Subcellular Localization of the b-Cytochrome Component of the Human Neutrophil Microbicidal Translocation during Activation Oxidase:

We describe a new method for subcellular fractionation of human neutrophils. Neutrophils were disrupted by nitrogen cavitation and the nuclei removed by centrifugation. The postnuclear supernatant was applied on top of a discontinuous Percoll density gradient. Centrifugation for 15 rain at 48,000 g resulted in complete separation of plasma membranes, azurophil granules, and specific granules. As determined by ultrastructure and the distribution of biochemical markers of these organelles, ~90% of the b-cytochrome in unstimulated cells was recovered from the band containing the specific granules and was shown to be in or tightly associated with the membrane. During stimulation of intact neutrophils with phorbol myristate acetate or the ionophore A23187, we observed translocation of 40-75% of the bcytochrome to the plasma membrane. The extent of this translocation closely paralleled release of the specific granule marker, vitamin B12-binding protein. These data indicate that the b-cytochrome is in the membrane of the specific granules of unstimulated neutrophils and that stimulus-induced fusion of these granules with the plasma membrane results in a translocation of the cytochrome. Our observations provide a basis for the assembly of the microbicidal oxidase of the human neutrophil. Neutrophils generate toxic oxygen derivatives such as superoxide anion (03), hydrogen peroxide (H202), and hydroxyl radicals (OH.) when stimulated by a variety of particulate and soluble stimuli (1-4). These oxygen species are generated through the cyanide-insensitive reduction of oxygen (5, 6) by electrons ultimately originating from oxidation of glucose in the hexose monophosphate shunt (6, 7). They are essential, either alone or in combination with myetoperoxidase and a halide, for optimal function of the neutrophil in the killing of bacteria and fungi (8-11), for lysis of antibody-coated target cells ( 12-14), and for inactivation of chemotactic factors (15) and of protease inhibitors ( 16, 17). The biochemical basis for this reduction of oxygen, known as the respiratory burst, is still a matter of debate. One candidate is an "enzyme" functioning as an NADPH-oxidase (18-20). This enzyme activity can be measured in plasma membrane preparations from activated normal neutrophils, but is not detectable in plasma membranes from resting neutrophils or from activated neutrophils from patients with chronic granulomatous disease (CGD) (21-23). Several lines of evidence support the view that the NADPH-oxidase may consist of an electron transport chain, one link of which is a b-type cytochrome (23-26). This cytochrome is present in substantial amounts in normal neutrophils and is reduced when intact neutrophils from normals, but not from CGD patients, are stimulated under anaerobic conditions (27-29). Moreover, the cytochrome is absent from the neutrophils of patients with the most common form of CGD, the classical X-linked type (30). The subcellular localization of this cytochrome is not yet agreed upon. An association with cytoplasmic granules has been indicated by Shinagawa et al. (24) in rabbit neutrophils and by Sloan et al. (31) in human neutrophils. In contrast, Segal et al. (32) have argued for a plasma membrane localization in human neutrophils based on the observation that all b-cytochrome is reduced when dithionite is added to intact TH~ JOURNAL OF CELL BIOLOGY . VOLUME 97 JULY 1983 52-61 5 2 © The Rockefeller University Press . 