Hormonal Control of Lysosomal Enzyme Release From Human Neutrophils

Abstract

Release of lysosomal enzymes from phagocytic leukocytes in the presence of various immune reactants is well documented (1-6). The tissue injury that results from the accumulation of, and phagocytosis by, granulocytes is probably a direct consequence of the extracellular release of granule constituents (7,8). Indeed, lysosome granules proteins are capable of mediating inflammation (9, 10) and cartilage degradation (11-13).The anti-inflammatory action of certain catecholamines in various animal models of acute (14, 15) and chronic inflammation (16, 17) has been reported. In fact, catecholamines have been thought to play a regulatory role in the inflammatory process (18). More recent studies have uncovered specific actions of catecholamines at the cellular and organelle level. For example, certain sympathomimetic amines were reported to inhibit the allergic release of histamine from sensitized human leukocytes (19) and lung fragments (20). In both of these studies it was suggested that the actions of the catecholamines were mediated by cyclic AMP. Release of enzymes from isolated lysosomes was also inhibited by epinephrine, norepinephrine, isoproterenol, theophylline, and cyclic AMP (21-24).In order to understand more completely the anti-inflammatory effects of catecholamines at the cellular level, several adrenergic and cholinergic amines and cyclic nucleotides were tested for their capacity to modify the release of /~-glucuronidase, a lysosomal marker enzyme, from and particle ingestion by human neutrophils during phagocytic uptake of serum-treated zymosan particles. In addition, the effects of autonomic neurohormones on neutrophil levels of cyclic AMP and cyclic GMP were determined in order to assess the possible involvement of cyclic nucleotides in mediating the actions of the neurohormones on phagocytic enzyme release. Preliminary reports of some of the present findings appeared earlier (25,26).

Mediation of Immunologic Discharge of Lysosomal Enzymes From Human Neutrophils by Guanosine 3',5'-Monophosphate

“…Previous reports from this laboratory indicated that acetylcholine enhanced whereas epinephrine inhibited the immunologic discharge of lysosomal enzymes from human mixed leukocytes (1) and purified neutrophils (3,4). However, the precise time-course of release of/3-glucuronidase under these conditions was not reported.…”

“…Cells (5 X 106) were preincubated in 1.0 ml of Hanks'-glucose at 37°C for 10 rain before the addition of 4 X 108 zymosan particles and then further incubated at 37°C for 10 min. Under these experimental conditions phagocytosis of particles occurred and/3-glucuronidase was discharged into the extracellular medium (4). Contact of cells with zymosan particles provoked a marked elevation of cyclic GMP levels but no change in cyclic AMP levels in neutrophils (Fig.…”

“…Isolation of Human Neutrophils.--Human neutrophils were isolated from fresh heparinized venous blood drawn from healthy volunteers by methods described previously (4). The final cell suspension (97-99% neutrophils) was prepared in Hanks' balanced salt solution containing 0.1% (wt/vol) glucose (Hanks'-glucose) and diluted to 5 × 106 cells/ml.…”

“…Preparation of Immunologic Reactants.--Untreated, serum-treated and rheumatoid arthritic (RA) serum-treated zymosan particles, and zymosan-treated serum were prepared by procedures described previously (4). Solutions of human IgG, suspensions of heat-aggregated (agg) IgG, suspensions of RA serum-treated agg IgG, and immobilized RA serum-treated agg IgG were prepared and utilized as described (2,3).…”

“…Incubations of cells with immobilized reactants were executed as described (3). Mter the incubations the samples were processed and measurements of f~-glucuronidase and lactate dehydrogenase discharge from, and determinations of cyclic GMP and cyclic AMP concentrations in, neutrophils were made according to methods outlined previously (4). Recoveries of/3-glucuronidase and lactate dehydrogenase activities from neutrophils and incubation media were determined in each of the experiments by procedures described previously (4).…”