The release of lysosomal enzymes from human polymorphonuclear leukocytes by human C3e

C3e is a leukocytosis-inducing peptide of degraded C3 that has the electrophoretic behavior of prealbumin and a MW of 10-12,000. Using a homogeneous preparation, the ability of C3e to promote lysosomal enzyme release from human polymorphonuclear leukocytes (PMNs) was examined by incubation of various concentrations of C3e with cytochalasin B-treated (5 micrograms/ml) human PMNs for 60 min at 37 degrees C. Amounts of extracellular beta-glucuronidase, myeloperoxidase, and lysozyme were determined in the cell-free supernatants and it was found that all three enzymes were released in significant amounts without concomitant release of the cytoplasmic enzyme lactate dehydrogenase (LDH). At a concentration of 25 micrograms/ml of C3e, 25 +/- 1.1% (SD) of lysozyme, 20 +/- 0.9% beta-glucuronidase, and 24.3 +/- 0.9% myeloperoxidase were released into the supernatant while the release of LDH remained within the 4-7% range throughout these studies. Furthermore the supernatants were found to contain a substance which was capable of a generating chemotactic fragment from isolated C5. The same range of C3e concentrations (5-25 micrograms/ml) was, however, incapable of effecting histamine release from basophils. These results suggest that generation of C3e in vivo may serve as a potent stimulus for the generation of the C5-cleaving enzyme from PMNs which in turn may function to recruit more neutrophils by a positive feedback mechanism.     

Biochemical characteristics of ATP-induced secretion of lysosomal enzymes from rabbit polymorphonuclear leukocytes

ATP induces a release of the lysosomal enzymes,β-glucuronidase and lysozyme, but not the cytoplasmic marker, lactic dehydrogenase, from rabbit peritoneal polymorphonuclear leukocytes. The release requires a low-ionic-strength medium but not divalent cations. It occurs to a greater extent in the presence of K⁺ than Na⁺, is greatly enhanced by cytochalasin B, is temperature dependent, and apparently requires a source of metabolic energy as indicated by its inhibition by deoxyglucose. The release is also inhibited by iodoacetate and the organic mercurial, salyrgan. DFP does not inhibit the release, indicating that esterase activation apparently is not involved. ADP and AMP do not sustain the release, nor do the ATP phosphorate analogs in which a methylene group is substituted for the oxygen between theα andβ phosphate groups or theβ andγ phosphates. A tentative explanation for these findings is that ATP induces lysosomal enzyme release in polymorphonuclear leukocytes by reacting directly with an ATPase which needs low ionic strength but not divalent cations for its activity. In doing so, ATP bypasses the proesterase activation and external Ca²⁺-mediated steps required in other forms of induced lysosomal enzyme release in the polymorphonuclear leukocyte but uses the same common final pathway.     

Ultrastructural alterations during ATP-induced secretion of lysosomal enzymes from rabbit polymorphonuclear leukocytes

Rabbit neutrophils incubated in low-ionic-strength media were stimulated by ATP to secrete lysosomal enzymes. This was greatly enhanced in the presence of cytochalasin B. ATP in these circumstances induced the cell to form large cytoplasmic extensions that were largely devoid of granules. In the presence of both ATP and cytochalasin B, however, the projections contained granules in close proximity to the cell membrane. Neutrophils in low-ionic-strength buffer were capable of binding to zymosan particles coated with C3b but not of phagocytizing them. Release of granule enzymes was observed and exocytosis of granules appeared to occur at sites distant from those portions of the plasma membrane adherent to the particle.Support was provided by U.S.P.H.S. grant A1-07007 and GMS 19322-03 and by U.S.P.H.S. Career Development Award 1-KO4GM-42567-05 to Dr. P.M. Henson. This is publication no. 873 from the Department of Immunopathology, Scripps Clinic and Research Foundation, La Jolla, California.Support was provided by U.S.P.H.S. grant A1-09648.     

Enhancement of in vitro immunoglobulin synthesis of human lymphocytes by lysosomal enzymes from polymorphonuclear leucocytes.