0021-9525/83/07/0052/10 $1.00 on N ovem er 7, 2017 jcb.rress.org D ow nladed fom cells, whereas the intragranular heme protein, myeloperoxidase, is only reduced by dithionite following disruption of the cells. However, subcellular fractionation indicated the presence of cytochrome b both in the plasma membrane and in more dense fractions (33). Millard et al. (34) reported a plasma membrane localization of the b-cytochrome in rat peritoneal leukocytes, and Gabig et al. (23) found a plasma membrane localization in phorbol myristate acetate (PMA)-stimulated human neutrophils. Our preliminary studies indicate that the traditional methods of subcellular fractionation, which make use of shear force to disrupt cells followed by either density gradient centrifugation or differential centrifugation, are unsatisfactory because artifacts are introduded by centrifugation on hypertonic sucrose gradients and because separation of the two main types of granules is impossible following differential centrifugation. We circumvented these problems as follows: the cells were disrupted by nitrogen cavitation. This technique has been shown by Klempner et al. (35) to induce minimal granule damage and proteolysis. Centrifugation of the postnuclear supernatant on discontinuous Percoll gradients results in a fast and efficient separation of granules and plasma membrane vesicles. MATERIALS AND METHODS Isolation of Neutrophils: 450 ml of blood was withdrawn from healthy donors who gave informed consent. The blood was anticoagulated with 25 mM sodium citrate and mixed with an equal volume of 3% Dextran T-500 (Pharmacia Fine Chemicals, Piscataway, N J) in 0.9% NaCI to ease the sedimentation of erythrocytes. After 45 min at room temperature, the leukocyterich supematant was siphoned off and the cells were pelleted in plastic tubes by centrifuging at 200 g for 10 rain. The cell pellets were resuspended in 0.9% NaCI. Mononuclear cells were separated from polymorphonuclear cells and residual erythrocytes by centrifugation through Ficoll-Hypaque (Ficoll; Pharmacia Fine Chemicals) (Hypaque, Winthrop Laboratories, New York, NY) as described by Boyum (36). The resulting granulocyte-ecythrocyte pellets were resuspended in ice-cold distilled H20 for 30 s to lyse the erythrocytes. Isotonicity was restored with an equal volume of 1.8% NaCI. The granulocytes were pelleted at 200 g for 6 min; the lysis of erythrocytes was repeated with 0.2% NaCI instead of distilled H20. After an additional wash in ice-cold saline, the cells were resuspended either in ice-cold relaxation buffer minus EGTA-100 mM KCI, 3 mM NaCI, 1 mM ATP(Na)2 (Sigma Chemical Co., St. Louis, MO), 3.5 mM MgC12, 10 mM PIPES, pH 7.3, if cavitation was to follow, or in Krebs Ringer-phosphate-119 mM NaCI, 4.7 mM KCI, 1.2 mM MgSO4, 0.75 mM CaCI2, 15 mM NaH2PO4/Na2HPO4, pH 7.4, containing 5.5 mM glucose, if the cells were to be stimulated before cavitation. More than 97% of the cells were polymorphonuclear leukocytes and more than 98% excluded trypan blue. Disruption of Cells: Purified neutrophils, 0.5-1.5 x 109 in 20 ml of ice-cold relaxation buffer minus EGTA, were pressurized with N2 for 20 min at 350 psi with constant stirring in a nitrogen bomb (Parr Instrument Company, Moline, 1L) at 4"C (35). The cavitate was then collected dropwise into EGTA, pH 7.4, sufficient for a final concentration of 1.25 mM. Subcellular Fractionation: Nuclei and unbroken cells were pelleted (P0 by centrifugation of the cavitate at 500 g for 10 rain at 4"C. The supernatant (S~) was decanted and loaded onto gradients precooled to 4"C. Density Centrifugation on Percoll Gradients: The tonicity of Percoll (Pharmacia Fine Chemicals) was adjusted by adding one-tenth the final volume of a 10 times concentrated relaxation buffer (1,000 mM KCI, 30 mM NaCI, 35 mM MgCI2, 10 mM ATP[Na]2, 12.5 mM EGTA, 100 mM PIPES, pH 6.8). For continuous Percoll gradients, the Percoll was adjusted to density 1.08 g/ml, and 30 ml was centrifuged for 10 rain at 20,000 rpm (48,000 g) in an SS34 rotor in a Sorvall RC-5B centrifuge (DuPont Co., Wilmington, DE) at 4"C. Thereafter, 8-10 ml of sample, S~, was applied on top of this preformed gradient and centrifuged for an additional 35 rain at 20,000 rpm. For discontinuous Percoll gradients, 14 ml of Percoll, density 1.120 g/ml, was layered under 14 ml of Percoll, density 1.050 g/ml, through a spinal needle. 