MODIFICATION OF ZYMOSAN-INDUCED RELEASE OF LYSOSOMAL ENZYMES FROM HUMAN POLYMORPHONUCLEAR LEUKOCYTES BY CYTOCHALASIN B

During the process of phagocytosis, polymorphonuclear leukocytes (PMN) release lysosomal enzymes into the extracellular medium. When the antibiotic cytochalasin B (CB) is present in the incubation medium along with phagocytable particles, enhanced recovery of enzyme activities from the incubation medium has been observed. These findings have led to the interpretation that CB enhances lysosomal enzyme release. Our results contradict this interpretation. The lysosomal enzymes acid phosphatase and β-galactosidase are unstable after they are released from cells. During the first 5–15 min of phagocytosis, significant amounts of both acid phosphatase and β-galactosidase can be recovered from the extracellular medium. After this, the recovery of enzyme from the medium declines, presumably because the rate of loss of lysosomal enzyme activity exceeds the rate of release at later time periods. In the presence of CB, the appearance of lysosomal enzymes in the extracellular medium of cells exposed to zymosan is retarded for 5–10 min, after which it begins and then continues for approximately 20 min. At the end of a 30-min incubation period, therefore, in the absence of CB, extracellular levels of lysosomal enzymes (especially those which are unstable) are declining toward low levels while, in the presence of CB, extracellular enzyme levels are continuing to rise. We also measured the lysosomal enzyme remaining within cells after exposure to zymosan. CB retarded the disappearance of enzyme from cells and resulted in significantly less total cell enzyme loss. Thus, in the presence of CB, a greater proportion of the lysosomal enzyme lost from cells is recovered in the extracellular medium. In contrast to the previous conclusions that CB enhances lysosomal enzyme release, our results indicate that CB delays and decreases the zymosan-stimulated release of lysosomal enzymes from PMN. Since CB inhibits phagocytosis by PMN, our results indicate that the antibiotic modifies the mechanism of release of lysosomal enzymes, resulting in zymosan stimulation of their release independently of phagocytosis.     

Release of beta-hexosaminidase after administration of different agents affecting the reticuloendothelial system. An experimental study in rats

Plasma levels of a lysosomal enzyme, beta-hexosaminidase (beta-N-acetylglucosaminidase, EC 3.2.1.30) were studied in Wistar rats after administration of 99mTc -sulfur colloid, 198Au colloid, gelatine (Haemaccel), alcohol, methylpalmitate and zymosan. The activity of beta-hexosaminidase was increased 10, 30 and 60 min after the zymosan injection. After 24 and 48 h, enzyme levels had returned to those at outset. The transient release of beta-hexosaminidase probably occurred only during the phagocytosis of zymosan which was evaluated by histological examination of lung, liver and spleen. After the injection of all other agents tested, no significant aberration of beta-hexosaminidase levels was seen. Activity distribution of the radio-labeled colloids revealed differences in organ uptake which were attributed to a difference in colloid particle size. Although the colloids tested have been used extensively for determination of reticuloendothelial function and histological studies suggest phagocytosis of the particles, their administration did not affect plasma beta-hexosaminidase levels. Since lysosomal enzymes are cleared from the blood predominantly by liver macrophages, the primary location of particle phagocytosis may explain the present findings.     

Lysosomal enzyme release from human monocytes by particulate activators is mediated by beta-glucan inhibitable receptors

