Oxidant-mediated phosphatidylserine exposure and macrophage uptake of activated neutrophils: possible impairment in chronic granulomatous disease

Transcription

1 Oxidant-mediated phosphatidylserine exposure and macrophage uptake of activated neutrophils: possible impairment in chronic granulomatous disease Mark B. Hampton,* Margret C. M. Vissers,* Jacqueline I. Keenan, and Christine C. Winterbourn* Departments of *Pathology and Surgery, Christchurch School of Medicine and Health Sciences, Christchurch, New Zealand Abstract: The removal of neutrophils from inflammatory sites is essential for the resolution of inflammation. Surface changes, including phosphatidylserine exposure, label neutrophils for phagocytosis by macrophages. Here, we demonstrate that externalization of phosphatidylserine and uptake by monocyte-derived macrophages occurred in human neutrophils ingesting Staphylococcus aureus. Both processes were dependent on oxidant production from the neutrophil NADPH oxidase. There was no requirement for myeloperoxidase, and H 2 O 2 was identified as the most likely trigger for PS exposure. We hypothesize that clearance of stimulated neutrophils would be delayed in chronic granulomatous disease (CGD) neutrophils, which lack a functional NADPH oxidase. To explore this possibility, heat-killed S. aureus were injected into the peritoneum of CGD and normal mice. Elevated neutrophil numbers were observed in the inflammatory exudate of the CGD animals, consistent with impaired recognition and clearance. J. Leukoc. Biol. 71: ; Key Words: phagocyte inflammation apoptosis hydrogen peroxide INTRODUCTION Neutrophils accumulate at sites of infection and play a prominent role in the destruction of invading microorganisms. Once their microbicidal capacity has been used, it is essential that neutrophils are removed from an inflammatory site, thereby reducing the risk of lysis and release of their cytotoxic, proteolytic, and proinflammatory mediators. Clearance occurs by apoptosis, where surface changes label the cells for phagocytosis by macrophages [1 3]. Several different surface changes and receptors are implicated in the interaction between apoptotic neutrophils and macrophages [4]. One of the best studied is phosphatidylserine (PS), which is transferred from the inner to the outer leaflet of the lipid bilayer of the neutrophil plasma membrane and is recognized by specific macrophage receptors as a signal for ingestion [5]. A recent study suggests that PS exposure is obligatory for the phagocytosis of apoptotic cells [6]. In an aging neutrophil population, PS exposure is accompanied by other conventional apoptotic markers, such as caspase activation, pyknotic nuclei, and DNA fragmentation [7]. This spontaneous apoptosis proceeds over h and appears crucial for the turnover of circulating neutrophils [3]. In contrast, neutrophils treated with the artificial stimulant phorbol myristate acetate (PMA) expose PS within 3 h, and this is not accompanied by caspase activation or the nuclear changes associated with apoptosis [7, 8]. As such, the stimulated model does not fit the conventional definition of apoptosis. However, it does embody the most important component of the apoptosis process, the labeling of the cell for clearance. PMA and other more physiological stimulants such as opsonized bacteria activate neutrophils to undergo a burst of oxygen consumption and superoxide production. This is a result of the rapid assembly of a reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex on the plasma membrane [9]. Although superoxide and its dismutation product H 2 O 2 have limited bactericidal properties, they are used by the neutrophil enzyme myeloperoxidase to produce HOCl, a potent oxidant and microbicidal agent [10]. Inhibition of the NADPH oxidase or myeloperoxidase in isolated neutrophils results in impaired killing of several species of bacteria [11 15]. Although there are few clinical symptoms associated with myeloperoxidase deficiency, patients with chronic granulomatous disease (CGD), whose neutrophils have a defective NADPH oxidase, suffer from recurrent life-threatening episodes of infection [16]. Although CGD is conventionally attributed to a defect in bacterial killing, the pathology of the disease appears more complex. The dominant feature is the formation and persistence of granuloma, seen in human patients and in mouse models of the disease [17 19]. The cause of granuloma formation is not clear and may not be related directly to impaired bacterial killing, because granulomas still form after wound sterilization with antibiotics [20]. They are also detected in CGD mice challenged with nonviable bacteria [17], suggest- Correspondence: Mark Hampton, Ph.D., Christchurch School of Medicine, P.O. Box 4345, Christchurch, New Zealand. mark.hampton@ chmeds.ac.nz Received January 5, 2002; revised January 5, 2002; accepted January 11, Journal of Leukocyte Biology Volume 71, May