The effect of human peripheral blood polymorphonuclear leucocyte (PMN) extracts and PMN granule lysates on in vitro immunoglobulin (Ig) synthesis by autologous peripheral blood mononuclear cells was studied. The mononuclear cells were cultured for 3 days with or without autologous plasma. Newly synthesized Ig in the culture supernatants was measured using 14C-labelled amino acids by an immune coprecipitation method. Upon addition of a PMN extract to plasma-free cultures Ig synthesis was stimulated, the mean stimulation index (SI) of cultures from thirteen individuals, including nine normals, three patients with rheumatoid arthritis and one with psoriatic arthritis being 1-8 +/- 0-2 in comparison with control cultures (P less than 0-05). By contrast, in 10% fresh autologous plasma, PMN extracts yielded a mean SI of 0-9 +/- 0-1 indicating inactivation of the active extracts by plasma inhibitors. In experiments using PMN granule lysates containing high concentrations of beta-glucuronidase and cultured in RPMI 1640, the mean stimulation index was 3-2 +/- 0-7. Stimulation of Ig synthesis was also produced by trypsin. Stimulation of Ig synthesis was also produced by trypsin. Stimulating factors in PMN extracts were inhibited by Trasylol, a protease inhibitor. These results indicate that trypsin and proteolytic lysosomal enzymes in PMN increase Ig synthesis of human peripheral blood mononuclear cells. They suggest a possible new role of PMN in the potentiation of immunoglobulin synthesis.     

Influence of human plasma kallikrein on lysosomal enzyme release from polymorphonuclear leukocytes

Stimulation of polymorphonuclear leukocytes with kallikrein demonstrated that enzyme acts selectively on the release of lysosomal enzymes of these cells. The release of collagenase, similarly to the release of lysozyme into the incubation medium increased proportionally to kallikrein concentration and the duration of incubation. Kallikrein had a small effect on -glucuronidase secretion. No effect on cytoplasm lactate dehydrogenase release was detected. These results suggest that kallikrein, as a soluble stimulus, predominantly induces degranulation of specific granules containing collagenase capable of degrading the connective tissue. Secretion of lysozyme and collagenase requires the presence of active kallikrein. Soybean trypsin inhibitor diminished the enzyme release.     

Cytochalasin B-dependent release of azurophil granule enzymes from human polymorphonuclear leukocytes

The dose-response effects of phorbol myristate acetate and cytochalasin B on secretion of azurophil and specific granule enzymes from viable human polymorphonuclear leukocytes have been examined. Secretion of the azurophil granule enzymes elastase and-glucuronidase from cells exposed to 50 ng/ml of phorbol myristate acetate is dependent on prior exposure of the cells to greater than 0.5 mg/ml of cytochalasin B. In contrast, the secretion of the specific granule enzyme lysozyme is not dependent on pretreatment with cytochalasin B. The concentration of phorbol myristate acetate needed to elicit maximal secretion of specific versus azurophil granule enzymes differs, being 5.0 ng/ml and 50 ng/ml, respectively. The results suggest that cytochalasin B-sensitive cellular components, possibly microfilaments, may selectively modulate some step in the exocytosis of azurophil granule enzymes from human polymorphonuclear leukocytes exposed to phorbol myristate acetate.     

Effect of growth hormone on the activity of some lysosomal enzymes in neutrophilic polymorphonuclear leukocytes of hypopituitary dwarfs

Elevated activities of beta-D-glucuronidase, myeloperoxidase, and lysozyme were found in polymorphonuclear leukocytes (PMNs) of both hypopituitary dwarfs and normal subjects after the administration of growth hormone (GH), as compared to the activities in PMNs from blood drawn immediately before the administration of GH. During in vitro incubation, GH was able to inhibit the release of lysosomal enzymes from resting PMNs. This inhibition may be one of the reasons for the elevated lysosomal enzyme activities observed in PMNs after the administration of GH. GH can also affect hexose monophosphate shunt (HMPS) activity and superoxide production by PMNs. The activity of HMPS is stimulated by GH in resting PMNs, while in PMNs incubated with zymosan the GH inhibits both HMPS and superoxide production.     

Different sensitivity of Pseudomonas aeruginosa exotoxin A and diphtheria toxin to enzymes from polymorphonuclear leukocytes

We demonstrate that exotoxin A (ExoA) of Pseudomonas aeruginosa is one to two orders of magnitude more sensitive than diphtheria toxin (DT) of Corynebacterium diphtheriae to lysosomal enzymes from polymorphonuclear leukocytes (PMN). It is especially sensitive to PMN elastase which inactivates its cell free enzymatic activity and its cytotoxicity as measured with the Chinese hamster ovary cell assay and the rabbit skin test. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed a rapid fragmentation of ExoA into small peptides at low PMN elastase concentrations, whereas DT remained largely uncleaved at PMN elastase concentrations 10 times higher. PMN elastase also removed the cell surface receptors for ExoA and DT on Chinese hamster ovary cells, suggesting that both toxins may be ineffective at local sites of severe inflammation. A comparison of fibroblasts from cystic fibrosis patients and normal healthy individuals revealed no differences in susceptibility to either DT or ExoA; this tends to exclude a genetic defect as an explanation for the absence of ExoA effects in cystic fibrosis patients.     

