Acidification activity of human neutrophils. Tertiary granules as a site of ATP-dependent acidification.

The acidification activity of human neutrophils, known to occur extracellularly and intraphagolysosomally, was studied in intact and in fractionated cells. The subcellular location of the acidification activity was investigated by rate zonal sedimentation of post-nuclear supernatants from resting cells on continuous sucrose gradients. The acidification measurements indicated a dominance of activity in gelatinase-rich tertiary granules. On the other hand, ATPase activities were located in plasma membrane and in the membranes of the cytoplasmic granules (specific, azurophilic, and tertiary). All of these activities were diminished by the inhibitors dicyclohexylcarbodiimide and diisothiocyanostilbene disulfonic acid; however, studies with other inhibitors, especially N-ethylmaleimide and duramycin, suggested ATPase enzymatic differences depending on location. The results taken together provide direct and strong indication of involvement of a proton pump ATPase in acidification inside neutrophils. Furthermore, the dominant location of acidification activity in tertiary granules that very readily degranulate presumably has significant implications for the importance of low pH in cidal events and the inflammatory process.

Neutrophils, the first line of defense in peripheral blood against infectious disease, first ingest and then destroy intruder organisms by releasing cidal factors and degradative enzymes against the entrapped target in the phagocytic vacuole (1,2). In this process, neutrophils generate reactive oxygen metabolites, mainly superoxide and hydrogen peroxide, that facilitate the killing of microbes (3). Although the digestive enzymes such as the cathepsins are known to require a low pH of 4-5, a dependency of bactericidal activity on acidification has not been established (2,4,5 ) . An acid pH per se is bactericidal for certain ingested microorganisms and acid pH does promote conversion of superoxide to hydrogen peroxide (6). Moreover, acidification may be required in neutrophils for the fusion of granules and the transfer of material from one compartment to another. Supporting this is the observation that protein and receptor recycling in other cells are sensitive to inhibitors of acidification (7,8). In view of the role of acidification in these biological processes, it is important to understand the mechanism and subcellular lo-* This research was supported by Grant A118410 from the National Institutes of Health The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ Recipient of a postdoctoral fellowship from the United States-Spanish Joint Committee for Scientific and Technological Cooperation. cation of acidification activity in neutrophils.
Various mechanisms of acidification in neutrophils have been proposed (a) an increase of lactic acid formation during phagocytosis (4,9), (b) the production by an NADPH oxidase of perhydroxyl radicals (. OOH) which subsequently split into superoxide (OF) and protons (lo), ( c ) a proton-translocating system in association with the oxidase system (11,12), (d) a carbonic anhydrase system (13,14), and ( e ) a proton pumping ATPase (15). Here we present evidence that is strongly indicative of a proton pump ATPase involvement in neutrophil function.
There are many precedents for the establishment and maintenance of an acid pH by a proton pump ATPase in a wide variety of subcellular fractions. Such an ATPase has been directly implicated in lysosomes (16, 17), clathrin-coated vesicles (18,19), synaptic vesicles (20), Golgi (21, 22), acrosomes (23), chromaffin granules (24, 25), and platelet granules (26)(27)(28). Although ATPase activity in human granulocytes has been reported previously (29, 30), its involvement in phagocytic vacuole acidification during phagocytosis has not been ascertained. We recently reported (31) the presence of a membrane-bound, MP-dependent ATPase activity in human neutrophils that was inhibited by N,N'-dicyclohexylcarbodiimide and suggested its function as a proton pump.
In the present extension of these studies, we show that tertiary granules, rather than the more familiar specific and azurophilic granules, are surprisingly dominant with respect to organelle acidification. The implications of this subcellular location and a role of acidification in microbicidal events are considered.

MATERIALS AND METHODS
Neutrophil Preparation-Neutrophils were prepared from fresh blood after removal of erythrocytes by sedimentation at unit gravity through dextran as described (31,32). Subsequent centrifugation into Ficoll-Hypaque gradients was performed as indicated following published protocols (33).