2 ing that there is dysfunctional regulation of the inflammatory response in CGD. We have found that NADPH oxidase activity is necessary for PS exposure in PMA-stimulated neutrophils [7]. This raises the possibility that in CGD defective PS exposure could result in impaired clearance of neutrophils, which are stimulated at sites of infection, and contribute to the granuloma formation characteristic of this disease. To explore this mechanism further, we have investigated whether PS exposure requires myeloperoxidase activity, whether exposure also occurs when neutrophils phagocytose bacteria, and whether inhibition of the NADPH oxidase impairs the uptake of stimulated neutrophils by macrophages. We show a clear requirement for oxidant production by the stimulated neutrophil in all of these processes. We also show greater accumulation of neutrophils in peritoneal exudates of CGD mice injected with heat-killed Staphylococcus aureus, which is indicative of a clearance defect. MATERIALS AND METHODS Materials Cell culture media and supplies were from Gibco-BRL (Grand Island, NY), supplied by Life Technologies (Auckland, New Zealand). S. aureus strain 502a (ATCC 27217) was obtained from the New Zealand Communicable Disease Centre (Porirua). The annexin V-fluorescein isothiocyanate (FITC) apotest kit was from Nexins Research B.V. (Hoeven, The Netherlands). HOCl was from Reckitt and Coleman (Auckland, New Zealand). All other biochemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Neutrophil isolation Neutrophils were isolated from the peripheral blood of healthy human donors and from a donor, previously determined in this laboratory to be myeloperoxidase-deficient [14], by Ficoll/Hypaque centrifugation, dextran sedimentation, and hypotonic lysis [21]. Neutrophils were also obtained from a 17-year-old female patient with a history of S. aureus and Aspergillus infections. Diagnosis was confirmed as CGD when there was no detectable reduction of cytochrome c by neutrophils stimulated with PMA. Neutrophil stimulation Isolated neutrophils (10 6 /ml) were incubated at 37 C in tissue culture plates with RPMI media, with 10% fetal bovine serum (FBS) for stimulation with PMA (100 ng/ml), or with 10% autologous serum for experiments that involved phagocytosis of bacteria. Neutrophils treated with PMA adhered to the tissue culture dishes quickly. After 3 h, they were detached with trypsin, pooled with any nonadherent cells, and then pelleted and washed. Although unstimulated cells did not adhere, they were treated in an identical manner. When required, inhibitors were added 10 min before the PMA. S. aureus were cultured overnight in nutrient broth, harvested by centrifugation, and washed in phosphate-buffered saline (PBS). Bacteria (10 9 /ml) were opsonized with 10% autologous serum before addition to the neutrophils at a final concentration of /ml to achieve a ratio of 20 bacteria per neutrophil. Culture dishes were agitated gently and then incubated at 37 C for 3 h. Trypsin was not required in harvesting neutrophils treated with bacteria. Incubation with oxidants Two procedures were used to examine the ability of H 2 O 2 to initiate PS exposure. Reagent H 2 O 2, standardized on the basis of A 240 (ε 43.6), was added as a bolus to 10 6 neutrophils in 1 ml media with 10% FBS at 37 C, or H 2 O 2 was generated continuously by the addition of glucose oxidase. Initial rates of H 2 O 2 generation, determined using the ferrous oxidation of xylenol orange assay [22], ranged from 2 to 8 nmol/ml/min. Cells were also treated with HOCl [standardized by A 292 (ε 350) of a dilution at ph 12] but while suspended in Hanks balanced saline solution (HBSS; 10 mm PBS, ph 7.4, containing 1 mm CaCl 2, 0.5 mm MgCl 2, and 1 mg/ml glucose) to avoid scavenging the HOCl by the media. After 5 min, the cells were resuspended in media and incubated for a further 3 h. PS exposure The exposure of PS was measured with an annexin V-FITC kit, according to the manufacturer s instructions. Flow cytometry was performed with a FACS (fluorescein-activated cell sorter) Vantage from Becton Dickinson (San Jose, CA). Cells were costained with propidium iodide (PI) to enable identification of necrotic cells. These cells were gated out of the final analysis. Mean fluorescence of the viable cells was recorded, using the geometric mean to account for the skewed population distributions. To account for the variability in labeling between neutrophil preparations, the