INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Neutrophilia provides a potential target in treating certain respiratory conditions including chronic obstructive pulmonary disease (COPD). Neutrophils provide a defense against infections that cause exacerbations of COPD, but persistent neutrophilia in the absence of infection in COPD reflects the chronic inflammatory state of the airways in this condition (1, 2).

Leukotriene (LT)B₄ is a potent chemoattractant and activator of neutrophils and eosinophils (3). Neutrophils rapidly release large amounts of LTB₄ in response to activating stimuli, and have a high density of cell-surface LTB₄ receptors (4). These characteristics provide the bases of autocrine stimulation of neutrophils by neutrophil-derived LTB₄. LTB₄ accumulation could be considerable at sites of inflammation in which neutrophils are in close proximity. In this context, sputum LTB₄ levels in patients with COPD can reach concentrations adequate for near saturation of cell-surface neutrophil LTB₄ receptors (5).

Neutrophils are nondividing cells that undergo spontaneous apoptosis. Neutrophil survival is increased significantly under conditions of neutrophil activation (5), providing prolonged neutrophil presence for host-defense activity. Granulocyte-macrophage colony-stimulating factor (GM-CSF) (8) can promote neutrophil survival alone, in common with other inflammatory stimuli such as lipopolysaccharide (LPS) (6). In this context, the response of neutrophils to corticosteroids appears paradoxical, since corticosteroids induce apoptosis in eosinophils (9) but survival in neutrophils (10). A consistent finding after exposure of neutrophils to GM-CSF is an increase in 5-lipoxygenase (5-LO) activity and LTB₄ production (11). This suggests a potential involvement of LTB₄ in the induction of neutrophil survival by GM-CSF and other stimuli. Evidence has been provided for maintenance of neutrophil survival by neutrophil-derived products, including LTB₄ (12).

The present study examined the potential for LTB₄ to be involved in the stimulation of survival by human neutrophils LPS, GM-CSF, and dexamethasone (DEX). We used inhibitors of leukotriene synthesis and leukotriene receptor antagonists to interrupt autocrine survival signalling by leukotrienes after exposure of neutrophils to LPS, GM-CSF and DEX. The findings of the study indicate a fundamental role for LTB₄ in maintenance of neutrophil survival responses to a range of survival stimuli.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

All reagents, except where stated, were obtained from Sigma Chemical, Ltd., Poole, UK. SB201146 and SKF104353 were provided by Dr. D. Hay of SmithKline Beecham, Inc., King of Prussia, PA. BWA4C was provided by Dr. L. Garland of the Wellcome Research Laboratories, Beckenham, Kent, UK. MK 886 was obtained from Tocris, Bristol, UK.

Subjects

Healthy subjects enrolled in the study had no history of either asthma or COPD. The inclusion criteria for patients with COPD were: age  40 yr, current smoking with a smoking history of  10 pack-years, FEV1/FVC ratio of  65%, and FEV1 of  70% predicted. All subjects with COPD were taking inhaled -agonists and anticholinergic bronchodilators.