Human peripheral blood monocytes ingest particulate activators and generate leukotrienes via a trypsin-sensitive, beta-glucan-inhibitable receptor. The incubation of monolayers of monocytes with from 4 X 10(5) to 2 X 10(8) zymosan or glucan particles resulted in a dose-dependent release of up to 9% +/- 1.9 and 17.8% +/- 5.3 (mean +/- SD, n = 3) of the lysosomal enzyme, N-acetylglucosaminidase, into the culture medium. Lysosomal enzyme release occurred throughout the 2-hr period studied, with the greatest rate of N-acetyl-glucosaminidase release occurring during the first hour; the presence of 5 micrograms/ml of cytochalasin B accelerated this process when zymosan was the agonist. The preincubation of monocytes with from 0.5 to 500 micrograms/ml of soluble yeast beta-glucan inhibited N-acetylglucosaminidase release by 4 X 10(7) zymosan and glucan particles in a dose-dependent manner, with 50% inhibition occurring with 50 micrograms/ml of soluble yeast beta-glucan (mean +/- SD, n = 3). Preincubation with as much as 5 mg/ml of yeast mannan had no inhibitory effect on N-acetylglucosaminidase release. The pretreatment for 30 min of monolayers of monocytes with 50 micrograms/ml of affinity-purified trypsin, which selectively inactivates the monocyte-phagocytic response to particulate activators, also fully inhibited lysosomal enzyme release induced by zymosan and glucan particles. The inhibitory effects of a soluble ligand, yeast beta-glucan, and of trypsin pretreatment on lysosomal enzyme release correspond to the inhibitory effect of these agents on monocyte phagocytosis of zymosan and glucan particles and thus indicates ligand specificity for the beta-glucan receptor in the release of stored intracellular mediators.     

Lysosomes Behave as Ca2+-regulated Exocytic Vesicles in Fibroblasts and Epithelial Cells

Lysosomes are considered to be a terminal degradative compartment of the endocytic pathway, into which transport is mostly unidirectional. However, specialized secretory vesicles regulated by Ca²⁺, such as neutrophil azurophil granules, mast cell–specific granules, and cytotoxic lymphocyte lytic granules, share characteristics with lysosomes that may reflect a common biogenesis. In addition, the involvement of Ca²⁺ transients in the invasion mechanism of the parasite Trypanosoma cruzi, which occurs by fusion of lysosomes with the plasma membrane, suggested that lysosome exocytosis might be a generalized process present in most cell types.

Here we demonstrate that elevation in the intracellular free Ca²⁺ concentration of normal rat kidney (NRK) fibroblasts induces fusion of lysosomes with the plasma membrane. This was verified by measuring the release of the lysosomal enzyme β-hexosaminidase, the appearance on the plasma membrane of the lysosomal glycoprotein lgp120, the release of fluid-phase tracers previously loaded into lysosomes, and the release of the lysosomally processed form of cathepsin D. Exposure to the Ca²⁺ ionophore ionomycin or addition of Ca²⁺containing buffers to streptolysin O–permeabilized cells induced exocytosis of ~10% of the total lysosomes of NRK cells. The process was also detected in other cell types such as epithelial cells and myoblasts. Lysosomal exocytosis was found to require micromolar levels of Ca²⁺ and to be temperature and ATP dependent, similar to Ca²⁺-regulated secretory mechanisms in specialized cells.

These findings highlight a novel role for lysosomes in cellular membrane traffic and suggest that fusion of lysosomes with the plasma membrane may be an ubiquitous form of Ca²⁺-regulated exocytosis.

     

On the mechanism of action of the phagocytosis-stimulating peptide tuftsin

Summary Incubation of human polymorphonuclear leukocytes (PMNL) or thioglycollate-stimulated mouse peritoneal macrophages with the phagocytosis-stimulating peptide, tuftsin (2.5 × 10^–7 M, at 37 °C), caused an increase of 89–90% in intracellular cGMP levels, accompanied by a decrease of 20–25% in intracellular cAMP levels. Significant changes in cyclic nucleotide levels were detectable after 4 min of incubation, were maximal at 10–20 min and persisted for at least 60 min. The concentration dependences of the stimulatory effect of tuftsin on modulation of intracellular levels of cyclic nucleotides and on phagocytosis are similar, suggesting a cause and effect relationship between the two phenomena. This notion is further supported by the finding that 8-Br-cGMP and 8-Br-cAMP elicit stimulatory and inhibitory effects on macrophage phagocytosis, respectively. Measurement of ⁴⁵Ca²⁺ influx into PMNL and macrophages in the presence and absence of tuftsin did not reveal any change in ⁴⁵Ca²⁺ uptake from the media. However, tuftsin did enhance release of ⁴⁵Ca²⁺ from cells preloaded with the isotope. Results suggest that modulation of both the amount of cell-associated ⁴⁵Ca²⁺ and the intracellular levels of cyclic nucleotides are key steps in the mechanism by which tuftsin augments phagocytosis.     

