Vol. 169, No. 1, 1990 May 31, 1990 A PROTEIN BIOCHEMICAL The Department Tel-Aviv March RESEARCH COMMUNICATIONS Pages 198-202 OF NEUTROPHIL GRANULES INTERFERES WITH OF NADPH OXIDASE IN A CELL-FREE SYSTEM Irit Received AND BIOPHYSICAL 26, Aviram and Anat ACTIVATION Faber of Biochemistry, Faculty of University, Tel-Aviv 69978, Life Sciences, Israel 1990 A soluble extract of neutrophil granules interfered with activation of the NADPH oxidase in a cell-free system . The extract had no effect on superoxide production by preactivated enzyme. The inhibitory activity was retained during dialysis and was lost upon exposure to proteinase K indicating that the active substance was a protein. The inhibitor exhibited a high stability at elevated temperatures . Chromatography of granules extract on ion exchangers implied that the inhibitor was a positively charged protein eluting from S Sepharose cation exchanger al990 Academic Press, Inc. above 0.4M concentration of NaCl. Superoxide kill ingested the superoxide to infections rare clinical production by stimulated neutrophils enables the cell to microorganisms (1,Z). The physiological significance of generating NADPH oxidase is evidenced by susceptibility of patients of chronic granulomatous disease (CGD) . This syndrome is characterized by a defective oxygen radical production by patients phagocytes (3,4). Since uncontrolled release of superoxide ions may damage body tissues, a careful regulation of the activity of NADPH oxidase is reguired. Latency of the enzyme in resting cells as well as pathways for deactivation of active NADPH oxidase in prestimulated PMN (5,6) represent different approaches for protection of body tissues against the effects of permanently active NADPH oxidase. NADPH oxidase can be activated also in a cell-free system consisting of neutrophil or macrophage plasma membranes, cytosolic components and an unsaturated fatty acid (e.g.arachidonate) or SDS (7,s ). In early reports on cell-free activation of NADPH oxidase in PMN , homogenates or unpurified cellular membranes were employed (7). Later, when only the light membrane fractions obtained by differential ( 9 ) or by density gradient centrifugation (10,ll ) were used for oxidase activation , specific activities improved . This observation suggested to us that in the granules fraction present in unpurified homogenates or membranes , an inhibitory substance might have been present. In the present report evidence for the existence of such an inhibitor of NADPH oxidase activation is presented. 0006-29lX'!JO $1.50 Copyright All rights 0 I990 by Academic Press, of reproduction in any form Inc. reserved. 198 Vol. 169, No,, 1, 1990 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS MATERIALS AND METHODS Fractionation & neutrouhils: Human neutrophils were isolated from fresh buffy coats by the standard procedure of dextgan sedimentation and Ficoll density gradient centrifugation. Cells (10 /ml) in 1OmM potassium buffered saline (PBS) containing 1mM EGTA , 7 mM phosphate - (pH 7.0) (PMSF) and 15 pg/ml leupeptin were disrupted by sonication as described (11). Sonicates were centr'ifuged for 10 minutes at BOOg and the pellets were discarded. Granules were sedimented 10 min at 10,OOOg and the supernatants were recentrifuged for 45 min at 204,000 g to give soluble cytosol and low density were resuspended in PBS / 0.34 M sucrose membranes . The membranes (sucrose-PBS). NADPH oxidase activation and assav: Arachidonate-dependent activation was performed in two succezve steps as described (11). Membranes and cytosol ( 1-2~10~ and 2-4~10~ cell equivalents respectively) in 0.1 ml sucrose-PBS containing 5 mM magnesium acetate and arachidonate (240-300 PM) were incubated 6 minutes at 30°C. After dilution with 0.7 ml sucrose-PBS / 0.2 mM NADPH / SO pg cytochrome c reduction rates of cytochrome c were measured at 550 nm before and aft& the addition of 30 pg of superoxide dismutase (SOD). In most experiments oxidase was activated in a single step assay using SDS (50 PM) . The reaction buffer (0.8 ml) consisted of lo mM pH 6.7 Hepes-buffered saline (HBS)/ 10 pM flavin adenine dinucleotide / a0 PM cytochrome c/ 1 mM EGTA . When