mean fluorescence of the resting cells was standardized at 1, and values for the treated cells were expressed relative to this. Monocyte-derived macrophages Human monocyte-derived macrophages were prepared by a modification of standard methods [1, 23]. The mononuclear cell layer was collected after the centrifugation of peripheral blood from healthy donors layered on Ficoll/ Hypaque. Cells were resuspended at /ml in HBSS with 0.1% autologous serum and incubated for 1 h on a 24-well tissue-culture plate. Nonadherent cells were removed by vigorous washing with HBSS, and the remaining cells were cultured in Iscove s modified Dulbecco s medium with 10% autologous serum. The medium was replaced after 4 days, and the cells were used at day 7. Final macrophage yields ranged from 1 to cells per well. Uptake of stimulated neutrophils by macrophages Neutrophils were stimulated with S. aureus as described, harvested after 3 h, and resuspended at per 200 l HBSS. The medium was removed from the macrophages and replaced with the neutrophils in HBSS. Mixtures were incubated for 30 min at 37 C, then the HBSS was aspirated, and the remaining cells were fixed for 10 min with 4% paraformaldehyde. Neutrophils were visualized with a myeloperoxidase stain by incubating the cells with 1 mm o-dianisidine and 0.3 mm H 2 O 2 in 50 mm sodium phosphate buffer, ph 6. Phagocytosis of neutrophils was quantified by counting the number of neutrophils ingested per 100 macrophages. Mouse model of inflammation CGD mice (C57BL/6-Cybb tmlx ), where the X-linked gp91 phox gene had been knocked out by targeted mutation [24], were purchased from the Jackson Laboratory (Bar Harbor, ME). Homozygous female and hemizygous male mice were bred to obtain the necessary numbers for this study. C57BL/6 mice were used as controls. Heat-killed S. aureus ( ) were injected intraperitoneally (i.p.), and at specific times, the mice were killed, and exudate was collected by successive lavages of the peritoneal cavity with PBS containing 0.1% bovine serum albumin. Cell counts were performed with a haemocytometer, and differential counts were undertaken on Giemsa-stained cytospins. Statistics Results were analyzed with the SigmaStat software package from Jandel Scientific (SPSS Science, Chicago, IL). Paired t-tests or Wilcoxon-signed rank tests on data that were not distributed normally were used to determine significant differences (P 0.05) between stimulated neutrophils in the presence or absence of selected inhibitors. RESULTS Oxidant involvement in PS exposure Neutrophils were isolated from the blood of healthy donors and stimulated with PMA. After 3 h, a large population of cells showed increased binding of annexin V-FITC without staining 776 Journal of Leukocyte Biology Volume 71, May

3 Fig. 1. PS exposure in PMA-stimulated neutrophils. Neutrophils were incubated alone (A) or with 100 ng/ml PMA (B) for 3 h before being harvested and stained with annexin-fitc and PI. A total of 10,000 cells were analyzed by flow cytometry. Any PI-positive necrotic cells were gated out, and annexin-fitc fluorescence is plotted from a representative experiment. The geometric mean fluorescence was 12.7 (A) and 88 (B). To account for variation between neutrophil preparations, the mean fluorescence of the resting cells was standardized at 1, and values for the treated cells were expressed relative to this [e.g., 6.9 (B)]. Where indicated, neutrophils were treated with 10 M DPI, 1 mm azide, or 100 g/ml catalase for 10 min before being stimulated with PMA. Results are the mean and SE of 3 8 experiments. MPO, Myeloperoxidase. *, P 0.05 significant difference from PMA-stimulated cells. for PI, indicating that they were viable cells with PS exposed on their outer surface (Fig. 1, A and B). As shown previously [7], inhibition of the production of superoxide and its secondary oxidants by the NADPH oxidase-inhibitor diphenyleneiodonium (DPI) blocked the increase in PS exposure completely (Fig. 1C). Although oxidant production was necessary for PS exposure, myeloperoxidase activity and HOCl were not. The myeloperoxidase inhibitor sodium azide did not diminish the response, and PS exposure in myeloperoxidase-deficient neutrophils was normal (Fig. 1C). The slight enhancement by azide could be a result of inhibition of intracellular catalase and consequent H 2 O 2 accumulation. Inhibition by added catalase suggests that H 2 O 2 was involved in the process leading to PS exposure. As observed with other PMA-stimulated events that require H 2 O 2, incomplete inhibition by catalase can probably be attributed to incomplete access to sites of H 2 O 2 production [25]. A role for H 2 O 2 is supported by the results of adding exogenous