Initial kinetics of lysosomal enzyme secretion and superoxide anion generation by human polymorphonuclear leukocytes

Human polymorphonuclear leukocytes (PMN) exposed to particulate and soluble stimuli secrete lysosomal enzymes. These stimuli cause prompt (< 10 sec) changes in membrane potential followed 30–45 sec later by superoxide anion (O ₂ ^– ) production. We describe a new technique utilizing flow dialysis apparatus which monitors the first stages of lysosomal enzyme release with a resolution of approximately 6 sec. Secretion of-glucuronidase from cytochalasin B-treated PMN could be detected 19±5 sec after exposure to the chemotactic peptideN-formylmethionylleucylphenylalanine (FMLP). The lag times for release of this enzyme were different for other stimuli: 35±8 sec (BSA/anti-BSA immune complex); 48±8 sec (serum-treated zymosan, STZ); 60±25 sec (calcium ionophore A23187). The lag times for lysozyme release were less dependent upon the stimulus presented (28±16 sec for FMLP, 28±8 sec for BSA/antiBSA, 32±10 sec for STZ, and 38±8 seconds for Con A); only A23187 had a long lag period: 74±27 sec. Lag periods for the onset of O ₂ ^– production (measured by the same mathematical criteria) were comparable to those for-glucuronidase release: 21±4 sec for FMLP, 43±14 sec for BSA/anti-BSA, 61±7 sec for Con A, and 50±13 sec for A23187. Changes in FMLP dose up to 100-fold affected the magnitudes of O ₂ ^– generation and-glucuronidase release, but did not alter the time required for the onset of these processes. A variety of agents, such as corticosteroids, colchicine, 2-deoxyglucose, andN-ethyl maleimide, also affected the magnitudes of the responses, but not the lag periods when FMLP was used as the stimulus. When BSA/anti-BSA immune complex was used as the stimulus, 2-deoxyglucose andN-ethyl maleimide increased the lag period for superoxide anion generation, but not for lysosomal enzyme release. This new flow dialysis technique has permitted us to demonstrate that O ₂ ^- production and lysosomal enzyme secretion are concurrent but dissociable processes which are subsequent to earlier responses of the granulocyte to ligandreceptor interactions as reflected by changes in membrane potential.Aided by grants (AM-11949, HL-19072, HI-19721, GM023211) from the National Institutes of Health, The National Foundation-March of Dimes, the National Science Foundation (76-05621), and the Arthritis Foundation.Recipient of Postdoctoral Fellowship from the Arthritis Foundation.     

Influence of neutrophil cationic proteins on generation of superoxide by human polymorphonuclear cells during phagocytosis

The cationic proteins from neutrophyl lysosomes have been shown to modulate phagocytic activity of granulocytes. The present study reports the effects of the cationic protein fractions on the generation of O ₂ ^– by human PMNs during phagocytosis. Human PMNs were reacted win different phagocytic stimuli in the presence and absence of lysosomal cationic proteins and the amount of O ₂ ^– generated was determined by superoxide dismutase inhibitable reduction of cytochromec. Total cationic protein extract from neutrophil lysosomes enhanced O ₂ ^– generated by PMNs during the phagocytosis of IgG-coated latex beads and opsonized zymosan particles. The analysis of the fractions of cationic proteins obtained from a Sephadex G-75 column showed that the O ₂ ^– generation-enhancing activity was associated with the proteins eluted in fractions III and IV. A protein fraction mainly eluted in void volume inhibited the cytochromec reduction by O ₂ ^– formed during phagocytosis. This was due to the presence of superoxide dismutase-like activity since O ₂ ^– generated by the xanthine-xanthine oxidase system was also inhibited by this fraction. The cationic protein fractions III and IV from the Sephadex G-75 column were further subfractionated. Although the O ₂ ^– -enhancing activity was eluted in the same fractions as chymotrypsin activity, there was no quantitative correlation between the amount of O ₂ ^– generation and chymotrypsin activity. Moreover, commercial chymotrypsin did not enhance O ₂ ^– generation. Electrophoretic analysis of the isolated protein fractions suggests that O ₂ ^– generation enhancing protein (SGEP) is different from lysozyme or chymotrypsin and probably represents previously undescribed protein.     