Neutrophil Isolation

Neutrophils were isolated from venous blood of healthy volunteers by dextran sedimentation of erythrocytes and density-gradient centrifugation of leukocytes. All procedures up to and including density-gradient centrifugation were conducted at 22° C unless stated otherwise. Blood was anticoagulated with acid-citrate-dextrose and centrifuged at 400 × g for 20 min to remove platelets. The platelet-rich plasma supernatant was removed and centrifuged at 2,000 × g for 30 min to prepare platelet-poor plasma (PPP). The platelet-depleted cell precipitate from the first centrifugation step was mixed with phosphate-buffered saline (PBS) (1:4). Dextran (Baxter, Warrington, UK) was added to a final concentration of 2% and erythrocytes were sedimented under gravity for 45 min at 15° C. The erythrocyte-depleted supernatant containing leukocytes was centrifuged at 400 × g for 10 min. The resulting leukocyte-enriched pellet was resuspended in PPP. The leukocytes were then layered over a discontinuous gradient of Percoll (4 ml of 61.5% and 4 ml of 76%) in a 15-ml polypropylene centrifugation tube. The Percoll densities were created from pure Percoll diluted with 10× PBS and PPP. Final percentages of PPP (vol/vol) in the Percoll solutions were 42.89% and 23.97% for the 61.5% Percoll layer and 76.0% layer, respectively. The Percoll densities and layered cells were then centrifuged at 400 × g for 20 min. After centrifugation, mononuclear cells were found at the plasma-61.5% Percoll layer interface, and neutrophils were found at the 61.5-76.0% Percoll layer interface. Neutrophils were removed and washed twice in RPMI-1640. Cells were counted with a hemocytometer and resuspended at 10⁶ cells/ml. Cells were plated with the compounds investigated in the study in RPMI-1640 containing 10% autologous-plasma-derived serum (PDS). PDS was obtained from nonanticoagulated cell-free plasma. Cell-free plasma was obtained by centrifugation of whole blood immediately after venipuncture. The cell-free plasma was incubated at 37° C and PDS was derived by removal of the protein clot. The purity of neutrophils was consistently greater than 99%.

Incubation with Survival Stimuli

Neutrophils were incubated for 20 h with the study substances in tissue culture-treated 96-well plates in an incubator in a humidified atmosphere of 95% air/5% CO₂ at 37° C before assessment of the proportion of cells in the apoptotic state. Compounds used to stimulate neutrophil survival (GM-CSF, LPS, and DEX) were added at the start of incubation and were present throughout. This was also the case for LTB₄ when it was included. Compounds used to inhibit leukotriene synthesis through 5-LO (BWA4C) or FLAP (MK886), or to antagonize leukotriene receptors to LTB₄ (SB201146) and cysteinyl leukotrienes (SKF104353), were applied to cells 60 min before addition of survival stimuli.

Assessment of Apoptosis

Cell apoptosis was assessed by three methods: examination of nuclear morphology with light- and electron microscopy, and cell-surface binding of phosphatidylserine by annexin V.

Light microscopy. Cells were cytospun onto slides before air drying. Cytospin preparations were exposed to undiluted Giemsa stain for 2 min before immersion in water for washing (30 s). Cells were examined for apoptosis under oil immersion, using the ×100 objective on a Nikon Eclipse E400 microscope (PLS, East Grinstead, UK). A single observer conducted all assessments in a blind fashion. Apoptotic cells were identified as having single-lobed nuclei with intense nuclear staining.

Electron microscopy. All solutions were diluted in 0.1 M cacodylate buffer (Agar Scientific, Stanstead, UK) and CaCl₂ (2.5 mM) (Buffer A). Neutrophils were fixed in 2.5% glutaraldehyde (Agar Scientific) at 4° C for 1 h and then washed three times in buffer and centrifuged at 2,500 × g for 10 min at 22° C in Buffer A after each wash. Neutrophils were refixed with 1% osmium tetroxide (Agar Scientific) for 1 h and rewashed twice in distilled water for 10 min each at 500 × g and 22° C before dehydration. This was followed by dehydration in 50% ethanol for 10 min, 70% ethanol for 15 min, 90% ethanol for 15 min, and twice in 100% (absolute) ethanol at 15-min intervals. Preparations were then embedded in Spurr's resin (Agar Scientific) at a resin-to-ethanol ratio of 1:1 for 30 min and at a resin-to-ethanol ratio of 3:1 for a further 30 min, and finally in 100% resin for 48 h. The fixed preparations were polymerized at 70° C for 12 to 24 h. Following this, preparations were first flattened with a heat pen (Agar Scientific) and then collected on carbon/formvar coated 200-mesh copper grids. Each preparation was then sliced with an ultramicrotome (Supernova; Reichert-Jung, Leica Microsystems, Milton Keynes, UK) and examined with a Hitachi H7000 transmission electron microscope (Nissei Sangyo, Wokingham, UK).