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.     

Modulation of the inflammatory response by products released from human polymorphonuclear leukocytes during phagocytosis

During phagocytosis of latex particles human polymorphonuclear leukocytes (PMNs) release a product that generates chemotactic activity from fresh human serum. Release of this product is maximal at 20–30 min of phagocytosis. It is present in resting PMNs, may be recovered from granule fractions, and functions at physiologic pH. Gel-filtration chromatography of the activated serum indicates that C5a accounts for most of the chemotactic activity generated. Studies utilizing preparations of C5, EDTA, magnesium-EGTA, CS-deficient serum, and serum heated for 20 min at 50°C demonstrate that generation of C5a results from activation of the complement system as well as from direct cleavage of C5. Activation of the complement system by this PMN-derived serum activator appears to proceed through both the alternate and classical pathways. In addition to this serum activator, an inactivator of C5a chemotactic activity is also released by the PMNs under certain conditions of phagocytosis. These studies suggest that phagocytizing PMNs have secretory functions that contribute to the localization and amplification of inflammatory responses.     

Chemiluminescent response to pathogenic organisms: normal human polymorphonuclear leukocytes

Chemiluminescence (CL) is a sensitive indicator of phagocytosis and intracellular killing; however, little is known of the normal CL response by human polymorphonuclear leukocytes to different pathogenic microorganisms. We investigated the luminol-enhanced CL response of normal polymorphonuclear leukocytes to a number of common bacterial pathogens and two yeasts. We analyzed the CL response to viable and heat-killed microorganisms at 25 and 37 degrees C. The CL response to all microorganisms was greater and more rapid at 37 degrees C. Variable responses were observed with viable and heat-killed microorganisms; some were unaffected, whereas other demonstrated reduced CL. Each microorganism caused a reproducible response pattern, which could be placed into two general categories. In the first category were those which caused a rapid exponential rise and decay in CL: Enterobacter cloacae, Salmonella typhimurium, Shigella flexneri, Staphylococcus aureus, Candida albicans, and zymosan. In the second category were those which rose slowly over a longer time course to a poorly defined peak: Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus mirabilis, and Streptococcus pyogenes. The CL response also reflected serum opsonic activity. The effect of inactivated complement, factor B, and removal of specific antibody were investigated. Increasing the concentration of zymosan gave a proportional rise in peak CL; however, a strain of E. coli caused a variation in peak time rather than peak height. Different CL kinetics were shown for three strains of K. pneumoniae, possibly a result of each having different membrane or cell wall characteristics. This study defines the nature and factors affecting the normal CL response to a variety of common pathogenic microorganisms.     

Enhancement of in vitro lymphocyte response by neuraminidase

In vitro blastogenic response of sensitized human lymphocytes to specific antigens was significantly enhanced by Vibrio cholerae neuraminidase treatment. No such enhancement was noted when the unsensitized lymphocytes were treated with this enzyme. An enhancement effect of neuraminidase on the lymphocyte response to concanavalin A or pokeweed mitogen (weak mitogens) was also noted; the enzyme had no effect on phytohaemagglutinin (strong mitogen)-induced blasto-genesis. The increased reactivity of lymphocytes is probably related to changed physical and functional properties of the cells after treatment with neuraminidase.     

Chemiluminescence response of polymorphonuclear leukocytes in atopic dermatitis

Atopic dermatitis (AD) is characterized by many signs of immunodeficiency. Our interest was to investigate if there are also alterations of the chemiluminescence (CL) response of polymorphonuclear leukocytes (PMN) as a measure of the release of toxic oxygen radicals. Isolated PMN of 13 patients with AD with mild to moderate disease activity were stimulated with a chemotactic peptide (f-met-phe), zymosan-activated serum (ZAS), zymosan particles and phorbolmyristate acetate. In the AD group, we found a significantly decreased response after stimulation with ZAS in comparison to the controls. With the other stimuli tested no significant difference was detected. The decreased response of PMN to stimulation with ZAS from patients with AD associated with a normal reactivity to the other stimuli could be due to specific desensitization of the PMN by C5a in vivo.     