Priming of human neutrophil functions by tumor necrosis factor: Enhancement of superoxide anion generation,degranulation, and chemotaxis to chemoattractants C5a and f-Met-Leu-Phe

Recombinant human tumor necrosis factor- (rTNF) stimulated increased generation of Superoxide anion (O ₂ ^– ) by human neutrophils in a concentration-dependent fashion. Preincubation of human neutrophils with rTNF (2.2–2200 units/ml) for 10 min enhanced the subsequent generation of O ₂ ^– in response to C5a and f-MetLeu-Phe(FMLP). Recombinant TNF did not enhance O ₂ ^– generation by neutrophils stimulated with phorbol myristate acetate (PMA). Recombinant TNF alone failed to induce release of myeloperoxidase (MPO) and lysozyme by neutrophils. However, it did enhance the release of MPO and lysozyme by neutrophils stimulated with C5a and FMLP, but not with PMA. Although rTNF alone (0.001–50,000 units/ml) was not chemotactic for neutrophils, preincubation of neutrophils with rTNF (0.001–0.1 units/ml) enhanced the chemotactic activity of suboptimal concentrations of C5a (0.1 nM) and FMLP (5 nM). Neutrophils treated with high concentrations of rTNF (100–10,000 units/ml) showed inhibition of random movement and of chemotaxis induced by C5a or FMLP. We conclude from these studies that rTNF primes neutrophils for enhanced responses to subsequent stimuli and thus may augment the inflammatory response by increased oxidant production and lysosomal enzyme release and promote down-regulation of chemotactic movement.     

Stimulation of neutrophil oxidative metabolism by indomethacin

Human neutrophils exposed to indomethacin demonstrate an enhanced capacity for superoxide ion (O ₂ ^– ) generation when stimulated with opsonized zymosan. Enhancement is not seen with indomethacin-treated cells exposed to solube oxidative stimuli. To further investigate this phenomenon, O ₂ ^– generation, chemiluminescence, and phagocytosis were assessed in human neutrophils preincubated with indomethacin. Zymosan-stimulated O ₂ ^– release was increased from 150 to 300% of controls in neutrophils exposed to 400 g/ml. indomethacin. Enhancement was not reversed by removal of indomethacin from the medium prior to addition of the stimulus and was dose-dependent at drug concentrations of 5 to 400 /ml. Neutrophils exposed to methacin alone also generated more O ₂ ^– than control cells, although this increment was not sufficient to account for the degree of enhancement seen when indomethacintreated cells were exposed to zymosan. Neutrophil cehmiluminescence induced by zymosan was also increased by exposure to indomethacin, and at a drug concentration of 400 g/ml (1.1 mM), enhancement randed from 253 to 333% of controls. As was observed with O ₂ ^– generation, chemiluminescence of neutrophils was increased in the presence of indomethacin alone, although, to a degree far less than was seen when drug-treated cells were stimulated with zymosan. Phagocytosis of radiolabeledS. aureus by neutrophils incubated with indomethacin was increased 13±5% over controls (P<0.01,n=5), but was unaltered by incubation of cells with the buffer used to solubilize the drug. The modest degree of enhancement of phagocytosis suggests that increased particle uptake is not the sole mechanism of oxidative enhancement. The data are in keeping with the hypothesis that indomethacin has a direct effect on the neutrophil plasma membrane and/or the O ₂ ^– -forming oxidase.     