H 2 O 2 to resting neutrophils. PS exposure was evident with H 2 O 2, added as a bolus of 1 mm or higher (Fig. 2A) or generated continuously by glucose oxidase (Fig. 2B). A small increase in annexin V-FITC binding was detected with 3 nmoles H 2 O 2 /ml/min, and this became more evident as the rate of generation increased to 5 and 7 nmoles H 2 O 2 /ml/min (Fig. 2B). PMA-stimulated neutrophils generate 3 5 nmoles H 2 O 2 / min/10 6 cells. PS exposure was also induced by the treatment of resting neutrophils with reagent HOCl (Fig. 2C). HOCl had no detectable effect at 15 M, but increased PS exposure was observed at 25 and 50 M. Up to 50% necrosis was occurring at this highest dose, indicating that the ability of HOCl to induce PS exposure is limited to a narrow concentration range. PS exposure in phagocytic neutrophils To determine whether oxidant-mediated PS exposure occurs after phagocytosis, neutrophils were incubated with opsonized S. aureus. In conventional phagocytosis assays, samples are mixed continually so that contact between neutrophil and bacteria is not a limiting factor, and phagocytosis and killing take place within a few minutes [26]. However, this caused considerable damage and PI uptake by the neutrophils, making it impossible to determine if PS exposure was occurring. To remedy this, neutrophils and bacteria were incubated in wells for 3 h without mixing. Neutrophil activation was apparent from the altered morphology (not shown), and an increase in annexin V-FITC binding without PI uptake was detected (Fig. 3). Although there was less PS exposure than with PMA, it showed the same characteristics dependence on NADPH oxidase but not myeloperoxidase activity (Fig. 3). Macrophage uptake of stimulated neutrophils Redistribution of PS is only one of several surface changes involved in the uptake and clearance of apoptotic neutrophils by macrophages [4]. To determine whether the changes associated with NADPH oxidase-dependent PS exposure are important for uptake of phagocytic neutrophils, neutrophils were incubated with S. aureus and then added to a population of adherent monocyte-derived macrophages. There was a visible loss in neutrophils from the media and signs of extensive macrophage activation (Fig. 4A). This was in distinct contrast to neutrophils stimulated in the presence of DPI (Fig. 4B) or resting neutrophils (not shown), which showed little interaction with macrophages. The neutrophils were washed before being Hampton et al. Clearance of stimulated neutrophils 777

4 than the background rate observed in the resting population (Fig. 5). The importance of a functional NADPH oxidase was confirmed by using neutrophils from a patient with CGD. After stimulation of the CGD neutrophils with S. aureus for 3 h, the uptake of these neutrophils resembled those of resting or DPI-treated neutrophils (Fig. 5). Mouse model of CGD Based on the in vitro studies described above, we predict that the phagocytosis and clearance of stimulated CGD neutrophils from an inflammatory site would be slower than that of normal neutrophils. To explore this hypothesis, experiments were carried out with CGD mice whose gp91phox subunit of the NADPH oxidase has been knocked out by a targeted mutation. These mice share the susceptibility to infection and pathological changes observed in the human condition [17, 24]. CGD and wild-type mice were injected i.p. with heat-killed S. aureus. Nonviable bacteria were used to control the killing defect in CGD neutrophils. Gram-stains of exudates from wild-type and CGD animals at 3 h showed neutrophils with intracellular bacteria (not shown). At 3, 6, and 24 h, the peritoneal exudate was collected, and cell counts were performed. Stained cytospins were used to quantify the cell types present. Considerably more neutrophils were present in the CGD exudates at all time points examined (Fig. 6). DISCUSSION Fig. 2. PS exposure with H 2 O 2. Neutrophils were treated with increasing concentrations of H 2 O 2 (A), glucose oxidase (B), or HOCl (C) and were incubated for 3 h before being harvested and stained with annexin V-FITC. The initial H 2 O 2 generation rates of the glucose oxidase are shown. HOCl treatment of cells occurred in HBSS for 5 min before cells were resuspended in fresh media. At 25 and 50 M HOCl, 25 and 50% necrosis was detected, respectively, resulting in decreasing cell numbers appearing in these traces. There were no significant increases in the number of necrotic cells with any of the concentrations of H 2 O 2 or glucose oxidase. Results are from 1 of 3 (A), 2 (B), or 3 (C) independent experiments that gave similar results. added to the macrophages, so the DPI was not affecting the macrophage itself. To