Microfilaments and microtubules in calcium ionophore-induced secretion of lysosomal enzymes from human polymorphonuclear leukocytes.

Human peripheral blood leukocytes (PMN) are induced to release lysosomal enzymes by the calcium ionophore A23187 in the presence but not the absence of extracellular Ca++. Whereas secretion induced by particulate or immune stimuli is accompanied by an increase in visible microtubules and is inhibitable by colchicine, secretion induced by A23187 and Ca++ was not accompanied by an increase in microtubule numbers and was not inhibited by colchicine. Ca++ did not appear to regulate microtubule assembly in these cells since resting PMN had a mean of 22.3 +/- 2.0 microtubules in the centriolar region as compared to 22.3 +/- 1.1 in ionophore-treated cells and 24.9 +/- 1.5 in cells exposed to ionophore and 1 mM Ca++. Bipolar filaments, 10 nm thick and 300--400 nm long, were numerous in the pericortical cytoplasm of cells exposed to both reagents. Microtubules in these cells were decorated with an electron-opaque fibrillar material. PMN exposed to A23187 and Ca++ were contracted in two directions at right angles to each other: (a) Contractions parallel to the plasma membrane resulted in extensive plication of the cell membrane. The cytoplasm subjacent to the plicae contained dense filamentous webs. Plication was prevented by cytochalasin B or reversed by subsequent exposure to an endocytic stimulus such as zymosan. (b) Contractions perpendicular to the plasma membrane, toward the cytocenter, resulted in the formation of vacuoles in normal PMN and of membrane invaginations in cytochalasin B-treated PMN. Whereas contractions parallel to the plasma membrane could occur in the absence of enzyme release (ionophore alone) and enzyme release could occur in the absence of such contractions (ionophore plus calcium plus cytochalasin B), contraction toward the cytocenter occurred in all experimental conditions in which significant enzyme release was obtained. Thus, lysosomal enzyme secretion in PMN involves contractile movements in the plasma membrane toward the lysosomes rather than the reverse. These calcium-mediated contractile events are mediated by cytochalasin B-insensitive microfilaments but not by microtubule assembly.     

Effect of oxygen tension on human peripheral blood leukocytes: lysosomal enzyme release and metabolic responses during phagocytosis

We found that nonlethal lysosomal enzyme release from human peripheral blood leukocytes during phagocytosis of opsonized zymosan in vitro was modified by the oxygen tension under which the cells were incubated; with decreasing Po(2), zymosan-induced release of lysosomal enzymes was potentiated. The effect on enzyme release could not be attributed secondarily to an effect on phagocytosis, because, as others have reported, Po(2) had little effect on that response. Metabolic responses that accompany phagocytosis were also modified by oxygen tension. Stimulation of oxidation by way of the pentose cycle was further enhanced by increasing Po(2). Conversely, anaerobic glycolysis was promoted by decreasing oxygen tension. ATP levels fell as a function of time and concentration of phagocytic stimulus, mirroring lysosomal enzyme release as modified by Po(2). Cyclic AMP levels fell during phagocytosis and lysosomal enzyme release, a change that could act to facilitate lysosomal enzyme release. However, the fall in nucleotide level was greatest with highest Po(2) (i.e., when lysosomal enzyme release was least). The inverse relationship between oxidative metabolism and enzyme release suggested that a product of oxidative metabolism might adversely influence enzyme release. Sulfhydryl antioxidants (Cysteine, glutathione) and scavengers of oxygen-derived reactants (superoxide dismutase, catalase, benzoate, hypoxanthine, xanthine, histidine, azide) all potentiated zymosan- stimulated enzyme release. These findings are consistent with the interpretation that one or more factors (e.g., superoxide anion, hydrogen peroxide, hydroxyl radical, singlet oxygen), generated in association with the burst of oxidative metabolism which accompanies phagocytosis, acts to inhibit lysosomal enzyme release.     