Lipoteichoic acid-antilipoteichoic acid complexes induce superoxide generation by human neutrophils

Human neutrophils (PMNs) which have been incubated with lipoteichoic acid (LTA) from group A streptococci generated large amounts of Superoxide (O ₂ ^– chemiluminescence and hydrogen peroxide when challenged with anti-LTA antibodies. Cytochalasin B further enhanced O ₂ ^* generation. The onset of Of generation by the LTA-anti-LTA complexes was much faster than that induced by BSA-anti-BSA complexes. LTA-treated PMNs generated much less O ₂ ^* when challenged with BSA complexes, suggesting that LTA might have blocked, nonspecifically, some of the Fc receptors on PMNs. PMNs treated with LTA-anti-LTA complexes further interacted with bystander nonsensitized PMNs resulting in enhanced Of generation, suggesting that small numbers of LTA-sensitized PMNs might recruit additional PMNs to participate in the generation of toxic oxygen species. Protelolytic enzyme treatment of PMNs further enhanced the generation of O ₂ ^– by PMNs treated with LTA-anti-LTA. Superoxide generation could also be induced when PMNs and anti-LTA antibodies interacted with target cells (fibroblasts, epithelial cells) pretreated with LTA. This effect was also further enhanced by pretreatment of the target cells with proteases. PMNs incubated with LTA released lysosomal enzymes following treatment with anti-LTA antibodies. The amounts of phosphatase,-glucoronidase,N-acetylglucosaminidase, mannosidase, and lysozyme release by LTA-anti-LTA complexes were much smaller than those released by antibody or histone-opsonized streptococci, suggesting that opsonized particles are more efficient lysosomal enzyme releasers. However, since the amounts of O ₂ ^– generated by the LTA complexes equaled those generated by the opsonized particles, it is assumed that the signals for triggering a respiratory burst and lysosomal enzyme secretion might be different.Supported by a research grant from Dr. S. M. Robbins of Cleveland Ohio, and by grants HL-288442, HL-31963 and GM-29507, from the National Institutes of Health, Bethesda, Maryland, and by grant IM-432 from the American Cancer Society.     

Altered function of synovial fluid granulocytes in patients with acute inflammatory arthritis

In rheumatoid arthritis (RA) a chronic inflammatory state exists in which the synovial fluid is periodically filled with large numbers of polymorphonuclear leukocytes (PMNs). Oxygen radicals produced by these cells have been implicated as mediators of tissue damage and may be directly involved in the pathogenesis of RA. We examined the production of oxygen radicals by synovial fluid PMNs (SFPMNs) and peripheral blood PMNs (PB-PMNs) by measuring chemiluminescence (CL) as well as Superoxide anion (O ₂ ^– ) release. Increased spontaneous CL in the presence of luminol and increased CL in response to phorbol myristate acetate (PMA) was observed in SF-PMNs when compared to PB-PMNs. When zymosan was used as the stimulus in the absence of luminol, a slightly lower CL response was observed in SF-PMNs as compared to PB-PMNs. No significant differences were observed in the generation of O ₂ ^– generation with any stimulus. Preincubation of normal PBPMNs in 10% synovial fluid enhanced the luminol-dependent spontaneous and PMA-stimulated CL as well as zymosan-stimulated CL. When O ₂ ^– release from normal PB-PMNs pretreated with 10% synovial fluid was compared to untreated controls, enhancement of spontaneous O ₂ ^– release was observed. PMA- and zymosan-stimulated responses did not differ significantly from controls. Increased spontaneous and PMA-stimulated release of myeloperoxidase (MPO) was also observed in normal PB-PMNs pretreated with synovial fluid. These findings may explain the increased luminol-dependent CL since this type of CL requires the presence of MPO. Our findings suggest that the enhanced chemiluminescence observed in normal PMNs treated with synovial fluids may be related to increases in spontaneous O ₂ ^– generation and myeloperoxidase release. Increased MPO release may account for enhanced CL observed in SF-PMNs.     