Decreased extracellular release of granule enzymes from in vitro-stimulated polymorphonuclear leukocytes in guttate psoriasis

In vitro degranulation of polymorphonuclear leukocytes, which were stimulated either with synthetic chemotactic peptide (N-formyl-methionyl-leucyl-phenylalanine, FMLP) or with C3b-opsonized zymosan as a promoter of phagocytosis, was studied in 66 patients with psoriasis, 18 lesion-free psoriatics, 18 healthy subjects, and 14 other dermatological disorder controls. Stimulated release of lysozyme (from specific granules and azurophil granules) and beta-glucuronidase (from azurophil granules) in the presence of both FMLP and serum-activated zymosan was markedly reduced in patients with actively spreading guttate psoriatic lesions, in whom relapse of lesions lasted for less than 1 month and papules involved about 13–25% of skin surface. In contrast, stimulated degranulation was within normal range in active plaque psoriasis, stationary plaque psoriasis, symptomless psoriatics, and patients with disseminated eczema. Spontaneous release of lysozyme and beta-glucuronidase (background) was found to be not different in all groups studied; however, patients with active guttate psoriasis had significantly lower total lysozyme activity than those with active and stationary plaque psoriasis as well as psoriatics in the remission. These data are in favor of in vivo activation of neutrophils in active guttate psoriasis by some factors related to the early relapse of the lesions. This results in a possible combination of the following phenomena: (1) in vivo partial degranulation of neutrophils; (2) induction of unresponsiveness state of these cells to subsequent in vitro stimulation; and/or (3) migration of highly responsive neutrophils to skin lesions, which leaves in the circulation the subpopulation less reactive to chemotactic and phagocytic stimuli.     

Chemiluminescence by polymorphonuclear leukocytes adhering to surfaces.

Stimulation of the plasma membranes of granulocytes results in an oxidative metabolic response. This response can be measured by measuring the reduction of oxidizable substrates, such as Nitro Blue Tetrazolium, as well as by measuring the energy released as light (chemiluminescence). While investigating the oxidative response of human granulocytes, we observed a marked variation in the chemiluminescence response when leukocytes were suspended in a balanced salt solution without gelatin or any other protein. We performed systematic study to investigate the role of protein in suspensions of human polymorphonuclear leukocytes. Final results were identical with human serum, albumin, fetal calf serum, and gelatin; gelatin was used as the protein source in most experiments. Polymorphonuclear leukocytes suspended in Hanks balanced salt solution without gelatin decreased in numbers during incubation at room temperature (approximately 50 percent after 60 min). Cell structures were observed on the walls of the tubes containing leukocyte suspensions without gelatin. Numbers of polymorphonuclear leukocytes were stable in suspensions containing gelatin. A chemiluminescence response which peaked at approximately 10 min and was sustained for at least 30 min was observed in suspensions of polymorphonuclear leukocytes without gelatin. This surface attachment-stimulated chemiluminescence occurred in the absence of either soluble or particulate stimuli. Chemiluminescence was inhibited by either superoxide dismutase or sodium azide and did not occur with suspensions of granulocytes from patients with chronic granulomatous disease. We postulate that both superoxide- and myeloperoxidase-dependent oxidative metabolic reactions are induced during the adherence of polymorphonuclear leukocytes to surfaces. Gelatin or other proteins in leukocyte suspending media are necessary when assays are performed to evaluate the metabolic responses of these cells to particulate or soluble stimuli.     

Human polymorphonuclear leukocytes produce cytokines in response to Leishmania major promastigotes

Polymorphonuclear leukocytes (PMN) release cytokines that may influence the development of the subsequent adaptive immune response. Little is known about cytokines produced by human PMN in response to Leishmania (L.). In this study, mRNA expression of Interleukin (IL)‐12p40, IL‐12p35, Interferon (IFN)‐γ, transforming growth factor (TGF)‐β, IL‐1, and IL‐4 in PMN of volunteers stimulated with L. major promastigotes has been investigated by real‐time PCR and the results were confirmed by flow cytometer. The results showed that L. major did not induce mRNA expression of IL12p40, IL12p35, IFN‐γ, and TGF‐β in PMN, while IL‐1 and IL‐4 mRNA were induced. Flow cytometry results confirmed no IFN‐γ production by PMN with or without stimulation. IL‐12p70 was present in untreated and L. major‐treated PMN, and these cells release IL‐12 following incubation with L. major. Significant amount of IL‐1 even without treatment with promastigotes was detected in PMN. Moreover, the proportion of PMN, which produce IL‐1 in response to L. major, was increased compared with the percent of unstimulated IL‐1‐producing PMN. The results showed the accumulation of small amounts of IL‐4 in PMN after stimulation. In conclusion, our results indicate that IL‐12 and IL‐1 are pre‐stored in human PMN, nor L. major induces IL‐1 and IL‐4, but not IL‐12, IFN‐γ, nor TGF‐β expression in these cells.