Relationship of prostaglandin secretion by rabbit alveolar macrophages to phagocytosis and lysosomal enzyme release

The phospholipids of rabbit alveolar macrophages were pulse-labelled with [(14)C]-arachidonic acid, and the subsequent release of labelled prostaglandins was measured. Resting macrophages released measurable amounts of arachidonic acid, the prostaglandins E(2), D(2) and F(2alpha) and 6-oxoprostaglandin F(1alpha). Phagocytosis of zymosan increased the release of arachidonic acid and prostaglandins to 2.5 times the control value. In contrast, phagocytosis of inert latex particles had no effect on prostaglandin release. Indomethacin inhibited the release of prostaglandin, and, at high doses (20mug/ml), increased arachidonic acid release. Analysis of the cellular lipids showed that after zymosan stimulation the proportion of label was decreased in phosphatidylcholine, but not in other phospholipids or neutral lipids. Cytochalasin B, at a dose of 2mug/ml, inhibited the phagocytosis induced by zymosan but increased prostaglandin synthesis to 3.4 times the control. These data suggest that the stimulation of prostaglandin synthesis by zymosan is not dependent on phagocytosis. Exposure to zymosan also resulted in the release of the lysosomal enzyme, acid phosphatase. Furthermore, cytochalasin B augmented the zymosan-stimulated release of acid phosphatase at the same dose that stimulated prostaglandin synthesis. However, indomethacin, at a dose that completely inhibited prostaglandin synthesis, failed to block the lysosomal enzyme release. Thus despite some parallels between the release of prostaglandins and lysosomal enzymes, endogenous prostaglandins do not appear to mediate the release of lysosomal enzymes. The prostaglandins released from the macrophages may function as humoral substances affecting other cells.     

Lysosomal enzyme release from human monocytes in response to particulate stimuli

Lysosomal enzyme release from human monocytes was evaluated in response to opsonized zymosan, opsonized sheep erythrocytes, and latex beads. Monocytes were found to release lysosomal enzymes immediately upon challenge with all three phagocytosable particles. Cytochalasin B enhanced beta-glucosaminidase release from mononuclear cells challenged with opsonized zymosan or opsonized red blood cells, but inhibited the response to latex particles. Lysosomal enzyme release was found to be independent of protein synthesis, and in the absence of cytochalasin B required the stimulus to be presented either as a phagocytosable particle or immobilized on a surface. The kinetics of enzyme release and phagocytosis were also examined and found to be different, lending support to the hypothesis that lysosomal enzyme release may be a physiologic response to a biologic stimulus in vivo and not simply an "accidental" consequence of an ongoing phagocytic event.     

Inactivation during phagocytosis of leucine aminopeptidase, an ecto-enzyme of polymorphonuclear neutrophils

Changes in enzyme activities of the plasma membrane makers were examined during phagocytosis using guinea-pig polymorphonuclear neutrophils. Incubation of neutrophils with fresh serum-opsonized zymosan particles showed a significant reduction in leucine aminopeptidase activity, whereas 5′-nucleotidase and alkaline phosphodieterase activities remained unchanged. Inactivation of leucine aminopeptidase activity was also observed by exposure of neutrophils to complement-opsonized zymosan particles, but not to non-opsonized zymosan, IgG-coated zymosan or polysterene latex particles. Pretreatment of neutrophils with cytochalasin B, which prevents phagocytosis but not surface binding of particles, provoked inactivation to the same degree as when the cells were allowed to phagocytose the particles. However, the inactivation during phagocytosis was protected by serine protease inhibitors. These findings suggest that loss of leucine aminopeptidase activity from phagocytosing cells may be mediated by certain serine protease inhibitor-sensitive factor(s) which are probably activated by the attachment of an opsonized zymosan particle to a specific membrane receptor, probably the C3b receptor.     