Occupancy of adenosine receptors on human neutrophils inhibits respiratory burst stimulated by ingestion of complement-coated particles and occupancy of chemoattractant but not Fc receptors

Chemoattractants are generated at inflammatory loci that not only induce neutrophils (PMNs) to leave the vasculature but also stimulate PMNs to release potentially toxic agents (e.g., H₂O₂, O ₂ ^– or OH). We have recently demonstrated that endothelium releases adenosine which, when bound to a specific receptor on the PMN surface, inhibits release of toxic oxygen metabolites from stimulated PMN. To determine whether occupancy of adenosine receptors modulates generation and release of oxygen metabolites, we have studied the effect of 2-chloroadenosine on O ₂ ^– generation and O₂ consumption in response to opsonized zymosan particles (STZ) and immune complexes (IC). 2-Chloroadenosine inhibits, in a dose-dependent fashion, Of generation by neutrophils that have been exposed to C3b-coated particles (STZ). Inhibition of Of generation is similar in the presence or absence of cytochalasin B (IC₅₀=53 ±19 and 16 ±5nM, respectively,P=NS). Since occupancy of adenosine receptors might inhibit only externalization but not generation of oxygen metabolites, we studied the effect of 2-chloroadenosine on oxygen consumption by activated neutrophils. 2-Chloroadenosine inhibited O₂ consumption stimulated by STZ and the surrogate bacterial chemoattractant FMLP; however, inhibition of O₂ consumption varied with the presence or absence of cytochalasin B. In contrast, when neutrophils were stimulated by immune complexes, 2-chloroadenosine only minimally inhibited O ₂ ^– release and O₂ consumption (10 ± 5 and 5 ± 4% inhibition, respectively). Thus, occupancy of adenosine receptors inhibits O₂ consumption in parallel with inhibition of O ₂ ^– release. These results support the hypothesis that ingestion of complement-opsonized particles stimulates the respiratory burst by a mechanism different from that by which the respiratory burst is stimulated after occupancy of Fc receptors. Moreover, these observations suggest that endothelium, by releasing adenosine, prevent activated neutrophils from damaging the microvasculature at inflammatory loci. In contrast, deposition of immune complexes in vessel walls leads to vascular damage because endothelial cells are incapable of preventing attack by immune complex-stimulated neutrophils.This research was supported by grants from the U.S. Public Health Service (AI-10343 and HL29034) and the Veterans Administration.Dr. Cronstein is the recipient of a Clinical Investigator Award (K11-AR-01490) and was a fellow of the Arthritis Foundation. Dr. Broekman was an Established Investigator of the American Heart Association.     

Alterations in complement-induced shape change and stimulus-specific superoxide anion generation by neonatal calf neutrophils

Increased susceptibility of neonates to infection may be related to defects in newborn neutrophil (PMN) functional activities, including altered responses to complement fragments (Cf) and defective microbicidal activity. We therefore compared the kinetics of newborn and adult bovine PMN membrane shape change responses following stimulation with zymosan-activated plasma (ZAP) as a source of Cf. Measurement of PMN membrane shape change was a rapid, sensitive, and reproducible measure of Cf stimulation within a population of PMNs; a maximum of 67–85% of the PMNs exhibited easily detectable membrane ruffling, lamellipodia formation, and polarity within 2 min. Newborn PMNs exhibited significantly increased (P<0.01) membrane shape change at 20, 30, 60, 120, and 300 sec after Cf stimulation. A maximum of 85.8±3.2% of newborn PMNs exhibited such C-finduced shape changes by 120 sec, which was significantly greater (P<0.01) than the maximum stimulation (67.7±4.3%) attained with adult PMNs. These data indicate enhanced kinetics of induced newborn PMN membrane shape change in response to Cf stimulation. We also compared stimulus-specific superoxide anion (O ₂ ^– ) generation as a measure of respiratory burst activity after incubation of new-born and adult PMNs with soluble (phorbol myristate acetate, PMA) and particulate (opsonized zymosan, OZ) stimuli. When PMA was used as the stimulus, newborn PMNs generated significantly less O ₂ ^– (9.3±0.5 nmol O ₂ ^– /10⁶ PMN,P<0.05) than did adult PMNs (12.4±0.3 nmol O ₂ ^– )/10⁶ PMN). This finding was reversed when OZ was used as the stimulus; newborn PMNs generated significantly more O ₂ ^– (7.7±0.4 nmol O ₂ ^– /10⁶ PMN,P <0.05) than did adult PMN (5.5 ±0.5 nmol O ₂ ^– /10⁶ PMN). These findings collectively document biochemical and morphological differences between newborn and adult PMNs as determined by stimulusspecific O ₂ ^– generation and C_f-induced membrane shape change. Such differences may be important to neonatal disease susceptibility.     