8-10 ml of sample, S~, was then applied on top and centrifugation was carried out at 4°C for 15 min at 20,000 rpm in an SS34 rotor. The density of the gradient was estimated from the bands of calibration beads of known density (Pharmacia Fine Chemicals) in gradients run in parallel. Continuous sucrose gradients were made from sucrose solutions of density 1.112 g/ml and 1.250 g/ml by a gradient mixer. The volume of gradients was 30 ml, and 8 ml of the sample, Sj, was carefully layered on top. The gradients were then centrifuged for 210 rain at 25,000 rpm ( 1130,000 g) in an SW28 rotor (Beckman Instruments, Inc., Palo Alto, CA). Fractions of ~ I ml were collected at 4"C by aspiration from the bottom of the gradients through a 50-ul disposable glass pipet attached to a polyethylene tube which was connected to a peristaltic pomp. Since the resolution into three distinct and well-separated bands was excellent on the discontinuous Percoll gradients, these bands, referred to as a, ~, and 3' in order of decreasing density, could alternatively be collected by hand through a pasteur pipette. Percoll was removed from pooled fractions by spinning at 35,000 rpm (180,000 g) for 120 rain in an SW41 rotor (Beckman Instruments, Inc.). To prevent trapping of biological material in the Percoll pellet, it is essential that the density of the pooled fractions in the centrifuge tubes is at least as high as that of the biological material. Therefore, Percoll in relaxation buffer, density 1.122, was added to fill and balance the centrifuge tubes. This is particularly important in the preparation of plasma membranes since these fractions near the top of the gradient contain the least amount of Percoll. After centrifugation, the sedimentable biological material was layered directly on a hard-packed pellet of Percoll from which it was easily separated by aspiration. Disruption of Granules: To separate granule membranes from granule contents, the granules from the B-band were resuspended in 1.5 ml of relaxation buffer and were subjected to freezing and thawing seven times (37). To deplete the membranes of adsorbed proteins, 6 ml of extraction buffer (1 M KC1, 3.5 M urea, 50 mM glycine, 10 mM Na2HPO4/NaH2PO4, pH 6.8) was added and the sample incubated for 45 min at 4"C (38). Membranes were then pelleted I~Y centrifugation, 45,000 rpm (220,000 g) for 90 min at 4"C in an SWS0.1 rotor (Beckman Instruments, Inc.). The supernatant was aspirated and the membrane pellet resuspended in relaxation buffer. Stimulation of Neutrophils: For each experiment, the isolated intact neutrophils from one donor were suspended in 20 ml of Krebs-Ringer phosphate buffer containing 5.5 mM glucose. Half of the cells were incubated with stimulus, either PMA (Sigma Chemical Co.) 5 ug/ml for 20 rain, or A23187 (Eli Lilly and Co., Indianapolis, IN), 1 /~M for 10 min, at 37"C in a water bath with shaking (90 strokes/rain). The incubation was terminated by adding 10 ml of ice-cold Krebs-Ringer phosphate buffer. The cells were then pelleted by centrifugation at 200 g for 6 min at 4°C. The supernatant, So, was removed for enzyme and spectral analyses and the cell pellet resuspended in 20 ml of ice-cold relaxation buffer minus EGTA and cavitated as described above. The other half of the cells, the control, was treated in exactly the same way except that only solvent, dimethyl sulfoxide (7 mM) for PMA, ethanol (18 mM) for A23187, was added. Spectroscopy: Absorption spectra from 400 to 600 nm were measured and recorded in the turbid sample compartment of a Perkin-Elmer 576 ST spectrophotometer (Coleman Instruments Division, Oak Brook, IL). Samples of 3 ml were divided into two plastic cuvettes and one, the sample, was reduced by adding solid dithionite ( 1-2 mg). The spectral scans were repeated following addition of 0.2% Triton X-I@) (Fisher Scientific Co., Fair Lawn, NJ) from a stock of 10% to both sample and reference cuvette. Cytochrome b was quantitated using an absorption coefficient of the 559-nm peak of 21.6 mM -j cm -t (39). Myeloperoxidase was quantitated using an absorption coefficient for the 472-nm peak of 75 mM -~ cm -~ (40). Enzyme Assays: Alkaline phosphatase (EC 3:1.3.1) was assayed with p-nitrophenyl phosphate (Sigma Chemical Co.) 1 mg,/ml as substrate in a 1 mM MgC12, 50 mM sodium barbital buffer, pH 10.5 (41). 