Canonical and Noncanonical G-Protein Signaling Helps Coordinate Actin Dynamics To Promote Macrophage Phagocytosis of Zymosan

Both chemotaxis and phagocytosis depend upon actin-driven cell protrusions and cell membrane remodeling. While chemoattractant receptors rely upon canonical G-protein signaling to activate downstream effectors, whether such signaling pathways affect phagocytosis is contentious. Here, we report that Gα_i nucleotide exchange and signaling helps macrophages coordinate the recognition, capture, and engulfment of zymosan bioparticles. We show that zymosan exposure recruits F-actin, Gα_i proteins, and Elmo1 to phagocytic cups and early phagosomes. Zymosan triggered an increase in intracellular Ca²⁺ that was partially sensitive to Gα_i nucleotide exchange inhibition and expression of GTP-bound Gα_i recruited Elmo1 to the plasma membrane. Reducing GDP-Gα_i nucleotide exchange, decreasing Gα_i expression, pharmacologically interrupting Gβγ signaling, or reducing Elmo1 expression all impaired phagocytosis, while favoring the duration that Gα_i remained GTP bound promoted it. Our studies demonstrate that targeting heterotrimeric G-protein signaling offers opportunities to enhance or retard macrophage engulfment of phagocytic targets such as zymosan.     

An alternative hypothesis of cellular transport of lysosomal enzymes in fibroblasts. Effect of inhibitors of lysosomal enzyme endocytosis on intra- and extra-cellular lysosomal enzyme activities

Recapture of lysosomal enzymes secreted by fibroblasts was inhibited by growing the cells in the presence of either free or immobilized antibodies against lysosomal enzymes or in the presence of phosphorylated carbohydrates known to interact with the cell-surface receptors for lysosomal enzymes. The following results were obtained. 1. Conditions that prevent recapture of released lysosomal enzymes increase the rate of extracellular accumulation of these enzymes up to twice that of controls. 2. Growing cells for 12 days in the presence of 0.5mm-mannose 6-phosphate, which decreases β-N-acetylglucosaminidase endocytosis to less than 10% of that of controls, has no effect on the intracellular activity of this and four other lysosomal enzymes. 3. Growing cells for 4 days in the presence of 50mm-mannose 6-phosphate, which is a 1000-fold higher concentration than that required for 50% inhibition of lysosomal enzyme endocytosis, leads to a 4-fold increase in extracellular β-N-acetylglucosaminidase accumulation and a decrease in intracellular enzyme. These results give evidence that, in fibroblasts, transfer of lysosomal enzymes into lysosomes does not require secretion before a receptor-mediated recapture [Hickman & Neufeld (1972) Biochem. Biophys. Res. Commun. 49, 992–999]. We propose that (a) lysosomal enzymes are present in a receptor-bound form in those vesicles that fuse with the cell membrane, (b) the major part of the lysosomal enzyme cycles via the cell surface in a receptor-bound form and (c) only a minor part of the lysosomal enzyme is released into the extracellular space during its life cycle.     

Differential augmentation by recombinant human tumor necrosis factor-alpha of neutrophil responses to particulate zymosan and glucan

Zymosan (Z) and its major insoluble carbohydrate component beta-linked glucan activate human neutrophils (PMN) through a trypsin-sensitive recognition mechanism. This mechanism is believed to involve the PMN CR3R. Both Z and glucan generated dose and time-dependent release of the secondary lysosomal granule marker vitamin B12 binding protein, leukotriene B4 (LTB4) and superoxide from PMN and were phagocytosed with similar dose-dependent kinetics. The PMN superoxide and LTB4 responses to glucan; however, were consistently greater than those to the same doses of Z. The phagocytosis of both particles was significantly reduced after partial digestion with beta-laminarinase but not beta-glucosidase or alpha-mannosidase suggesting a recognition mechanism dependent on intact beta-1,3-glucosidic bonds in both particles. TNF-alpha (rhTNF-alpha) promoted a time- and dose-dependent increase in the expression of PMN CR3 up to 60 min. The increased expression of CR3 was paralleled by the release of the secondary lysosomal granule marker vitamin B12-binding protein. This granule contains a population of CR3R in its boundary membrane and it is the fusion of this membrane with the plasma membrane that may represent the mechanism by which CR3 expression is increased. Preincubation of PMN with 10(-9)M rhTNF-alpha augmented phagocytosis, LTB4, and superoxide generation by PMN in response to activation by Z. In contrast, none of the responses to glucan was significantly increased after incubation with rhTNF-alpha. These differences suggest a lack of absolute homology between the recognition mechanisms for zymosan and glucan and that there is a component of the recognition mechanism for zymosan that is independent of that for glucan and is up-regulated after rhTNF-alpha pretreatment.     