Inhibition of neutrophil response by curcumin

Blood neutrophils, when exposed to appropriate stimuli, aggregate, degranulate and generate superoxide anion. Curcumin, a non-steroidal antiinflammatory agent, modulated these functions, depending upon the kind of stimulus used. It inhibited monkey neutrophil aggregation induced by chemotactic peptide fmlp and zymosan activated plasma (ZAP) but did not affect that induced by serum treated zymosan (STZ) and arachidonic acid (AA). Generation of O ₂ ^– radical was inhibited by curcumin, when cells were stimulated by AA, STZ and fmlp. Curcumin inhibited the release of myeloperoxidase, an azurophilic granule marker enzyme. Release of lysozyme was less susceptible to inhibition by curcumin. The results suggest that curcumin interferes with neutrophil responses to various physiological stimuli and a part of its antiinflammatory action is mediated via inhibition of neutrophil function. Inhibition of neutrophil function by curcumin appears to be mediated via calcium dependent mechanisms.     

Control of leukocyte functions

An activable membrane-bound neutral esterase which responds to many of the known leukocyte cytotaxins and appears to be involved in several membrane functions of these cells is described. Activation of this enzyme is associated with excretion of H⁺, O ₂ ^– , and lysosomal enzymes; aggregation of intact cells and isolated membranes; activation of glucose oxidation; and changes in cellular motility. Conditions which alter the activity of this enzyme, e.g., addition of H⁺ or of synthetic hydrophobic peptides or esters (whose hydrolysis liberates H⁺ within or on these membranes) or by repeated washing of the cells (which partially removes this enzyme from the cells), all markedly alter rates of the above cellular functions. Although the native membrane substrate for this enzyme is not yet identified, the enzyme appears not to be involved in-glutamyl transpeptidase reactions. Rather the data support the concept that hydrolysis by the enzyme of membrane components (proteins) results in H⁺ and O ₂ ^– efflux (as well as other cation fluxes) with resultant rise in intracellular pH. Under these conditions cell movement ceases. Agents, e.g., low concentrations of chemotactic peptides or proteins which inhibit H⁺ and O ₂ ^– excretion accelerate motility. The presence of external hydrophobic substrates for this enzyme, whose hydrolysis liberates H⁺ in or on these membranes and also inhibits efflux of H⁺ and O ₂ ^– , also optimizes motility. The esterase(s) thus appears to control many functions of these cells by controlling H⁺ efflux from these cells.     

Role of myeloperoxidase in respiratory burst of human polymorphonuclear leukocytes

A comparative study of the respiratory burst [monitored as superoxide (O₂ ^–) production] of normal and myeloperoxidase (MPO) -deficient poiymorphonuclear leukocytes (PMNs) was carried out on 11 MPO-deficient subjects that represent the largest sample of this kind ever studied. The rate of O₂ ^– production by isolated PMNs and whole blood from normal and MPO-deficient subjects was comparable during the initial 30–40 min of incubation with serum-treated zymosan (STZ). Afterwards, the amount of O₂ ^– produced became progressively higher in MPO-deficient cells at least until 120 min incubation with STZ. On the contrary the rate of O₂ ^– production by both cell types in response to 4--phorbol-12-myristate-13-acetate (PMA) was the same. The PMNs of four MPO-deficient subjects were tested for their ingestion ability by counting the number of ingested particles on toluidine blue-stained sections of epoxy-embedded PMN suspensions. Both cell types ingested STZ particles at a comparable rate at early postphagocytic times, whereas on prolonged incubation MPO-deflcient PMNs ingested more STZ particles than normal PMNs. These results suggest that the ingestion capacity of normal cells may undergo a more rapid deterioration than that of MPO-deficient cells during incubation with STZ. Evidence for a higher deterioration of normal PMNs with respect to MPO-deficient PMNs was obtained also from studies on the effect of storage on O₂ ^– generation. After standing at melting ice temperature for 3 h, normal PMNs produced less O₂ ^– than MPO-deficient PMNs in response to PMA, and the difference in O₂ ^– production by the two cell types in response to STZ was evident at earlier postphagocytic periods than with freshly isolated cells. Taken all together these results suggest that normal PMNs and MPO-deficient PMNs do not intrinsically differ in O₂ ^– generating potential and that the difference in the respiratory burst observed during phagocytosis may be accounted for by a more marked deterioration, in normal PMNs, of one or more functions related to the respiratory burst.     

