Respiratory Burst Enzyme in Human Neutrophils. Evidence for Multiple Mechanisms of Activation
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Respiratory burst enzyme in human neutrophils. Evidence for multiple mechanisms of activation. L C McPhail, … , P M Henson, R B Johnston Jr J Clin Invest. 1981;67(3):710-716. https://doi.org/10.1172/JCI110087. Research Article Alteration of the surface of human neutrophils with the nonpenetrating, protein-inactivating agent p-diazobenzenesulfonic acid (DASA) was found to prevent activation of the respiratory burst by some stimuli, but not others. Production of superoxide anion (O2-) stimulated by concanavalin A or the chemotactic peptide formyl-methionyl-leucyl-phenylalanine FMLP was inhibited by DASA pretreatment, whereas O2- production stimulated by phorbol myristate acetate (PMA), sodium fluoride. or the ionophore A23187 was not inhibited by DASA. Pretreatment with DASA inhibited oxygen uptake stimulated by FMLP, but not oxygen uptake stimulated by PMA. DASA reproducibly inhibited activities of two known surface enzymes Mg++-ATPase and alkaline phosphatase, by 45-55% and 60-70%, respectively. The inhibition by DASA of O2- production did not appear to be caused by interference with binding of the affected stimuli, since pretreatment with DASA did not inhibit release of the lysosomal enzymes lysozyme and myeloperoxidase induced by concanavalin A or FMLP. Membrane-rich particulate fractions from neutrophils have been shown to contain NADPH-dependent oxidative activity that is presumably responsible for the phagocytosis-associated respiratory burst of intact cells. The PMA-activated enzyme was susceptible to inhibition of directly exposed to DASA in this particulate fraction. These findings suggest that more than one mechanism exists for activation of the respiratory burst oxidase in human neutrophils, and that the neutrophil possesses at least one oxidase that is not an ectoenzyme. Find the latest version: https://jci.me/110087/pdf Respiratory Burst Enzyme in Human Neutrophils EVIDENCE FOR MULTIPLE MECHANISMS OF ACTIVATION LINDA C. MCPHAIL, PETER M. HENSON, and RICHARD B. JOHNSTON, JR., Department of Pediatrics, National Jewish Hospital and Research Center, and University of Colorado School of Medicine, Denver, Colorado 80206 A B S TR A C T Alteration of the surface of human neu- gen metabolites are essential to the microbicidal func- trophils with the nonpenetrating, protein-inactivating tion of the neutrophil (1, 2), and the biochemical basis agent p-diazobenzenesulfonic acid (DASA) was found of their generation is of great interest. The enzyme gen- to prevent activation of the respiratory burst by some erally believed to initiate this respiratory burst is an stimuli, but not others. Production of superoxide anion oxidase which utilizes as electron donor NADPH, (O°) stimulated by concanavalin A or the chemotactic NADH, or both (1, 3). It has not yet been purified or peptide formyl-methionyl-leucyl-phenylalanine well characterized, and its location in the neutrophil is FMLP was inhibited by DASA pretreatment, whereas still controversial (3, 4). °2 production stimulated by phorbol myristate Activation ofthe respiratory burst enzyme is initiated acetate (PMA), sodium fluoride, or the ionophore by perturbation of the plasma membrane and does not A23187 was not inhibited by DASA. Pretreatment with require phagocytosis. A number of soluble compounds DASA inhibited oxygen uptake stimulated by FMLP, are effective stimuli, including chemotactic factors, de- but not oxygen uptake stimulated by PMA. DASA tergents, lectins, certain lipids, and ionophores (5). reproducibly inhibited activities of two known sur- Activation of the respiratory burst appears to involve face enzymes, Mg++-ATPase and alkaline phospha- calcium ion with some of these stimuli but not with tase, by 45-55% and 60-70%o, respectively. The inhibi- others (6, 7), raising the possibility that different stimuli tion by DASA of °2 production did not appear to be activate the oxidase by different mechanisms. One caused by interference with binding of the affected possible approach to this question would be to test stimuli, since pretreatment with DASA did not inhibit the effects of chemically altering the plasma mem- release of the lysosomal enzymes lysozyme and mye- brane on the ability of various stimuli to activate loperoxidase induced by concanavalin A or FMLP. oxidative metabolism. Membrane-rich particulate fractions from neutro- One chemical agent previously used to inhibit sur- phils have been shown to contain NADPH-dependent face membrane proteins is p-diazobenzenesulfonic oxidative activity that is presumably responsible for the acid (DASA)1 (8). Pretreatment of neutrophils by DASA phagocytosis-associated respiratory burst of intact cells. was reported to inhibit the ability of the lectin concana- The PMA-activated enzyme was susceptible to inhibi- valin A (Con A) to stimulate °2 production (9). This tion if directly exposed to DASA in this particulate was widely interpreted as evidence that the O2 form- fraction. These findings suggest that more than one ing