Lysosomal enzyme secretion from human neutrophils mediated by cyclic CMP: inhibition of cyclic GMP accumulation and neutrophil function by glucocorticosteroids.

The effects of several glucocorticosteroids on cyclic GMP accumulation, guanylate cyclase activity, calcium influx, lysosomal enzyme secretion, and phagocytosis were studied in human neutrophils. Contact between neutrophils and serum-treated zymosan particles, in the presence of calcium at pH 7.4, triggered these cellular events within five minutes. Each of these neutrophil functions was markedly inhibited by methylprednisolone sodium succinate, triamcinolone acetonide hemisuccinate and paramethasone acetate but was unaffected by two mineralo-corticosteroids. Human neutrophil soluble guanylate cyclase activity was not changed by the glucocorticoids. Inhibition of phagocytosis by, and lysosomal enzyme secretion from, neutrophils by glucocorticosteroids may be the result of a reduction in cyclic GMP accumulation within these cells. The data suggest that glucocorticosteroids inhibit cyclic GMP accumulation in neutrophils by reducing the influx of extracellular calcium into the cells, thereby limiting the availability of intracellular calcium for metabolic processes associated with the accumulation of cyclic GMP.     

Release of lysozyme from hemolymph cells of Mercenaria mercenaria during phagocytosis

Activity of the lysosomal enzyme, lysozyme, has been quantitatively determined in the serum and cells of the hemolymph of Mercenaria mercenaria which had been exposed to known quantities of Bacillus megaterium and also in the serum and cells of hemolymph which had not been exposed to bacteria. The results indicate that the level of enzyme activity is greater in serum of hemolymph that had been exposed to B. megaterium and concurrently, there is an equivalent decrease in the level of activity in the cells. This evidence indicates that the amount of lysozyme released from cells into serum is enhanced during phagocytosis of the bacteria.It has also been demonstrated that the release of lysozyme from cells occurs during the process of phagocytosis and is not a delayed phenomenon.Enzyme release by secondary phagosomes is reflected morphologically by what is commonly referred to as degranulation. This process does not involve the rupture of the plasma membrane of the hemolymph cells since biochemical studies have revealed that there is no release of the cytoplasmic enzyme, lactate dehydrogenase.     

Exocytosis of acid sphingomyelinase by wounded cells promotes endocytosis and plasma membrane repair

Rapid plasma membrane resealing is essential for cellular survival. Earlier studies showed that plasma membrane repair requires Ca²⁺-dependent exocytosis of lysosomes and a rapid form of endocytosis that removes membrane lesions. However, the functional relationship between lysosomal exocytosis and the rapid endocytosis that follows membrane injury is unknown. In this study, we show that the lysosomal enzyme acid sphingomyelinase (ASM) is released extracellularly when cells are wounded in the presence of Ca²⁺. ASM-deficient cells, including human cells from Niemann-Pick type A (NPA) patients, undergo lysosomal exocytosis after wounding but are defective in injury-dependent endocytosis and plasma membrane repair. Exogenously added recombinant human ASM restores endocytosis and resealing in ASM-depleted cells, suggesting that conversion of plasma membrane sphingomyelin to ceramide by this lysosomal enzyme promotes lesion internalization. These findings reveal a molecular mechanism for restoration of plasma membrane integrity through exocytosis of lysosomes and identify defective plasma membrane repair as a possible component of the severe pathology observed in NPA patients.     