Transmission electron micrographs were taken with Kodak 4489 electron microscope film, developed in Kodak D-19 solution, and fixed with Kodak unifix fixer (Agar Scientific). Magnification was from ×3,000 to ×4,000. Under electron microscopy, the apoptotic state was observed primarily as condensation of the nucleus into a single-lobed, electron-dense nucleus, accompanied by cell shrinkage and smoothing of the plasma membrane (Figures 1-3).

View larger version (156K): [in this window] [in a new window]   Figure 1.   Electron microscopic examination of neutrophil survival. Plate I (original magnification: ×4,000), freshly isolated cells.

View larger version (164K): [in this window] [in a new window]   Figure 2.   Cells exposed to LPS (0.5 mg/ml). Original magnification: ×3,000.

View larger version (141K): [in this window] [in a new window]   Figure 3.   Cells exposed to LPS and SB201146. Arrow indicates an apoptotic cell. Original magnification: ×3,000.

Examination of neutrophil apoptosis by flow cytometry. Neutrophils were stained with annexin V and propidium iodide for dual labeling in order to detect apoptosis and necrosis, respectively. Stained cells were examined with a FACscan flow cytometer (Beckton Dickinson, Oxford, UK), using Cellquest software. In flow-cytometric analysis with annexin V staining, two regions of fluorescence were observed under basal conditions, demonstrating distinct populations of neutrophils exhibiting discrete densities of cell-surface phosplatidylserine (PS). A high level of annexin V staining indicated a high level of apoptosis.

Measurement of LTB₄

Cells were exposed to GM-CSF under identical conditions to those used for examination of apoptosis. LTB₄ levels were measured by enzyme immunoassay (Cayman Chemicals, Ann Arbor, MI) after a 20-h incubation.

Examination of LTB₄ Binding to Neutrophils

[³H]-LTB₄ (2 nM) (Amersham, Ltd., Amersham, UK) was incubated with intact neutrophils with and without unlabeled LTB₄ at 4° C for 60 min with constant shaking in incubation solution (TRIS, 20 mM; NaCl, 0.154 M; ethylenediamine tetraacetic acid, 1 mM; pH 7.4). Nonspecific binding of [³H]-LTB₄ was assessed by inclusion of unlabelled LTB₄ (1 µM). Reactions were terminated by rapid filtration through Whatman GF/C membranes (Whatman, Maidstone, UK), using a 24-place Brandel Harvester (Semat, St. Albans, UK), followed by three washes with incubation solution at 22° C. Binding was assessed by counting filtration membranes in a beta-scintillation counter (Packard, Reading, UK).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Neutrophils underwent spontaneous apoptosis with basal rates of apoptosis reaching approximately 50% apoptotic cells in the total cell population over a period of 20 h of incubation (Figure 4). As with the cells observed under light and electron microscopy, the population exhibiting a high PS content, indicative of apoptosis, constituted approximately 50% of the population under basal conditions (Figure 5A).

View larger version (12K): [in this window] [in a new window]   Figure 4.   Concentration-response relationship for effect of inhibition of leukotriene synthesis on neutrophil survival. Neutrophils were incubated with the respective compounds for 20 h. Inhibition of leukotriene synthesis was produced with inhibitors of 5-LO (BWA4C: 109 to 105 M) and of FLAP (MK886: 109 to 105 M). Basal survival (open triangles), GM-CSF (open squares), BW4C (closed circles), and MK886 (open circles). **p < 0.01 compared with basal apoptosis rate; ++p < 0.01 compared with GM-CSF alone.

View larger version (11K): [in this window] [in a new window]   Figure 5.   Flow-cytometric analysis of the effect of SB201146 on LPS-induced neutrophil survival. Cells were exposed to LPS for 20 h in the presence and absence of SB201146. Cells were then prepared for flow-cytometric analysis.