Effect of deuterium oxide on neutrophil oxidative metabolism,phagocytosis, and lysosomal enzyme release

We have previously shown that deuterium oxide (D₂O) enhances the oxidation of methionine, a myeloperoxidase (MPO)-mediated reaction, by human neutrophils during phagocytosis. However, D₂O has no effect on the oxidation of methionine by the purified MPO-H₂O₂-Cl^– system. To explain this observation, we studied the effect of D₂O on the oxidative metabolism, phagocytosis, and lysosomal enzyme release by human neutrophils. D₂O stimulated the hexose monophosphate shunt (HMS) activity of resting neutrophils in a dose-response fashion. In the presence of latex particles or phorbol myristate acetate (PMA), D₂O brought about an exaggerated stimulation of the HMS activity. This enhancement of the HMS activity by D₂O was markedly reduced when neutrophils form two patients with X-linked chronic granulomatous disease (CGD) were used, either in the presence or absence of latex particles or PMA. Superoxide and H₂O₂ production by neutrophils in the presence of latex particles or PMA were also stimulated by D₂O. In contrast, D₂O inhibited the ingestion of latex particles. D₂O enhanced the extracellular release of MPO, but not lactate dehydrogenase, by neutrophils only in the simultaneous presence of cytochalasin B and latex particles. The enhancement of HMS activity and MPO release by D₂O was partially inhibited by colchicine. Our results suggest that enhancement of neutrophil oxidative metabolism by D₂O may in part explain the stimulation of methionine oxidation by phagocytosing neutrophils.     

Influence of corticosteroids on human polymorphonuclear leukocyte function in vitro

In an experimental system in which phagocytosis or adherence of cells to surfaces were excluded as variables, we have investigated the possibility that corticosteroids may inhibit release of lysosomal constituents from viable human polymorphonuclear leukocytes into the extracellular environment. Release of-glucuronidase and lysozyme from cytochalasin B-treated cells exposed to serum-treated zymosan, heat-aggregated human IgG, and the complement component C5a was significantly reduced by pretreatment with hydrocortisone sodium succinate and methylprednisolone sodium succinate (5×10^–4 M). Both steroids also reduced superoxide production by these cells. These in vitro studies provide evidence that corticosteroids influence membrane-dependent responses of intact, viable polymorphonuclear leukocytes to immune reactants. Inhibition of these responses (lysosomal enzyme release, superoxide production) may explain, in part, both the antiinflammatory actions of steroids and their deleterious effects on host defenses.This research was supported by grants from the National Institutes of Health (AM-18531, AM-11949, and HL-15140) and by the Whitehall Foundation.Career Scientist of the Irma T. Hirschl Trust.     

Characteristics ofN-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 associated-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 for-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 of-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.     