Measurements of pH Changes in Neutrophil Suspensions-All the incubations were made in a solution containing 150 mM NaC1,5 mM KCI, 1.2 mM MgClz, 1.3 mM CaC& adjusted to pH 7.0. Neutrophils (9 X 10' cells) were preincubated with 5 pg/ml cytochalasin B and the respective agent for 5 min in a total volume of 2 ml, and the suspension was mixed with a magnetic stirrer. The pH changes were detected with a pH meter attached to a recorder. The pH was brought to 8.0 with sodium hydroxide and the buffering capacity of the suspension determined by addition of 50-100 nmol of sodium hydroxide. Proton extrusion was calculated from the slope of the recorded pH curve during the first minute after addition of the stimulus (5 pg/ ml phorbol myristate acetate or lo" M N-formyl-L-methionyl-Lleucyl-L-phenylalanine). Stock solutions of DCCD' (10 mM) and quercetin (10 mg/ml) were prepared daily in dimethyl sulfoxide. The effects on proton extrusion of these compounds were compared to The abbreviations used are: DCCD, N,N'-dicyclohexylcarbodiimide; DIDS, diisothiocyanostilbene-2,2'-disulfonic acid.

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Acidification Activity of Neutrophils controls containing an equivalent (1%) amount of dimethyl sulfoxide. Subcellular Fractionation-Cell suspensions were diluted with T S (0.25 M sucrose, 10 mM Tris so,, pH 8) and reisolated by centrifugation a t 800 X g for 3 min. The packed cells, about l g, wet weight, were resuspended in TS to a final volume of 2 ml. Disruption was accomplished by addition of 10 ml of cold water and, after 2.5 min on ice, homogenization in a Dounce tissue grinder using a type A (tight) pestle (IO strokes). Iso-osmolarity was rapidly restored by addition of 1.25 ml of 2 M sucrose. The homogenate was centrifuged a t 2000 X g for 4 min to obtain nuclear and postnuclear supernatant or extract fractions. For rate zonal gradient analyses, 9 ml of the extract was layered on a 25-ml, 14.3-34.5% (w/w) continuous sucrose gradient, above a I-ml cushion of 60% sucrose. Gradients were centrifuged a t 20,000 rpm (70,000 X g) for 15 min a t 4 "C in a Beckman ultracentrifuge using a SW 27 rotor. For all gradients, 8 ml of the sample layer (fraction 1) and seven subsequent 4-ml fractions were collected from the top by pumping 60% sucrose into the bottom.
Gelatinase activity was determined by slight modification of the described method (35). Samples were pretreated for 15 min at 37 "C with inhibitors, 3.7 mM diisopropylphosphofluoridate and 0.1 mM Ep-475, in 100 mM Tris-S04, pH 8, containing 0.05% Triton X-100, in order to inhibit serine and thiol proteinases, respectively (36). Then 1.7 mM p-aminophenylmercuric acetate was added to activate the latent form of gelatinase (37). After 1 h a t room temperature, radioactive gelatin was added and the assay performed.
ATPase Assay-ATPase activity was measured at neutral p H with Mg,ATP as substrate (16). Released inorganic phosphate was quantitated spectrophotometrically using the ascorbate/molybdate method.

RESULTS
ATPase Activity of Neutrophils-Extracts from resting human neutrophils hydrolyze ATP at a rate of 28 f 4 nmol/ min/mg of protein (n = 22), when assayed at neutral p H in the presence of magnesium. This activity was inhibited by DCCD as previously reported (31), with half-maximal inhibition at 60 p~ DCCD. The ATPase activity of neutrophils appeared to be stable as it was not disminished after storage a t -20 "C for 1 month or a t 4 "C for 1 week. Subcellular Distribution of ATPase and Acidification Activities- Fig. 1 shows the fractionation studies that permitted identification of the subcellular locations of ATPase activity in neutrophils. Extract fractions obtained from resting cells were separated by rate zonal sedimentation under conditions that resolved cytosol (glucose-6-phosphate dehydrogenase), plasma membrane (5'-nucleotidase), tertiary (gelatinase), specific (lactoferrin and lysozyme), and azurophilic (myeloperoxidase) granules. This 15-min centrifugation showed plasma membrane and granule locations for the ATP hydrolytic activity, as the ATPase activity profiles (total and DCCD-sensitive) were broad. One area paralleled the distribution of specific granules (fraction 6), another that of tertiary granules (fraction 4), and another the distribution of plasma membrane (fraction 2). We demonstrated in a previous report (31) that DCCD-sensitive, Mg2+-dependent ATPase activity was intrinsic to the tertiary granule, enriched in cytochrome b and ubiquinone, with sedimentation properties intermediate between those of the specific granules and plasma membrane. Quantitative analysis of Fig. 1 as well as of the previous results indicate that about 30% of the total ATPase activity is localized in plasma membrane. Interestingly, azurophilic granules, which are marked by myeloperoxidase and which contain the major proportion of acid hydrolases in neutrophils, accounted for only 10% of the total DCCD-sensitive ATPase activity. The specific activities of the total ATPase and DCCD-sensitive ATPase in the various particulate fractions are shown in Table I. The highest specific activities were found in tertiary and specific granules.