visualize and quantify neutrophil uptake, the media and majority of uningested neutrophils were aspirated, and the adherent macrophages and remaining neutrophils were fixed and stained for myeloperoxidase with o-dianisidine. The neutrophils appear as a dark, orange/brown color, and the macrophages are yellow (Fig. 4, C E). Many of the stimulated neutrophils associated with macrophages showed diffuse staining patterns (Fig. 4, C and D). This was caused by digestion and leakage of myeloperoxidase into the macrophage cytoplasm and indicates that complete ingestion was occurring. Far fewer of the DPI-treated neutrophils were associated with macrophages, and the majority was morphologically intact (Fig. 4E). By counting the number of macrophage-associated neutrophils, we were able to determine that stimulated neutrophils were ingested in sixfold higher numbers than resting neutrophils (Fig. 5). Impaired uptake of neutrophils stimulated in the presence of DPI was evident, and their uptake was no different Neutrophils use the NADPH oxidase enzyme complex and myeloperoxidase to generate large amounts of reactive oxidants for the purpose of killing bacteria [10]. Bacteria are internalized by the neutrophils, thereby protecting host tissue from collateral damage, but the neutrophils themselves are subject to an extreme oxidative stress. To prevent the uncontrolled leakage of their contents, they are cleared by inflammatory macrophages. In this paper, we have shown that oxidants from the neutrophil NADPH oxidase also play a crucial role in triggering the clearance of neutrophils after stimulation. Oxidants have been implicated previously in the apoptosis associated with aging neutrophils [27 30]. Agents that stimulate low-level oxidant production appear to accelerate this process [31 33]. In contrast, when fully activated neutrophils generate an intensive burst of oxidizing species over several minutes, these oxidants appear to prohibit the use of the redox-sensitive caspases and promote a specialized form of apoptosis [7]. With PMA, conventional nuclear and morphological changes cannot be detected, in part because of the extensive intracellular vacuolation and cytoskeletal changes associated with stimulation [7, 8]. However, a prominent feature is the externalization of PS, which is common to the vast majority of apoptosis models and is considered important in the recognition and uptake of apoptotic cells. We showed that PS exposure occurs within3hofincubating neutrophils with S. aureus and that exposure with this physiological stimulus is dependent on a functional NADPH oxidase. Others have shown apoptosis in neutrophils incubated with phagocytic stimuli [34, 35], and neutrophils from CGD 778 Journal of Leukocyte Biology Volume 71, May

5 Fig. 3. PS exposure with S. aureus. Neutrophils were treated with S. aureus at a ratio of 1:20 in the presence of 10% autologous serum for 3 h before being harvested and stained with annexin V-FITC and PI. Results are expressed as in Figure 1 and are the mean and SE of five independent experiments. *, P 0.05 significant difference from S. aureus-stimulated cells. patients do not undergo apoptosis after phagocytosis of opsonized beads [35]. Measurement of apoptosis in these studies was based on morphological and nuclear changes, and PS exposure in phagocytic neutrophils has not been demonstrated. The appearance of PS on the outer surface of the neutrophil is not related to physical changes associated with phagosome formation and degranulation, because exposure was blocked completely by DPI, which only affects NADPH oxidase function [12]. Our results with PMA-stimulated neutrophils indicate that myeloperoxidase activity is not necessary for PS exposure, and H 2 O 2 generation appears sufficient. Although exogenous HOCl was able to induce PS exposure and may play a role in stimulated neutrophils where a large proportion of the H 2 O 2 is converted to HOCl, the process occurred normally in its absence. This situation is in distinct contrast to the killing of the ingested bacteria, where H 2 O 2 appears to act as a precursor of the major microbicidal oxidant HOCl and makes little direct contribution to killing [10]. Other studies have identified the ability of H 2 O 2 to induce apoptosis in neutrophils [36, 37] and documented that catalase can reduce the rate of spontaneous apoptosis [27, 28, 30, 36]. In one study, metal chelators were Fig. 4. Incubation of neutrophils with monocyte-derived macrophages. Neutrophils were incubated with S. aureus as described in Figure 3, in the absence (A, C, D) or presence (B, E) of DPI before being harvested and layered onto monocyte-derived macrophages. An inverted-phase contrast light microscope was used to capture images after 30 min (A, B). The media was removed at this time, and the cells were fixed, stained for myeloperoxidase, and rephotographed (C E). M, Macrophage; EN, extracellular neutrophil; PN, phagocytosed neutrophil. Fig. 5. Ingestion of neutrophils by monocyte-derived macrophages. The number of neutrophils per 100 macrophages was calculated from the experiments described in Figure 4, and the means and SE from six independent experiments are plotted. *, P 0.05 significant difference from S. aureus-stimulated cells. A single experiment with CGD neutrophils was performed as above. Hampton et al. Clearance of stimulated neutrophils 779