Extra- and intracellular Ca2+ requirements for lysosomal enzyme secretion in human neutrophils

Ca²⁺-EGTA combinations were utilized in Hank's buffers to fix extracellular free Ca²⁺ concentrations [Ca²⁺ _f] for the study of human neutrophil lysosomal enzyme secretion. Ca²⁺-dependent neutrophil secretion initiated by formyl-methionyl-leucyl-phenylalanine (FMLP) and C5a required small amounts of [Ca²⁺ _f] (1–3×10^–6 M) while that caused by ionophore A23187 required 10^–5 M or greater [Ca²⁺ _f]. The inhibition of FMLP- and C5a-induced lysosomal enzyme secretion by the intracellular Ca²⁺ antagonist, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) was additive to lowering extracellular [Ca²⁺ _f] from 10^–4 M to 10^–6 M or blocking plasma membrane Ca²⁺ flux with verapamil. These results suggest that extracellular and intracellular Ca²⁺ flux may be coupled in the initiation of neutrophil secretion caused by FMLP and C5a.     

Selective enrichment of NADPH oxidase activity in phagosomes from guinea pig polymorphonuclear leukocytes

Recent studies have demonstrated that the activated NADPH oxidase, the enzyme responsible for the stimulation of O₂ consumption with O ₂ ^– formation during phagocytosis, is located in the plasma membrane of leukocytes. The present work deals with whether the activation induced by phagocytosis involves the enzyme of the entire membrane or only that of the portion of the membrane that interacts with the phagocytosable particle and forms the phagosome. The results presented show that the activity of the NADPH oxidase of phagosomal membrane, isolated by centrifugation of homogenates on discontinuous sucrose gradients, is increased 12.6-fold with respect that of homogenate. In contrast, the activities of 5-nucleotidase and of acidp-nitrophenyl phosphatase, enzyme markers of the plasma membrane not activated during phagocytosis and uniformly distributed on the entire membrane, are increased only about threefold with respect to that of homogenate. These results indicate that during phagocytosis the activation of NADPH oxidase is a segmentary response that involves only the enzyme that forms the phagocytic vacuole. This fact is relevant for the function of toxic intermediates of oxygen reduction that are discharged in direct contact with the engulfed agent.     

Polyl-histidine

Poly-l-histidine (PHSTD) of molecular weight 26,000 induced the generation of large amounts of Superoxide (O ₂ ^– ) and hydrogen peroxide (H₂O₂) in human neutrophils (PMNs). Despite its low solubility at neutral pH, PHSTD was bound very rapidly to the PMN surfaces. Maximal generation of O ₂ ^– took place with 4–5 ×10^–6 M of PHSTD, starting after a lag of about 25 sec and proceeding for 15–17 min at a rate of 150 nmol/10⁷ PMNs/min, suggesting that this polycation is one of the most potent stimulators of O ₂ ^– generation known, PHSTD was found to be non-toxic for PMNs even at millimolar concentrations. Generation of O ₂ ^– by PHSTD depended on extracellular calcium; it was inhibited by calcium channel blockers and by trifluoperazine, and it triggered a sharp rise in intracellular calcium as determined by the Quin 2 fluorescence technique. The generation of both O ₂ ^– and H₂O₂ by PHSTD was partially inhibited by cytochalasin B or (CYB, CYE). On the other hand, CYB markedly enhanced the generation of both O ₂ ^– and H₂O₂ following stimulation of PMNs either by PHSTD, polyarginine, histone, or by antibody-opsonized group A streptococci. Electron microscopic analysis and NBT reduction tests revealed that both PHSTD and PHSTD-opsonized streptococci were avidly phagocytosed by PMNs. Since CYB totally inhibited internalization of both PHSTD and the PHSTD-opsonized streptococci, it was suggested that these agents stimulated oxygen radical generation mainly on the leukocyte surfaces. Complexes (CX) formed between PHSTD and polyanethole sulfonate (a strong polyanion) or between histone and the polyanion mimicked immune CX in their ability to trigger the generation of large amounts of O ₂ ^– which were inhibited by CYB. Generation of O ₂ ^– and chemiluminescence either by PHSTD or by PHSTD-opsonized streptococci were markedly inhibited by poly-l-glutamate, suggesting that PHSTD acted as a cationic agent which interacted via electrostatic forces with some negatively charged sites in the leukocyte membrane. Generation of H₂O₂ by PHSTD was also markedly inhibited by deoxyglucose, KCN, DASA, as well as by the lipoxygenase inhibitors nordihydroguaiaretic acid, phenidone, and propylgallate. On the other hand, cyclooxygenase inhibitors such as aspirin, indomethacin, and piroxicam were inactive, suggesting that arachidonic acid metabolism via lipoxygenase pathway might have been involved in the activation by PHSTD of the NADPH oxidase in PMNs. PHSTD may mimic the effects of antibodies both as an opsonin and as a potent stimulator of the respiratory burst in PMNs and may thus serve as a model for further study of leukocyte-bacteria interactions in infectious and inflammatory sites and of the pathogenicity of immune complexes.Supported by a research grant from Dr. S. M. Robbins of Cleveland, Ohio.     