column fractions eluted at high NaCl concentration were added to the activation mixture, controls of NADPH oxidase activity at identical salt concentration were run in parallel. Extraction & aranules: Granules pellet was resuspended in 20 mM TrisHCl pH7.5 buffer/EGTA/leupeptin/PMSF at a density corresponding to 2X10a cell equivalents/ml and sonicated (3x20 set). The sonicate was centrifuged 15 minutes at 27,000g and the supernatant representing the soluble granules extract containing 0.9-1.3 mg/ml protein was used as the source of the inhibitory material. m exchanae chromatoaranhv of the inhibitor : Granules extract (1-3ml) was applied to a 0.8x1.5 cm DE-52 cellulose (Whatman) column equilibrated with 20 mM Tris-HCl pH 7.5. The flow-through containing all the inhibitory activity was loaded on a o.axl.ocm S Sepharose (Sigma ) column equilibrated with 20 mM Hepes pH 7.0 buffer. The column was washed with the equilibration buffer containing 0.15 M Nacl and eluted with a linear 0.15-0.8M NaCl gradient (20 ml total volume). Fractions of 1.0 ml were collected. Protein concentration: was evaluated by the method of Lowry ( 12 ) . Columns fractions were monitored at 28Onm . RESULTS AND DISCUSSION Inhibition of NADPH oxidase activation & aranules extract. Arachidocell-free activation of the NADPH oxidase was carried out nate-promoted in two steps to permit separation of the activation and activity phases increasing amounts of the granules extract were present (11 ). When inhibition of during the first (activation) phase , a dose dependent Introduction of the inhibitory enzymic activity was observed (Fig.1). dose after completion of the activation (6 minutes) followed by an additional 6 minutes of incubation had no effect on superoxide production, indicating that the inhibitory substance interfered with activation step only and did not act as a superoxide scavenger. Since otherwise the experimental conditions at which the components of oxidase this finding implied also were exposed to the inhibitor were identical, 199 Vol. 169, No. 1, 1990 BIOCHEMICAL 5.0 AND BIOPHYSICAL 10.0 15.0 granule 20.0 extract 25.0 RESEARCH COMMUNICATIONS 30.0 (pg) Fig-l. !Phe effect of soluble granules extract on the activity of NADPH oxrdase activated by arachidonate in a cell-free system.(o)-the extract was present during the initial step (6 minutes) of arachidonate-mediated was added after the initial 6 minutes activation : (0) -extract and arachidonate and incubated for preincubation of membranes , cytosol an additional 6 minutes before final dilution. that the latter did not act as a non specific protease . It follows that the inhibitory substance seems distinct from the NADPH oxidaseinactivating proteinase of azurophilic granules mentioned by Borregaard (13)* Granules extract could be Pronerties of the inhibitory substance. concentrated by ultrafiltration (Diaflo ultrafilter, Amicon, equipped with yMl0 membrane ) and when chromatographed on a Sephadex G-25 column, the active substance eluted in the void volume (results not shown): both findings indicated that the inhibitor was a macromolecule. At pH 7.5 the inhibitor present in the extract of granules was not retained by a DE-52 cellulose anion exchange column . Flow-through of 0.4 . 0.3 I 4 0.2 z 4- 0.1 0.0 0 5 10 15 FRACTION 20 25 Nr. Fig.2. Chromatography of the NADPIi oxidase inhibitory substance on a S Sepharose cation exchange column. Soluble extract of neutrophil granules (6.0 ml of 0.8 mg protein/ml) was loaded on a DE-52 cellulose column ; flow-through containing all the inhibitory activity ( 0.5mg/ml protein) was transferred to an S Sepharose column and eluted by salt gradient. 0.1 ml of each fraction was added to SDS-supported NADPH oxidase activation assay. 