6 Fig. 6. Neutrophil numbers in the peritoneal exudate of S. aureus-inoculated mice. Mice were inoculated i.p. with heat-killed S. aureus, and the inflammatory exudates were collected 3, 6, and 24 h later. The percentage of cells that were neutrophils (ranging from 10 60%) was determined from stained cytospins of wild-type and CGD exudates and was converted to absolute numbers in the exudate by multiplying by the total number of white cells harvested from each peritoneum. Results are the means and SE of three different mice at each time point. *, P 0.05 significant difference between control and CGD exudates. able to block induction of neutrophil apoptosis by H 2 O 2, suggesting hydroxyl radical involvement [36]. The oxidant-sensitive targets that initiate PS exposure are yet to be elucidated, but one possible mechanism is direct oxidation of PS leading to its externalization [38]. The ability of DPI to prevent PS exposure led us to hypothesize that oxidant production would be necessary to trigger the uptake of phagocytic neutrophils by macrophages. It was important to test this directly, because PS exposure is not necessarily the only surface change on apoptotic cells that is critical for uptake by macrophages [4], and although PS exposure was detected in phagocytic neutrophils, other factors critical for uptake by macrophages may have been absent. Also, our flow cytometry results indicated that stimulation may give rise to populations of high and low PS-exposing neutrophils that may alter their ability to be taken up by macrophages. Further studies are required to determine the relationship between PS exposure and macrophage ingestion. However, we are able to conclude that a functional NADPH oxidase is necessary to trigger both processes. Based on these findings, we would expect impaired uptake of stimulated CGD neutrophils by macrophages. This could result in delayed clearance from inflammatory sites and contribute to the pathology of the condition. Our preliminary findings are consistent with this mechanism. We observed increased neutrophil numbers in CGD mice injected with heat-killed S. aureus. Because altered killing cannot account for the differences, these findings confirm a defect in regulation of the inflammatory response of CGD animals. Although these results are consistent with a clearance defect, it is equally likely that increased neutrophil recruitment could be the cause of elevated neutrophil numbers. Further mouse studies are required to elucidate the relative contributions of either mechanism. Granuloma formation at sites of infection is a key feature of CGD. Direct evidence is limited, but it is usually attributed to the release of multiplying organisms from CGD neutrophils, leading to a continuing cycle of neutrophil recruitment [18]. However, it is not simply a consequence of impaired microbial killing. This was first highlighted when inflammatory exudates of skin abrasions showed a slower decline in neutrophil numbers in CGD patients as compared with normal patients [39]. More recently, studies with the knockout mouse models showed increased neutrophil numbers during thioglycollateinduced peritonitis in the CGD mouse [19, 24]. In both of these studies, there was no gross evidence of abnormal neutrophil apoptosis, and enhanced recruitment was proposed as the cause of increased neutrophil numbers. However, it is difficult to accurately quantitate apoptotic neutrophils in exudates because of their rapid clearance, and our studies suggest that the clearance defect is maximal with activated neutrophils. The skin window and thioglycollate models may not stimulate a neutrophil population to the same extent as bacterial infection. It is also of interest that anti-inflammatory glucocorticoids, which have been shown to clear obstructive granulomas in CGD [40], can act on macrophages to promote the uptake of apoptotic neutrophils [41]. Our results may also shed light on the apparent conundrum of myeloperoxidase deficiency, which is not associated with