Time course of superoxide generation by leukocytes—The MCLA chemiluminescence system

This study was performed to examine the pattern of Superoxide (O ₂ ^– ·) generation from leukocytes using the O ₂ ^– · specific chemiluminescence (CL) method.Cypridina luciferin analog, 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-alpha]pyrazin-3-one (MCLA) was used as a CL probe. The appropriate conditions of the MCLA method was first determined for the evaluation of the time course of O ₂ ^– · generation by leukocytes. The time course of O ₂ ^– · generation obtained by the MCLA-CL system was compared with that by the luminol-dependent CL, electron spin resonance (ESR)/spin trapping, and cytochromec systems. Following stimulation by three different stimulants (PMA, OZ, FMLP), leukocytes continuously generated O ₂ ^– · for up to 5 h in the MCLA-CL system, irrespective of the kind of stimulation. The curves obtained by generation ceased more rapidly in the luminol-CL, ESR/spin trapping, and cytochromec systems. A 50% activity of the initial value was observed at 70 min in the MCLA-CL system, but 30, 10 and 35 min in the other systems, respectively. The CL or O ₂ ^– · generation value decreased to less than 1% (possible termination) at 300, 90, 120 and 180 min, respectively. With the exception of ESR studies with OZ, the cell viability was not significantly affected in any of the trials. These results indicate that leukocytes can generate O ₂ ^– · much longer than previously estimated and that the MCLA-CL-system is the most suitable system for the measurement of the O ₂ ^– · generation by leukocytes.     

Unique characteristics of superoxide production by human eosinophils in eosinophilic states

Eosinophils from patients with peripheral blood eosinophilia and human neutrophils from normal subjects and patients with neutrophilia produced superoxide anion (O₂ ^–) in vitro at similar rates in the absence of stimulation and exhibited comparably increased rates of O₂ ^– production during the initial 1 h of incubation with opsonized zymosan. In the presence of opsonized zymosan, the rate of O₂ ^– production by eosinophils was constantly high for 3 h, whereas the rate of production by neutrophils fell by more than 65% after 1 h. Consequently, the amount of superoxide produced by phagocytizing leukocytes was twofold higher for eosinophils than for neutrophils at 3 h. O₂ ^– production by cell-free sonicates of zymosan-stimulated eosinophils and neutrophils exhibited the same preference for NADPH over NADH. One mM sodium azide significantly decreased the generation of O₂ ^– by phagocytizing eosinophils, but lacked an effect on neutrophils. The prolonged release of O₂ ^– by eosinophils engaged in phagocytosis may contribute both to their unique microbicidal profile and to the capacity of eosinophils to injure host tissues in some eosinophilic syndromes.     

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 (O ₂ ^– ) 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 O ₂ ^– generation. Preincubation of neutrophils with cytochalasin B prior to their interaction with A23187 results in a significant enhancement of O ₂ ^– production. The activity of the O ₂ ^– -generating system was maximum at 37°C and was significantly curtailed at lower and higher temperatures. A23187-induced O ₂ ^– 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 O ₂ ^– -generating system.