Acidification analyses of the gradient fractions showed that ATP-dependent activity occurred in several fractions, being most abundant in those fractions rich in gelatinase activity (fraction 4, Fig. 1). It thus appears that the tertiary granules, which constitute a small fraction of the total granule population in resting neutrophils, are a principal site of acidification. In view of the different distribution of acidification and ATPase activities, it was desirable to assess the sensitivity of acidification activity to inhibitors.
Effect of Inhibitors on the ATPase and Acidification Activities-As shown in Table 11, a number of inhibitors were tested with the ATPase activities present in different fractions isolated from human neutrophils. Ouabain and vanadate, well known inhibitors of (Na+,K')-ATPase (38), had no significant effect on the ATPase activity of any of the particulate fractions. These results are consistent with previous suggestions that a minor fraction of the ATPase activity in neutrophils is due to the presence of (Na+,K')-ATPase (15,30). Conversely, DCCD and DIDS, nonspecific inhibitors of proton-translocating ATPases (39), inhibited greatly all the particulate ATPases. Incubation with duramycin, an inhibitor of coated vesicle ATPase (40), resulted in a partial inhibition of the plasma membrane ATPase, whereas the other organelle ATPases were activated.
N-Ethylmaleimide, an inhibitor of the coated vesicle proton pump (19), partially affected the plasma membrane ATPase activity at 0.1 mM and had less of an effect on the other ATPases. At 1 mM, N-ethylmaleimide inhibited completely all acidification activity and inhibited about 40% of the tertiary granule ATPase activity. It is worth noting a previous report (30) in which the M$'-ATPase activity of human granulocytes was inhibited 50% by 0.2 mM N-ethylmaleimide.
Oligomycin used a t 0.5 pg/ml (0.6 pM), which inhibits 90% of the mitochondrial ATPase (41), had little effect on neutrophil ATPase activity, 3% inhibition (31). This insensitivity to oligomycin is in agreement with the relative scarcity of mitochondria in mature granulocytes (42) and demonstrates that the ATP hydrolytic activity in neutrophils is not due to mitochondrial contamination. High concentrations of oligomycin (9 pg/ml or 11 p~) known to cause nonspecific inhibition of ATPases (43), affected in different degrees the particulate ATPases. A low mitochondrial contamination was found to be located between specific and azurophilic granules in rate zonal gradients (31), and this fraction was relatively sensitive to oligomycin. However, even incubation of the mitochondrial fraction with 9 pg/ml oligomycin resulted in only 40% inhibition of the ATPase activity. This lack of more than 40% inhibition indicates that the mitochondrial fraction contains other ATPases.
Incubation of the different fractions with azide, a mitochondrial inhibitor, showed an inhibitory effect on the granule ATPases, this being higher in the azurophilic granules. In contrast, plasma membrane ATPase was not affected by azide under the conditions of the assay. 5,5'-Dithiobis(2-nitrobenzoic acid), a t 0.3 mM, had negligible effects on the ATPase activity in neutrophils.
Acidification activity was affected by ATPase inhibitors as shown in Table 111. Dicyclohexylcarbodiimide, N-ethylmaleimide, quercetin, and DIDS were effective inhibitors, whereas vanadate was not. In general the results of acidification assays parallel those of ATPase activity, especially with regard to sensitivity to the inhibitors DCCD and DIDS and insensitivity to vanadate.