susceptibility to infection, although the rates of bacterial killing by CGD and myeloperoxidase-deficient neutrophils are impaired similarly in vitro [14]. We speculate that in both disorders, neutrophils at an inflammatory site ingest pathogens but kill them more slowly than normal by predominantly nonoxidative mechanisms. However, in the case of myeloperoxidase deficiency, PS exposure as a result of H 2 O 2 production should occur normally, enabling these cells to be ingested and cleared by fully functional macrophages. A recent study described delayed PS exposure in PMA-stimulated neutrophils from myeloperoxidase-deficient mice, although 3 h after stimulation, there appeared to be no observable difference [42]. In conclusion, H 2 O 2, generated by stimulated neutrophils, plays two critical roles. It is used by myeloperoxidase to generate a lethal microbicidal agent, and it also activates a pathway that ensures clean disposal of neutrophils. Both mechanisms would be defective in CGD and could combine to cause the accumulation of neutrophils at an inflammatory site and promote granuloma formation. ACKNOWLEDGMENTS This research was supported by a postdoctoral fellowship (M. B. H.) from the New Zealand Foundation for Research, Science and Technology, and Health Research Council of New Zealand. Technical support for this project was provided by Lisa Haring, Sarah Cuddihy, and Tessa Mocatta. We also acknowledge Dr. Tony Kettle and Assoc. Prof. Stephen Chambers for their valuable input and thank A. M. and P. L. for the donation of blood. REFERENCES 1. Newman, S. L., Henson, J. E., Henson, P. M. (1982) Phagocytosis of senescent neutrophils by human monocyte-derived macrophages and rabbit inflammatory macrophages. J. Exp. Med. 156, Savill, J. S., Wyllie, A. H., Henson, J. E., Walport, M. J., Henson, P. M., Haslett, C. (1989) Macrophage phagocytosis of aging neutrophils in inflammation. Programmed cell death in the neutrophil leads to its recognition by macrophages. J. Clin. Investig. 83, Savill, J. S., Haslett, C. (1994) Fate of neutrophils. In Immunopharmacology of Neutrophils (P. J. Hellewell, T. J. Williams, eds.), London, Academic, Savill, J. S., Fadok, V. A. (2000) Corpse clearance defines the meaning of cell death. Nature 407, Journal of Leukocyte Biology Volume 71, May

7 5. Fadok, V. A., Bratton, D. L., Rose, D. M., Pearson, A., Ezekewitz, R. A. B., Henson, P. M. (2000) A receptor for phosphatidylserine-specific clearance of apoptotic cells. Nature 405, Fadok, V. A., de Cathelineau, A., Daleke, D. L., Henson, P. M., Bratton, D. L. (2001) Loss of phospholipid asymmetry and surface exposure of phosphatidylserine is required for phagocytosis of apoptotic cells by macrophages and fibroblasts. J. Biol. Chem. 276, Fadeel, B., Åhlin, A., Henter, J., Orrenius, S., Hampton, M. B. (1998) Involvement of caspases in neutrophil apoptosis: regulation by reactive oxygen species. Blood 92, Takei, H., Araki, A., Watanabe, H., Ichinose, A., Sendo, F. (1996) Rapid killing of human neutrophils by the potent activator phorbol 12-myristate 13-acetate (PMA) accompanied by changes different from typical apoptosis and necrosis. J. Leukoc. Biol. 59, Babior, B. M. (1999) NADPH oxidase: an update. Blood 93, Hampton, M. B., Kettle, A. J., Winterbourn, C. C. (1998) Inside the neutrophil phagosome: oxidants, myeloperoxidase, and bacterial killing. Blood 92, Ellis, J. A., Mayer, S. J., Jones, O. T. G. (1988) The effect of the NADPH oxidase inhibitor diphenyleneiodonium on aerobic and anaerobic microbicidal activities of human neutrophils. Biochem. J. 251, Hampton, M. B., Winterbourn, C. C. (1995) Modification of neutrophil oxidant production with diphenyleneiodonium and its effect on bacterial killing. Free Radic. Biol. Med. 18, Mandell, G. L. (1974) Bactericidal activity of aerobic and anaerobic polymorphonuclear neutrophils. Infect. Immun. 9, Hampton, M. B., Kettle, A. J., Winterbourn, C. C. (1996) Involvement of superoxide and myeloperoxidase in oxygen-dependent killing of Staphylococcus aureus by neutrophils. Infect. Immun. 64, Klebanoff, S. J., Hamon, C. B. (1972) Role of myeloperoxidase-mediated antimicrobial systems in intact leukocytes. J. Reticuloendothel. Soc. 12, Segal, B. H., Leto, T. L., Gallin, J. I., Malech, H. L., Holland, S. M. (2000) Genetic, biochemical, and clinical features of chronic granulomatous disease. Medicine 79, Morgenstern, D. E., Gifford, M. A. C., Li, L., Doerschuk, C. M., Dinauer, M. C. (1997) Absence of respiratory burst in X-linked chronic granulomatous disease mice leads to abnormalities in both host defence and inflammatory response to Aspergillus fumigatus. J. Exp. Med. 185, Forehand, J. R., Nauseef, W. M., Johnston Jr., R. B. (1989) Inherited disorders of phagocyte killing. In The Metabolic Basis of Inherited Disease (C. R. Scriver, A. L. Beauder, W. S. Sly, D. Valle, eds.), New York, McGraw-Hill, Jackson, S. H., Gallin, J. I., Holland, S. M. (1995) The p47 phox knock-out model of chronic granulomatous disease. J. Exp. Med. 182, Gallin, J. I. (1983) Recents advances in chronic granulomatous disease. Ann. Intern. Med. 99, Boyum, A. (1968) Isolation of mononuclear cells and granulocytes from human blood. Isolation of mononuclear cells by one centrifugation and of granulocytes by combined centrifugation and sedimentation at 1 g. Scand. J. Clin. Investig. 