GM-CSF (5 ng/ml), LPS (0.5 ng/ml), DEX (30 nM), and LTB₄ (1 µM) increased neutrophil survival above the level observed for unstimulated neutrophils by 32%, 38%, 67%, and 52%, respectively. Examination of the potencies of the survival-stimulating agents permitted the designation of a high but submaximal concentration of each agent for use in further experiments to examine the action of leukotrienes in mediating the actions of GM-CSF, LPS, and DEX.

LTB₄ induced neutrophil survival in a concentration-dependent manner, and produced a biphasic concentration-effect curve (Figure 6). The EC₅₀ of LTB₄ for neutrophil survival was 2.3 nM (range: 1.3 to 4.0 nM). The region of lower potency but higher efficacy corresponded with the region of cell-surface binding by [³H]-LTB₄ (Figure 6). The K_d of bound LTB₄ was 2.1 nM (range: 1.3 to 3.4 nM). It is noteworthy that the effects of LTB₄ on neutrophil survival were observed under conditions different from those of LTB₄ binding to cell-surface receptors. This could account for the presence of a low efficacy/ high potency effect of LTB₄ on survival, but this was not observed as a high-affinity receptor in examination of the cell-surface binding profile.

View larger version (10K): [in this window] [in a new window]   Figure 6.   LTB4 binding to intact neutrophils and effect on neutrophil survival. Neutrophils were incubated with [3H]-LTB4 for 60 min at 4° C. For assessment of its effect on survival, LTB4 was incubated with neutrophils for 20 h at 37° C. The results are from 10 experiments for binding of LTB4 and from 12 experiments for the effects of LTB4 on survival. Apoptosis is shown by open circles and [3H]-LTB4 binding by closed circles.

Activation of neutrophils by LPS was observed by changes in cell morphology under electron microscopy. LPS induced a reduction in the proportion of cells exhibiting nuclear condensation and plasma-membrane smoothing characteristic of apoptosis (Figures 1-3). In addition, the effect of LPS on survival was observed through flow cytometry. The biphasic annexin V staining profile became monophasic with the loss of higher-level annexin V staining and therefore with a reduction in cell-surface PS and apoptosis (Figure 5B).

To investigate the role of leukotriene synthesis in mediating neutrophil survival, we applied BWA4C and MK886 to cells 1 h before exposing them to GM-CSF and DEX. BWA4C and MK886 reversed GM-CSF-induced neutrophil survival to basal levels at concentrations of 0.1 µM for both compounds (Figure 4). BWA4C and MK886 abolished GM-CSF-induced neutrophil survival in a concentration-dependent manner (1 nM to 10 µM), with inhibitory concentrations producing 50% of maximal effect (IC₅₀, geometric mean with 95% confidence interval [CI]) of 182 nM (range: 84.3 to 392 nM) and 63.1 nM (range: 60.5 to 65.8 nM), respectively. BWA4C and MK886 reversed DEX-induced neutrophil survival by 90% and 86%, respectively.

To examine the role of cell-surface LTB₄ receptors in autocrine stimulation of neutrophil survival, we applied the LTB₄ receptor blocker SB 201146 to neutrophils 1 h before exposing them to LPS, GM-CSF, or DEX. The effect of SB201146 on LPS-induced neutrophil survival was confirmed by electron microscopy and flow cytometry. The findings derived from electron-microscopic analysis of the reversal of LPS-induced neutrophil survival by SB201146 are summarized in Figure 7. Antagonism of LTB₄ receptors with SB201146 reversed GM-CSF-, LPS-, and DEX-induced neutrophil survival by 54%, 100%, and 85%, respectively (Figures 1-3, 5, and 7-10). With flow cytometry, the effect of SB201146 on LPS-induced neutrophil survival was observed as an increase in annexin V fluorescence intensity above that observed in the presence of LPS alone (Figure 5). SB201146 at 3.0 nM reversed LPS- induced neutrophil survival to the basal level. SB201146 (10 pM to 1 µM) abolished LPS-induced neutrophil survival in a concentration-dependent manner, with an IC₅₀ of 1.9 nM (range: 0.247 to 14.73 nM) (Figure 8).