oxidase was an ectoenzyme, but the experiments mechanism exists for activation of the respiratory burst also raised the possibility that another step in the path- oxidase in human neutrophils, and that the neutrophil way of activation used by that stimulus was affected. possesses at least one oxidase that is not an ectoenzyme. We found that DASA pretreatment did not inhibit stim- ulation of O2 production by the surface-active lipid INTRODUCTION phorbol myristate acetate (PMA). This prompted us to survey the influence of DASA pretreatment on activa- Phagocytosis by human neutrophils induces a dramatic stimulation of their oxidative metabolism, during which oxygen is converted to superoxide anion (O°), 'Abbreviations used in this paper: Con A, concanavalin A; hydrogen peroxide, and hydroxyl radical (1). These oxy- DASA, p-diazobenzenesulfonic acid; DMSO, dimethyl sulfox- ide; FMLP, formyl-methionyl-leucyl-phenylalanine; KRP-D, Krebs-Ringer phosphate buffer with dextrose; LDH, lactate Receivedfor publication 17 December 1979 and in revised dehydrogenase; PBS-D, phosphate-buffered saline with dex- form 22 September 1980. trose; PMA, phorbol myristate acetate. 710 J. Clin. Invest. X The American Society for Clinical Investigation, Inc. - 0021-9738181/0310710/07 $1.00 Volume 67 March 1981 710-716 tion of the respiratory burst by a variety of soluble varied somewhat depending on the stimulus used. The assay stimuli. The results, described below, suggest that buffer was KRP-D in experiments with the stimuli PMA, Con more one mechanism for activating the respiratory A (Sigma Chemical Co., St. Louis, Mo., Lot 16C-1790; Phar- than macia Fine Chemicals, Uppsala, Sweden, Lot DB-5333), and burst exists in human neutrophils. the ionophore A23187 (kindly supplied by Dr. Robert L. Hamill of Eli Lilly, Inc., Indianapolis, Ind.). Con A was dis- METHODS solved in KRP-D before use. Ionophore A23187 was stored at -70°C as a 0.1 mM stock solution in DMSO. Equivalent Isolation ofneutrophils and particulatefractions. Human amounts of O- were produced by normal cells stimulated neutrophils were isolated from heparinized venous blood by with either Sigma or Pharmacia Con A; experiments with dextran sedimentation and Ficoll-Hypaque centrifugation DASA were performed using Sigma Con A. Assays with the (2). Cells were counted in a hemocytometer and suspended stimulus FMLP (Vega-Fox Biochemicals, Div. Newbery at the appropriate concentration in Krebs-Ringer phosphate Energy Corp., Tucson, Ariz.) used phosphate-buffered saline buffer, pH 7.34, containing 0.2% dextrose (KRP-D). Prepara- containing 0.2% dextrose (PBS-D), and were performed in the tions routinely contained 97-99%o granulocytes. presence of 1 mM CaCl2. FMLP was used from a 0.1 mM Particulate fractions were isolated as described (10, 11). stock in distilled water kept at -70°C. The use of NaF (Fisher Briefly, neutrophil suspensions at 5 x 107/ml were incubated Scientific Co., Fairlawn, N. J.) as a stimulus required a special for 3 min at 37°C with 1 mM KCN or 1 mM NaN3 (12) and buffer based on Dulbecco's phosphate-buffered saline, but PMA (Consolidated Midland Corp., Brewster, N. Y.) dissolved containing only 0.117 M NaCl and 0.31 mM CaCI2 (15) to cor- in dimethylsulfoxide (DMSO) at a final concentration of 100 rect for the electrolytes added with the NaF and to prevent ng/ml, or with an equivalent amount of DMSO. In some cases the precipitation of CaF2. In assays where less than 20 mM (as noted in Results), particulate fractions were prepared from NaF was used, NaCl was added to balance electrolytes. All cells incubated in a similar manner for 3 min with opsonized assays with NaF were performed in plastic tubes to prevent zymosan (10), at a final zymosan concentration of 11 mg/ml but adsorption of the stimulus onto glass. NaF was used from a in the absence of KCN or NaN3. Treated cell suspensions were freshly made 0.6 M stock in distilled water. diluted with an equal volume of cold KRP-D and were centri- NADPH-dependent 0° production by isolated particulate fuged in the cold for 8 min at 180 g. Cells were resuspended fractions was measured as reduction of cytochrome c (11). in cold 0.34 M sucrose to 2.5 x 107/ml and sonicated immedi- The assay mixture consisted of 0.05 M potassium phosphate ately in an ice-water bath for 1.5 min at a setting of 60 W for buffer, pH 7.0, 0.2 mM NADPH, 0.08 mM cytochrome c, and 30-s intervals alternating with cooling (Branson Sonifier, 0.1-0.3 mg/ml particulate fraction. Half of the mixture was Model W185; Branson Sonic Power Co., Danbury, Conn.). placed in the sample cuvet and half in the reference cuvet, Sonicates were centrifuged at 500 g for 10 min; supernates which also contained 50 ,ug/ml superoxide dismutase. The as- were then centrifuged at 27,000 g for 15 min. The pellets from say was performed at ambient temperature and was followed this final centrifugation (particulate fractions) were resus- continuously on a Cary 219 double-beam spectrophotometer pended in 0.34 M sucrose to a protein concentration of 1-2 (Varian Associates, Industrial Equipment Group, Palo Alto, mg/ml (13), and stored at -70°C.