50-#1 samples were assayed in duplicate. Samples were incubated for 30 rain at 37°C in a total volume of 1 ml and the reaction was terminated by addition of 1 ml of icecold barbital buffer. Sodium hydroxide, which normally terminates this assay, was found to induce flocculence in Percoll-containing samples. The absorbance at 410 nm was read immediately after the assay was terminated, and the enzyme activity was calculated using an absorption coefficient for p-nitrophenol of 18.6 mM -~ cm -~ (42). For samples containing Percoll, an identical assay with omission of p-nitrophenyl phosphate was always run in parallel to estimate the light scattering at 410 nm induced by the presence of Percoll. These values were subtracted from the enzyme assay values. 1 U of enzyme liberates 1 umol product per minute. /~-glucuronidase (EC 3.2.1.31) was assayed in duplicates as described (43) by liberation of phenolphthalein from 1 mM phenolphthalein/~-monoglucuronic acid (Sigma Chemical Co.) in 100 mM sodium acetate buffer, pH 4.4, at 37°C for 4 h. The assay was terminated by adding 120 mM glycine, pH 10.5. The activity was calculated using an absorption coefficient for phenolphthalcin of 33 mM -1 cm-! at 550 nm (44). I U of enzyme liberates 1 umol of substrate per minute. Samples containing Percoll became very turbid during incubation at the low pH but clarified immediately upon addition of the glycine buffer. However, it was found that the enzyme activity in samples to which Percoll BORREGAARD ET AL. Cytochrome b in Human Neutrophils 53 on N ovem er 7, 2017 jcb.rress.org D ow nladed fom was added was inhibited in proportion to the content of Pereoll. We observed a 40% inhibition when Pereoll was added to samples to give the same contem as that in the a-peak. Cytochrome c oxidase was assayed as described by Cooperstein and Lazarow (45). Cytochrome c was purchased from Sigma Chemical Co. Lysozyme (EC 3.2. I. 17) was assayed kinetieally by following the decrease in turbidity measured at 450 nm of 0.2 mg/ml Micrococcus lysodeikticus (Sigma Chemical Co.) in a 67 mM NaH2PO,/Na2HPO4 buffer, pH 6.2, at room temperature (46). Egg white lysozyme (Sigma Chemical Co.) was used as standard. Vitamin B~2-binding protein was measured in duplicates on 25-, 50-, and 100-~l samples essentially as described by Gonlieb et al. (47). To 1,0 ml of saline was added 500 ~1 STCo-vitamin B,~ (New England Nuclear, Boston, MA), 4 ng/ml, sp act l0 s cpm/ng. The sample was then added and, after mixing, 1 ml of albumin-coated charcoal was added. The samples were centrifuged for 15 min at 1,000 g at room temperature and I ml of the supernatant was aspirated and counted in a Packard auto-gamma scintillation counter (Packard Instrument Co., Inc., Downers Grove, IL) to determine the amount of bound STCo-B~5. Protein was determined as described by Lowry (48) using bovine serum albumin (Sigma Chemical Co.) as a standard. Addition of Percoll to a concentration comparable to that of the samples had no effect on color development in the standards. DNA was measured as described by Giles and Myers (49) with standard obtained from Sigma Chemical Co. Electron Microscopy: Electron microscopy was done on samples fixed for 12 hours in relaxation buffer containing 4% formalin, 1% glutaraldehyde. After rinsing, the samples were fixed in 1% osmium tctroxide for l h and stained in 0.1% uranyl acetate. After dehydrating by incubating in ethanol, 70°/o. 95%, and 100%, the samples were covered with propylene oxide, infiltrated in Epon 812 propylene oxide, l:l, and imbedded in Epon 812. Sections of 400 A were cut and stained with uranyl acetate and viewed on a Zeiss electron microscope.