View larger version (25K): [in this window] [in a new window]   Figure 7.   Electron microscopic examination of neutrophil survival. Cells were exposed to LPS for 20 h in the presence and absence of the LTB4 receptor antagonist SB201146. Cells were then prepared for electron microscopy. Results are derived from 10 different experiments. **p < 0.001 compared with basal level; ++p < 0.001 compared with LPS alone.

View larger version (13K): [in this window] [in a new window]   Figure 8.   Concentration-response relationship for effects of antagonism of receptors to LTB4 on LPS-induced neutrophil survival and GM-CSF-induced eosinophil survival. Neutrophils were incubated with the respective compounds for 20 h. LTB4 receptor antagonist (SB201146) or cysteinyl leukotriene antagonist (SKF104353) was applied to neutrophils 1 h before exposure to LPS. The results are derived from 12 experiments. Basal apoptosis rate is shown by opened triangles, effect of SKF104353 by closed circles, and SB201146 by opened circles. **p < 0.0001 compared with basal level; ##compared with LPS alone; *p < 0.05 compared with LPS alone.

View larger version (45K): [in this window] [in a new window]   Figure 9.   Examination of the effect of leukotriene receptor antagonism and leukotriene synthesis inhibition on basal and GM-CSF- and LPS-induced neutrophil survival. Neutrophils were incubated with the respective compounds for 20 h. Inhibition of leukotriene synthesis was achieved with inhibitors of 5-LO (BWA4C: 106 M) and FLAP (MK866: 106 M). Leukotriene receptor antagonism was achieved with LTB4 receptor antagonists (SB201146: 106 M) and with cysteinyl leukotrienes (SKF104353: 106 M). Results are derived from 12 experiments. **p < 0.001 compared with basal rate; ##p < 0.001 compared with GM-CSF alone; ++p < 0.001 compared with LPS alone; *p < 0.05 compared with SB201146 alone.

View larger version (45K): [in this window] [in a new window]   Figure 10.   Effect of leukotriene receptor antagonists and leukotriene synthesis inhibitors on DEX-induced neutrophil survival. Cells were incubated with the respective compounds for 20 h. Antagonism of receptors to LTB4 was achieved with SB201146 at 106 M and of cysteinyl leukotriene receptors with SKF104353 at 106 M. Inhibition of leukotriene synthesis was achieved with the 5-LO inhibitor BW4AC (106 M) and the FLAP inhibitor MK886 (106 M). Results are derived from six experiments. **p < 0.001 compared with basal apoptosis; ++p < 0.0001 compared with DEX alone.

The cysteinyl receptor antagonist SKF104353 (pobilukast) was used to examine the potential of neutrophil-derived cysteinyl leukotrienes to produce autocrine stimulation of neutrophil survival. SKF104353 exhibited a significantly lower potency (by two orders of magnitude) (Figure 8) and efficacy than did SB201146 in inhibiting LPS-induced neutrophil survival. Exposure of neutrophils to SB201146 and SKF104353 together did not provide a greater inhibition of GM-CSF-, LPS-, or DEX-induced neutrophil survival than did SB201146 alone (Figures 9 and 10).

To examine the potential cell specificity of leukotriene receptor antagonism in inhibition of neutrophil survival, we exposed eosinophils to GM-CSF (5 ng/ml) with and without the LTB₄ receptor antagonist SB201146. LTB₄ receptor antagonism was relatively ineffective against GM-CSF-induced survival of eosinophils as compared with neutrophils, with a potency three-orders of magnitude lower than with neutrophils (data not shown). In comparison, SB201146 at 1 µM reversed GM-CSF-induced eosinophil survival to basal levels (data not shown), but did not do so at concentrations below this.

To examine the effect of leukotriene receptor antagonism and leukotriene synthesis inhibitor on the survival of neutrophils from COPD subjects, we added SB201146, SKF104353, BWA4C, or MK886 to neutrophils 1 h before exposing them to LPS (1 mg/ml). SB201146, SKF104353, BWA4C, and MK886 all reversed LPS-induced neutrophil survival (Figure 11).