21 (Suppl. 97), Wolff, S. P. (1994) Ferrous ion oxidation in presence of ferric ion indicator xylenol orange for measurement of hydroperoxides. Methods Enzymol. 233, Musson, R. A. (1983) Human serum induces maturation of human monocytes in vitro. Changes in cytolytic activity, intracellular lysosomal enzymes, and nonspecific esterase activity. Am. J. Pathol. 111, Pollock, J. D., Williams, D. A., Gifford, M. A. C., Li, L., Du, X., Fisherman, J., Orkin, S. H., Doerschuk, C. M., Dinauer, M. C. (1995) Mouse model of X-linked chronic granulomatous disease, an inherited defect in phagocyte superoxide production. Nat. Genet. 9, Lundqvist, H., Follin, P., Khalfan, L., Dahlgren, C. (1996) Phorbol myristate acetate-induced NADPH oxidase activity in human neutrophils: only half the story has been told. J. Leukoc. Biol. 59, Hampton, M. B., Vissers, M. C. M., Winterbourn, C. C. (1994) A single assay for measuring the rates of phagocytosis and bacterial killing by neutrophils. J. Leukoc. Biol. 55, Hannah, S., Mecklenburgh, K., Rahman, I., Bellingan, G. W., Greening, A., Haslett, C., Chilvers, E. R. (1995) Hypoxia prolongs neutrophil survival in vitro. FEBS Lett. 372, Oishi, K., Machida, K. (1997) Inhibition of neutrophil apoptosis by antioxidants in culture medium. Scand. J. Immunol. 45, Narayanan, P. K., Ragheb, K., Lawler, G., Robinson, J. P. (1997) Defects in intracellular metabolism of neutrophils undergoing apoptosis. J. Leukoc. Biol. 61, Kasahara, Y., Iwai, K., Yachie, A., Ohta, K., Konno, A., Seki, H., Miyawaki, T., Taniguichi, N. (1997) Involvement of reactive oxygen intermediates in spontaneous and CD95(Fas/APO-1)-mediated apoptosis of neutrophils. Blood 89, Kettritz, R., Falk, R. J., Jennette, J. C., Gaido, M. L. (1997) Neutrophil superoxide release is required for spontaneous and fmlp-mediated but not for TNF -mediated apoptosis. J. Am. Soc. Nephrol. 8, Gamberale, R., Giordano, M., Trevani, A. S., Andonegui, G., Geffner, J. R. (1998) Modulation of human neutrophil apoptosis by immune complexes. J. Immunol. 161, Harper, L., Ren, Y., Savill, J., Adu, D., Savage, C. O. S. (2000) Antineutrophil cytoplasmic antibodies induce reactive oxygen-dependent dysregulation of primed neutrophil apoptosis and clearance by macrophages. Am. J. Pathol. 157, Watson, R. W. G., Redmond, H. P., Wang, J. H., Condron, C., Bouchier- Hayes, D. (1996) Neutrophils undergo apoptosis following ingestion of Escherichia coli. J. Immunol. 156, Coxon, A., Rieu, P., Barkalow, F. J., Askari, S., Sharpe, A. H., von Andrian, U. H., Arnaout, M. A., Mayadas, T. N. (1996) A novel role for the b2 integrin CD11b/CD18 in neutrophil apoptosis: a homeostatic mechanism in inflammation. Immunity 5, Rollet-Labelle, E., Grange, M., Elbim, C., Marquetty, C., Gougerot-Pocidalo, M., Pasquier, C. (1998) Hydroxyl radicals as a potential intracellular mediator of polymorphonuclear neutrophil apoptosis. Free Radic. Biol. Med. 24, Lundqvist-Gustafsson, H., Bengtsson, T. (1999) Activation of the granule pool of the NADPH oxidase accelerates apoptosis in human neutrophils. J. Leukoc. Biol. 65, Kagan, V. E., Fabisiak, J. P., Shvedova, A. A., Tyurina, Y. Y., Tyurin, V. A., Schor, N. F., Kawai, K. (2000) Oxidative signaling pathway for externalization of plasma membrane phosphatidylserine during apoptosis. FEBS Lett. 477, Gallin, J. I., Buescher, E. S. (1983) Abnormal regulation of inflammatory skin responses in male patients with chronic granulomatous disease. Inflammation 7, Chin, T. W., Stiehm, E. R., Falloon, J., Gallin, J. I. (1987) Corticosteroids in treatment of obstructive lesions of chronic granulomatous disease. J. Pediatr. 111, Liu, Y., Cousin, J. M., Hughes, J., Van Damme, J., Seckl, J. R., Haslett, C., Dransfield, I., Savill, J. S., Rossi, A. G. (1999) Glucocorticoids promote nonphlogistic phagocytosis of apoptotic leukocytes. J. Immunol. 162, Tsurubuchi, T., Aratani, Y., Maeda, N., Koyama, H. (2001) Retardation of early-onset PMA-induced apoptosis in mouse neutrophils deficient in myeloperoxidase. J. Leukoc. Biol. 70, Hampton et al. Clearance of stimulated neutrophils 781