Phagocytosis-induced release of arachidonic acid from human neutrophils

The phospholipids of human neutrophils were labeled with [³H] arachidonic acid and [¹⁴C] palmitic acid. Phagocytosis of opsonized zymosan resulted in rapid release of free arachidonic acid but not of palmitic acid. Arachidonic acid was not released when the cells were exposed to unopsonized zymosan, zymosan-activated serum, or phorbol myristate acetate. These observations suggest that phagocytosis of opsonized zymosan results in the activation of a phospholipase A₂.     

A macrophage receptor for (mannose/glucosamine)-glycoproteins of potential importance in phagocytic activity

Lung macrophages, in the absence of serum factors in vitro, strongly bound and ingested yeast cells ($\text{Candida}\text{krusei}$ and zymosan). Binding was temperature-and calcium-dependent, and was inhibited by the presence of D-mannose, D-glucosamine, horseradish peroxidase and beta-glucuronidase. Pretrypsinization of the macrophages also prevented binding of yeast cells. Binding was not affected by D-mannitol, D-glucose, D-galactose nor L-fucose. I suggest that macrophage binding of yeast cells is mediated by a mannose/glucosamine receptor on the cell membrane. This receptor may be responsible for opsosin-independent phagocytosis of activators of the alternative complement pathway and, as well, the phagocyte-dependent clearance of certain lysosomal enzymes.     

THE MOBILIZATION AND EXTRACELLULAR RELEASE OF GRANULAR ENZYMES FROM HUMAN LEUKOCYTES DURING PHAGOCYTOSIS

The importance of granular (lysosomal) enzymes from neutrophils in producing the tissue damage of acute inflammation has been suggested by much indirect and some direct evidence. This study has investigated the kinetics of release and subsequent fate of granular enzymes from phagocytizing human leukocytes The following observations are made: (a) During phagocytosis, the granular enzyme lysozyme is released from leukocytes into the extracellular medium. (b) Release of lysozyme increases as phagocytic challenge increases, but attains a maximum. (c) Release of lysozyme accompanies phagocytosis and is not a delayed event. (d) The lack of release of a nongranular enzyme, lactic dehydrogenase, indicates that cell damage is not a necessary condition of enzyme release. (e) Like lysozyme, β-glucuronidase is released from phagocytizing leukocytes. Acid α-naphthyl phosphatase and cathepsin also appear to be released, but are not found in appreciable amounts in the extracellular medium, in part because of their lability in solution. These results support the concept that extracellular release of granular enzymes may be a useful secretory function of inflammatory leukocytes which becomes damaging to the host in certain circumstances.     

Effect of cryogenic preservation (−80 °C) on polymorphonuclear neutrophil integrity as determined by biochemical analysis

The effect of cryopreservation on plasma membrane and granule associated enzymes of polymorphonuclear neutrophils (PMNs) was studied. The activity of PMNs to generate superoxide anions during phagocytosis was very sensitive to cryopreservation and exhibited approximately 60% inhibition in 24 hr. The total enzyme activity was not as affected during 1-month cryopreservation as that observed with the extracellular release of enzymes. Acid p-nitrophenyl phosphatase and peroxidase were released slightly from frozen and thawed PMNs. However, the extracellular release of LDH, a cytosol marker, and β-glucuronidase and lysozyme, granuleassociated enzymes, increased with cryopreservation time. The degree of release of these enzymes was LDH > β-glucuronidase > lysozyme. A considerable amount of LDH was extracellularly released after 1-month storage. Frozen and thawed PMNs became sensitive to hypotonic solutions, although fresh, nonfrozen PMNs were very resistant to hypotonic lysis. The hypotonic fragility increased even after 1 hr of cryopreservation.Addition of ATP to the preservation medium did not improve enzyme activity, enzyme release, or stimulated superoxide anion generation but increased the hypotonic fragility of PMNs. However, albumin showed protective effects against cryopreservation injury to the O₂^?-generating system, the extracellular enzyme release, and osmotic fragility.