-deazaadenosine (3-DZA) and L-homocysteine thiolactone (HCTL), alone or in combination, had no effect on LTB4-elicited degranulation. The protein synthesis inhibitor, cycloheximide, was inactive, Neutrophils pretreated with LTR4 or 5(S), 12(R), 20-trihydroxy-6, 14-cis-8,10-trans-eicosatetraenoic acid (20-OH-LTB4, an ω-oxidation metabolite of LTB4) were desensitized to the subsequent exposure to LTB4. Cross-desensitization was also demonstrated between LTB4 and 20-OH-LTB4. The stimulus specific nature of LTB4-induced desensitization of neutrophil degranulation was demonstrated by the fact that cells exposed to 1-O-hexadecyl/octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC) orN-formyl-methionyl-leucyl-phenylalanine (FMLP) were capable of inducing granule exocytosis from LTB4-pretreated neutrophils. Enzyme release from LTB4-treated cells was suppressed with the phospholipase inhibitor, 4-bromophenacyl bromide (4-BPB), the cyclooxygenase/lipoxygenase inhibitor, ETYA, and the 5-lipoxygenase inhibitor, U-60, 257. However, the cyclooxygenase inhibitor, flurbiprofen, exerted a weak suppressive effect on LTB4-induced degranulation.

Effects of an anion channel blocker, 4, 4′-diisothiocyano-2,2′-disulfonic acid stilbene (DIDS), on human neutrophil function

We investigated the capacity of DIDS to influence degranulation and superoxide anion (O-2) generation by human neutrophils exposed to soluble, surface-active stimuli. DIDS caused a concentration-related (25-200 microM) inhibition of myeloperoxidase (MPO) release from N-formyl-methionyl-leucyl-phenylalanine (FMLP), 5(S), 12(R)-dihydroxy-6, 14-cis-8, 10-trans-eicosatetraenoic acid (LTB4), 1-0-hexadecyl/octadecyl-2-0-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC) and phorbol myristate acetate (PMA)-stimulated neutrophils. In contrast, DIDS had no effect on O-2 production by FMLP and PMA activated cells, whereas it enhanced LTB4 and AGEPC-elicited O-2 generation. The respective effects of DIDS on these neutrophil activities were reversed by washing the cells prior to exposure to stimulus. Thus, DIDS represents a useful pharmacologic probe for investigating the role(s) of anions in the mechanisms of neutrophil activation with structurally and chemically dissimilar stimuli.

Properties of concanavalin A-elicited granule exocytosis from human polymorphonuclear neutrophils

Exposure of human neutrophils to concanavalin A (Con A) resulted in a time- and concentration-dependent extracellular release of granule-associated lysozyme but notΒ-glucuronidase or cytosolic lactate dehydrogenase. Maximum extrusion of lysozyme occurred 30 min after cell contact with Con A. The percent of total granule enzyme activity discharged is insignificant when cells are not preincubated with cytochalasin B prior to being exposed to Con A (5–80Μg/ml). Granule enzyme release from Con A-treated cells is markedly inhibited byα-methyl-d-mannoside. Con A-elicited extrusion of lysozyme is reduced significantly, but not abolished, in the absence of extracellular calcium. However, contact between neutrophils and EGTA in calcium-free medium had no effect on Con A-stimulated release of granule enzymes. 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), an antagonist of intracellular calcium, caused a dose-dependent inhibition of lysozyme discharge from Con A-treated neutrophils. The activity of TMB-8 could be abrogated with the addition of calcium, but not magnesium, to the extracellular medium. Therefore, Con A and TMB-8 should serve as useful tools for elucidating the mechanism of granule enzyme release from neutrophils.

Pepstatin A-induced degranulation and superoxide anion generation by human neutrophils

Abstract Exposure of human neutrophils to pepstatin A, a chemotactic pentapeptide of bacterial origin, resulted in a time- and concentration-dependent release of the granule-associated enzymes, β-glucuronidase and lysozyme, and the generation of superoxide anion (O2−). Maximum discharge of granule enzymes and O2− production occurred 15 sec and 15 min, respectively, after cell contact with pepstatin A (0.1–10 μM). The percentage of total enzyme activity released is insignificant when cells are not preincubated with cytochalasin B prior to interaction with pepstatin A, whereas O2− generation by neutrophils was reduced significantly but not abolished in the absence of cytochalasin B. The rate and amount of enzymes released and O2− produced by pepstatin A-stimulated neutrophils were enhanced appreciably in the presence of extracellular calcium. Incubation of neutrophils with EGTA in calcium-free medium, however, had no effect on the induction of these cellular functions by pepstatin A. Carbobenzoxy-phenylalanyl-methionine (CBZ-Phe-Met), an antagonist of N-formylmethionyl peptide receptors, caused a dose-related suppression of pepstatin A-elicited granule exocytosis and O2− generation. Thus pepstatin A represents a new probe for studies of the human neutrophil-degranulating and O2− generating mechanisms.