However, in regard to the Acidification Activity of Neutrophils  release of protons. It therefore is of interest to consider the degree to which tertiary granule components are present in the cell membrane. Accordingly, gelatinase release and the reactivity of intact cells with impermeant reagents were tested as probes of tertiary granules. The data in Table  IV indicate that gelatinase activity, known to mark tertiary granules (37), was readily released from neutrophils. This presumably involves fusion of tertiary granules with the cell membrane, release of soluble gelatinase, and insertion of granule membrane into the cell surface. Activation by addition of phorbol ester lead to 65% release, in considerable excess of lysozyme from specific granules. It is noteworthy that specific granules are known to join the were prepared and assayed as in Table I phagosome during the early phases of phagocytosis (44). The data suggest that tertiary granules join the phagosome at least as fast as the specific granules. If membrane fusion occurs during activation of neutrophils, an increase in surface protein available for reaction with impermeant reagents might be expected. Accordingly, the impermeant reagent diazobenzenesulfonate was used to label resting and phorbol myristate acetate-activated cells. Resting cells were labeled to a significant extent when incubated with diazobenzenesulfonate, indicating the presence of exposed components, presumably membrane proteins (Table V). Pretreatment of neutrophils with phorbol myristate acetate caused an increased reactivity (Table V). In addition, sonication of the cells during reaction with diazobenzenesulfonate resulted in more than an additional 10-fold greater reactivity (data not shown), indicating that a relatively small proportion Reactivity of resting and activated neutrophils with the impernwant reagent, diatobenzenesulfonate Freshly prepared neutrophils pretreated as indicated were reacted with 0.4 mM radioactive diazobenzenesulfonate in saline buffered at pH 9 with 3 mM sodium carbonate (58). After reaction for 1-2 min, chilling, and thorough washing, the cells were analyzed for radioactivity and protein. Of the 40 X 10' cpm of [35S]diazobenzenesulfonate available for reacting, an average of 9000 cpm reacted with the resting cells (resting cell specific radioactivity about 7000 cpm/mg of protein).

Pretreatment
Relative specific reactivity of cellular components reactive to diazobenzenesulfonate are exposed in intact cells. Thus, enzyme release and impermeant labeling, used, as probes for the granules, indicate that membrane fusion occurs during activation. Therefore, it seems reasonable to suggest that the increased proton secretion that occurs in activated neutrophils is due to a significant extent to proton pump activity derived from the tertiary granules as a consequence of fusion between the granule and cell membranes.

DISCUSSION
The ATPase and Proton Pumping Activities of Neutrophils-The present results further demonstrate both a plasma membrane and granule localization of the ATPase activity in human neutrophils. This is in agreement with previous reports (15,31,45). About 30% of the total ATPase is localized in the plasma membrane, and, interestingly, only about 10% is located in the azurophilic granules, even though these contain most of the acid hydrolases that would presumably require an ATP-driven proton pump to maintain an acid pH favorable to hydrolytic action. The remainder of the ATPase activity is recovered on sucrose gradients in the region of tertiary and specific granules. In all cases, inhibition of the Mg+-dependent ATPase by the inhibitors DCCD and DIDS, suggests the presence of a proton pump functioning in phagocytic vacuole acidification. Inhibition of ATPase and acidification activities by millimolar concentrations of N-ethylmaleimide is further evidence for the presence of a uvacuolar" type of proton pump, similar to that of lysosomes and coated vesicles (18,19).
Some differences between the different membrane populations emerge from the results obtained (a) using inhibitors and (b) measuring acidification. The tertiary and specific granules differ from plasma membrane in that their ATPase activity is more sensitive to azide inhibition and less'sensitive to N-ethylmaleimide. Interestingly, duramycin inhibits partially the ATPase located in gradient fractions containing plasma membrane. As duramycin is an inhibitor of coated vesicle ATPase (40), the effect may be due to the presence of this ATPase either in plasma membrane per se or in coated vesicles that co-fractionated with plasma membrane.