200 Vol. the 169, No,, 1, 1990 DE-52 Column BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS containing all activity detected in the soluble extract , was loaded on a Sepharose S cation exchanger . The inhibitor was adsorbed by the resin and could be eluted as a broad peak between 0.4-0.7 M NaCl concentration (Fig.2 ). The inhibitory substance (in the crude extract and in partially purified fractions from S Sepharose column) exhibited a high stability to heat: its activity was not reduced by IO minutes incubation at 90°C This property was taken advantage of in experiments devised (Table I). to test whether it was susceptible to cleavage by proteinase K, namely, whether it was a protein . The granules extract was incubated I5 minutes with proteinase K (Merck). Before the resulting extract could be assayed for its effect on oxidase activation it was necessary to inactivate proteinase K, since the latter abolished activation of the oxidase This was accomplished by exposure of the mixture of granule (Table I). extract and proteinase K to 90° , a treatment which inactivates proteinase K (Table I). The extract treated with proteinase K lost its inhibitory activity, indicating that the active substance was a protein, cleaved by the protease. In conclusion t we described a novel activity of neutrophil granules which interferes with activation of the NADPH oxidase in a cell-free system. The inhibitory substance is a positively charged highly thermostable protein . It is conceivable that the inhibitory protein might have been defined previously but its regulatory function with respect to activation of the NADPH oxidase remained unrecognized The present study gives no indication regarding the mechanism action of the inhibitor nor its exact location within the different subpopulations of granules (I3,14) . It is noteworthy that Clark al., (15) and Borregaard (13) claimed that most of activatable, membrane-bound NADPH oxidase of resting , disrupted neutrophils Table Effects of None DE-52 flow-through (17.5 fig) heateda DE-52 flow-through proteinase X-treated r heateda proteinase Kc heated proteinase Ka : O~(nmol/min*mg DE-52 flow-through inhibitor et is to protein) 450 155 160 400 30 400 '70 jbg protein in 0.1 ml 20 mM Tris-HCl pIi 7.5 was incubated temperature with 40 pg proteinase K and heated for 10 minutes 25~1 of this mixture was added to the oxidase assay. %Opg protein. 2Ul . of I heat and proteinase X on the capacity of the interfere with activation of NADPIi oxidase Additions , at room at 90 OC. Vol. 169, No. 1, 1990 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS located in specific granules . This would suggest that the inhibitor described and partially charaterized in this study is confined to other compartments of the cell e.g. azurophilic granules. The existence of a negative regulatory control of activation of the NADPH oxidase might have been anticipated. Most constituents of granules exhibit however antimicrobial activity (14) : in this respect , a possible antiinflammatory activity of a protein activation of the superoxide generating pathway studies will be necessary to define the nature of mode of its activity within the cell. Acknowledqment: Binational Science This research Foundation. was supported in that interferes is unusual. the inhibitor part by with Further and the U.S.-Israel REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. Babior, B.M. (1984) Blood 64, 959-966. Biophys . Acta 853, 65-89. Rossi, F. (1986) Biochim. Curnutte, J.T. and Babior, B.M. (1987) Adv. Hum. Genet.16, 229-297. Segal, A.W. (1989) J. Clin. Invest. 83, 1785-1793. Cohen, H.J. , Chovaniec, M-E., Wilson, M.K. and Newburger, P.E. (1982) Blood 60, 1188-1194. Akard, L-P., English, D., and Gabig, T.G. (1988) Blood 72, 322-327. Curnutte, J.T. (1985) J. Clin. Invest. 75, 1740-1743. Y. and Pick, E. (1985) J. Biol. Chem. 260, 13538- 13545. Bromberg, Fujita, I., Takeshige, K. and Minakami, S. (1987), Biochim.Biophys. Acta 932, 41-48. Curnutte, J-T., Kuver, R., and Scott, P.J. (1987) J. Biol. Chem. 262, 5563-5569. Aviram. I. and sharabani M. (1989) Biochem. J. 261, 477-482. Lowry,O.H., Rosebrough, N.J., Farr, A.L. and Randal1,R.J. (1951) J. Biol. Chem. 193, 265-275. Borregaard, N. (1988) J. Bioenerg. Biomembr. 20, 637-651. Henson, P.M., Henson, J.E., Fittschen, C., Kimani, G., Bratton, and Riches, D.W.H. (1988) in 1nflammation:basic D-L., principles and clinical correlates (J.I. callin, I.M. Goldstein and R. Snyderman, eds.) Raven Press pp.363-390. Clark, A.L.,Leidal, K.G., Pearson, D.W., and Nauseef, W.M. J.Biol. Chem. 262, 4064-4074. 202