View larger version (33K): [in this window] [in a new window]   Figure 11.   Examination of the effect of leukotriene receptor antagonism and leukotriene synthesis inhibition on LPS-induced survival of neutrophils from subjects with COPD. Neutrophils were incubated with the respective compounds for 20 h. Inhibition of leukotriene synthesis was achieved with inhibitors of 5-LO (BWA4C: 106 M) and FLAP (MK886: 106 M). Leukotriene receptor antagonism was achieved with receptor antagonists of LTB4 (SB201146: 106 M) and cysteinyl leukotriene (SKF104353: 106 M). Results are derived from eight experiments. **p < 0.01 compared with basal level; ##p < 0.001 compared with LPS alone.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

This study demonstrated the capacity of GM-CSF, LPS, and DEX to increase neutrophil survival. Interruption of leukotriene synthesis by inhibition of 5-LO or FLAP, and blockade of neutrophil-derived LTB₄ activity with receptor antagonism, reversed LPS-, GM-CSF-, and DEX-induced neutrophil survival. Antagonism of cysteinyl leukotriene receptors was relatively ineffective as compared with LTB₄ receptor antagonism. LTB₄ receptor blockade alone also reduced unstimulated basal neutrophil survival. The capacity of LTB₄ receptor antagonism and leukotriene synthesis inhibition to reverse neutrophil survival responses was also demonstrated in cells from patients with COPD.

These findings could be significant with regard to conditions with associated neutrophilia and their potential treatment with LTB₄ receptor antagonists or leukotriene synthesis inhibitors. The potency of LTB₄ for inducing neutrophil survival demonstrated in this study suggests that sputum LTB₄ concentrations in COPD (10 to 100 nM) (5) in the absence and presence of exacerbations of the disease are sufficient to induce significant increases in neutrophil survival.

In this study we used a cytokine (GM-CSF), a bacterial protein (LPS), and a noninflammatory compound (DEX) as stimuli of neutrophil survival. GM-CSF production is increased in chronically inflamed airways (13), and bacterial LPS is a major promoter of inflammation during bacterial infection. Corticosteroids are used as immunomodulators in treating both asthma and COPD, albeit with a relatively lower clinical efficacy in COPD. The potency of GM-CSF for inducing neutrophil survival is consistent with the affinity of GM-CSF for GM-CSF receptors (14). Work associated with the present study also showed the capacity for GM-CSF to increase neutrophil LTB₄ production (data not shown). This is consistent with previous findings of upregulation of neutrophil 5-LO activity and LTB₄ synthesis in response to GM-CSF and other neutrophil-activating stimuli (15). The potency of the leukotriene synthesis inhibitors, BWA4C and MK886 against GM-CSF-induced neutrophil survival was consistent with their relative potencies for inhibiting LTB₄ production (16- 18). The potency of the LTB₄ receptor antagonist SB201146 against LPS-induced neutrophil survival was consistent with affinity values for SB201146 for LTB₄ receptors (19).

We used near maximally stimulating concentrations of LPS and GM-CSF for promoting neutrophil survival. Although the concentration of DEX was 30-fold less than that at which maximal survival responses are observed in neutrophils (10), the survival response to DEX was greater than with either LPS or GM-CSF. Inhibition of leukotriene synthesis or antagonism of LTB₄ binding sites is therefore effective in abolishing both the near maximal activity of two distinct promoters of neutrophil survival and large survival responses to DEX.

SB201146 lacked potency in reversing GM-CSF-induced survival in eosinophils. This indicates that reversal of neutrophil survival by LTB₄ receptor antagonism is relatively cell specific. This finding is consistent with LTB₄ as a major product of neutrophils but not eosinophils. The relative absence of an effect of SB201146 on eosinophils also indicates that the action of SB201146 on neutrophil survival was not mediated through nonspecific toxicity.