The acidification activity of the different membranes of resting neutrophils is clearly greatest in gradient fractions containing the gelatinase-rich tertiary granules (Fig. 1). The acidification activity of all active fractions exhibited a similar sensitivity to inhibitors, however, and thus provide no direct evidence for the existence of different proton pump systems in different membranes. The presence of distinct ATPases in plasma membrane must be taken into account and may contribute to the different behavior of the ATPase activity located there. Thus, the presence of a (Na+,K+)-ATPase (15, 46) and of a Ca2+ pump (47, 48) in the plasma membrane of neutrophils has been reported. As noted above, plasma membrane fractions from the gradients exhibited relatively more ATPase than acidification activity (Fig. 1). Some of this difference can be attributed to the presence of ATPases that do not pump protons. In addition, unsealed plasma membrane vesicles are perhaps present and these would be expected to show maximum ATPase activity without acidification activity, as proton gradients cannot be established in leaky vesicles. In regard to disrupted membranes, it is also necessary to point out that the membrane fragments derived from disrupted granules might also be located in the upper region of the rate zonal gradients with the plasma membrane.
The inhibition of proton release from activated cells by DCCD and quercetin suggest the involvement of a proton pump. Although nonspecific, DCCD does inhibit proton translocating ATPases (49) and has been shown to inhibit the M$+-dependent ATPase activity of human neutrophils (31). It is significant that the release of protons by activated neutrophils is not affected by vanadate inasmuch as vanadate inhibits not only the (Na+,K+)-ATPase but also certain plasma membrane proton pumps, e.g. that of fungal plasma membrane (50). The vanadate result indicates that a covalent phosphorylated intermediate is not involved (51, 52) and provides further evidence that neutrophils contain a distinct ATP-driven proton pump.
The lack of inhibition of proton release from resting neutrophils by DCCD and quercetin agrees with the idea that this release, unlike that of stimulated cells, can be attributed to lactic acid production (53). The DCCD results also suggest that the stimulated proton release from activated cells is due to the fusion of granules with plasma membrane and concomitant insertion of proton pumps in the plasma membrane. In this context, it may be significant that DCCD is able to inhibit enzyme release from granules when neutrophils are activated with phorbol myristate acetate (31).
A Relationship between Acidification and Respiratory Burst Activities-Although the importance of acidification in the killing and digestive aspects of the microbicidal process in neutrophils is not fully understood, it is clear that acidification driven by an ATP-dependent proton pump is not required for activation of the respiratory burst activity. Thus, the presence of 20 PM monensin, 1.5 PM carbonyl cyanide ptrifluoromethoxyphenylhydrazone, , 10 PM carbonyl cyanide m-chlorophenylhydrazone, 0.1 mM 2,4-dinitrophenol, 0.6 PM oligomycin, or 0.9 ptM valinomycin had no effect on the respiratory burst activity induced by treatment of cells with phorbol myristate acetate. Furthermore, exposure of neutrophils to DCCD, 50-500 PM, may even trigger respiratory burst activation (Ref. 54 and Fig. 2).
The relationship of ATP-dependent proton release and phagolysosomal acidification to the transient alkalinization of the phagolysosomal compartment observed at short intervals after stimulation requires comment. First, the alkalinization is presumably driven by the reactions of the active oxygen intermediates, e.g. dismutation of superoxide consumes protons (12). Second, if both processes are triggered simultaneously, they would offset one another and diminish any pH change. Accordingly, one should consider whether regulation is complex with a hierarchy of events that allow sequential triggering of the respiratory burst and alkalinization and subsequent triggering of the proton pump ATPase and acidification. The answers to these possibilities will be facilitated when we can selectively affect the ATPase(s) of the involved compartments.
A Probable Relationship between Membrane Fusion and Activation of the Respiratory Burst-The gelatinase activity of the tertiary granule is readily released from neutrophils. To our knowledge, it is not possible to activate the respiratory burst activity of neutrophils in the absence of gelatinase release. This applies also to enucleated neutrophils inasmuch as the release of gelatinase occurs during the process of enucleation, a process that involves extensive treatment of the cells at 37 'C (55), conditions favorable for membrane fusion. However, the observations that release can occur prior to activation, as in enucleated cells (55) and in cells pretreated with low amounts of various stimuli (56), indicate that membrane fusion events alone are not sufficient for activation of the respiratory burst. Accordingly, the concept of a "priming" step in activation has evolved (56). We interpret our results, especially those on gelatinase release, as additional evidence for the priming of neutrophils. Furthermore, we suggest that such fusion events are obligatory for activation of the respiratory burst. It is noteworthy that cells defective in degranulation are unable to exhibit the burst (57).