The cysteinyl leukotriene receptor antagonist SKF104353 (pobilukast) also inhibited LPS-induced neutrophil survival, but with a significantly lower efficacy and relatively low potency (Figure 8). There is the possibility that at higher concentrations (1 µM), SKF104353 interacted with the low-density population of cysteinyl leukotriene receptors observed on human neutrophils (20), resulting in interruption of a relatively minor autocrine survival signal. This concept is not consistent, however, with the apparent absence of a survival signal in human neutrophils in response to LTC₄ (21). Furthermore, the combination of SKF104353 with SB201146 did not enhance the inhibition of survival over that with SB201146 alone. It is also possible that SKF104353 at higher concentrations exhibits antagonism at receptors for LTB₄, and that this action could contribute to the effect of this compound on neutrophil survival. The primary implication of these findings is, therefore, that LTB₄ rather than cysteinyl leukotrienes is the primary leukotriene responsible for maintaining neutrophil survival.

The involvement of leukotrienes in the survival of carcinosarcoma cells has previously been demonstrated (22). We have found evidence for the involvement of cysteinyl leukotrienes in GM-CSF- and fibronectin-induced eosinophil survival (23). The latter findings are consistent with GM-CSF receptor- coupled leukotriene production (15) and the existence of leukotriene-supported survival in a range of cell types. The mechanism underlying the marked prolongation of neutrophil survival by DEX is not understood. The possibility should be considered that leukotrienes provide a fundamental aspect of neutrophil survival, whether such survival is stimulated by inflammatory or noninflammatory stimuli. This possibility is supported by the significant LTB₄ receptor antagonist effect of SB201146 on both DEX-stimulated and basal, unstimulated neutrophil survival in the present study.

The concept of induction of apoptosis as a therapeutic antiinflammatory goal to reduce excessive cell population density or activity appears to be consistent with endogenous processes of resolution of inflammation. The engulfment of apoptotic cells by macrophages and other "nonprofessional" phagocytic cells before the necrotic stage occurs efficiently, without release of inflammatory signals from the macrophage (24, 25). The concept of leukotriene antagonists mediating the resolution of inflammation is novel. Leukotriene antagonists interfere with leukocyte trafficking, and the cysteinyl leukotriene receptor antagonists exhibit bronchodilator activity. Both characteristics are currently utilized in treating asthma with leukotriene synthesis inhibitors and leukotriene receptor antagonists. In another investigation, we also observed a potent and efficacious action of the cysteinyl leukotriene receptor antagonist pobilokast against eosinophil survival (26). This is consistent with autocrine maintenance of eosinophil survival by the major eosinophil leukotriene product LTC₄. The possibility must therefore be considered that the benefit derived from cysteinyl leukotriene receptor antagonists in treating asthma could involve an effect on cell survival, particularly that of the eosinophil. This concept also has relevance to the potential development of LTB₄ receptor antagonists for treating COPD. The findings in the present study suggest that both leukotriene synthesis inhibitors and LTB₄ receptor antagonists have potential for reversing activated neutrophil survival responses. An LTB₄ antagonist has produced a reduction of neutrophil influx into airways of asthmatics individuals at 24 h after airway antigen challenge (27). This action was attributed to interruption of LTB₄-derived chemotactic activity, but could also involve a reduction of neutrophil survival.

It is therefore conceivable that prolonged neutrophilia could be addressed by undermining neutrophil survival by targeting leukotriene activity. An antiinflammatory approach to treating COPD is not without precedent, since corticosteroids are administered in this condition despite the potential resultant undermining of host defense activity (28).

In conclusion, leukotriene production involving LTB₄ provides a positive feedback autocrine pathway that supports neutrophil survival promoted by both endogenous and exogenous stimuli of neutrophil activation. Inhibition of LTB₄ activity therefore provides a potent and efficacious route for undermining neutrophil survival. These findings warrant further investigation in the context of resolving chronic inflammation associated with neutrophilia.