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The Journal of Immunology, 2002, 168: 3570-3576.
Copyright © 2002 by The American Association of Immunologists

Modulation of Leukotriene B4 Receptor-1 Expression by Dexamethasone: Potential Mechanism for Enhanced Neutrophil Survival1

Jana Stankova, Sylvie Turcotte, Jennifer Harris and Marek Rola-Pleszczynski2

Immunology Division, Department of Pediatrics, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Quebec, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Glucocorticoids can down-regulate many inflammatory and immune responses and constitute a powerful therapeutic tool in a number of diseases. However, they have a somewhat paradoxical effect on neutrophils, in that they prolong their survival. Because leukotriene B4 (LTB4) can also extend neutrophil survival, we proposed that glucocorticoids could prevent neutrophil apoptosis by up-regulating their expression of the high-affinity LTB4 receptor (BLT1). Here we show that, indeed, dexamethasone (DEX) up-regulates the steady-state levels of BLT1 mRNA in human neutrophils. The effect was time and concentration dependent, being maximal at 4 h and at 10–100 nM DEX. The effect was also dependent on transcriptional activity, whereas BLT1 mRNA stability was not affected. DEX-induced up-regulation of BLT1 expression was prevented by pretreatment with the LTB4 antagonist LY255283. Moreover, LTB4 itself up-regulated the expression of BLT1 mRNA. BLT1 protein expression on neutrophils exposed to DEX for 24 h was also up-regulated 2- to 3-fold, and DEX-treated as well as LTB4-treated cells showed enhanced responsiveness to LTB4 in terms of intracellular Ca2+ mobilization and chemotaxis. Whereas DEX and LTB4 alone decreased neutrophil apoptosis by ~50%, neutrophils treated with both LTB4 and DEX showed >90% survival at 24 h. Moreover, BLT1 antagonists prevented the increased neutrophil survival induced by DEX as well as by LTB4. Taken together, our results suggest that DEX-induced up-regulation of BLT1 expression in neutrophils may be one mechanism through which glucocorticoids can prolong neutrophil survival, namely by enhancing cell responses to the antiapoptotic effect of LTB4.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Leukotriene B4 (LTB4)3 is a potent lipid mediator of allergic and inflammatory reactions, as well as a modulator of immune responses (1). It is rapidly synthesized by phagocytic cells, principally neutrophilic polymorphonuclear leukocytes (PMNs) (2) and alveolar macrophages (3), upon challenge with a variety of stimuli, including LTB4 itself (4).

LTB4 is one of the most powerful agents for chemokinesis (5) and chemotaxis (6) of PMNs. In addition, it can induce neutrophil aggregation (5), degranulation (7), cation fluxes (8), and enhanced binding to endothelial cells (9). Finally, it has been shown to exert antiapoptotic activity on neutrophils (10). LTB4 has been detected in significant concentrations in various inflammatory conditions, including inflammatory synovial exudates (11, 12), psoriatic skin lesions (13, 14), and sputum from chronic obstructive pulmonary disease (15). LTB4 has also been shown to play a role in experimental models of asthma (16), ulcerative colitis (17), and postischemic tissue injury (18, 19). The development of LTB4 antagonists and specific LT synthesis inhibitors and, more recently, the production of 5-lipoxygenase (5-LOX) (20) and 5-LOX-activating protein (21) knock-out mice allowed a more precise assessment of the potential role of LTB4 in a number of conditions, including inflammation, immune responses, and host defense against infection (22, 23).

Two types of plasma membrane receptors for LTB4 have been described on human neutrophils (24). The high-affinity receptor mediates aggregation, chemotaxis, chemokinesis, and increased adherence to surfaces, whereas the low-affinity receptor mediates degranulation and increased oxidative metabolism. In 1997, Yokomizo et al. (25) successfully cloned and expressed a high-affinity human leukocyte LTB4 receptor (BLT1). It is a member of the G protein-coupled receptor (GPCR) family, in a subfamily of GPCRs that includes receptors for chemokines and other chemotactic factors. Recently, a second, lower affinity receptor for LTB4 (BLT2) has also been cloned (26), with a broader ligand specificity for various eicosanoids (27). BLT1 mRNA is expressed in leukocytes and to a lesser degree in spleen and thymus. The recent development of mice with disrupted BLT1 gene (28, 29) indicates a major role for BLT1 in acute inflammation and immediate hypersensitivity, as well as in leukocyte functions such as chemotaxis and firm adhesion to endothelium in response to LTB4.

Neutrophils are nondividing phagocytes that undergo spontaneous apoptosis. Their survival can be increased, however, by diverse stimuli, including glucocorticoids (30, 31) and LTB4 (10). This may constitute an important contribution to maintenance or enhancement of host defenses. In contrast, dexamethasone (DEX) induces apoptosis in other leukocyte populations, namely eosinophils and lymphocytes (32, 33). However, the mechanisms underlying the increase in PMN survival by DEX or LTB4 are not understood. Recently, endogenous LTs were suggested to play a role in PMN survival after stimulation by DEX, GM-CSF, or LPS (34). The present study was undertaken to test the hypothesis that glucocorticoids could up-regulate BLT1 expression and thus provide a mechanism for enhanced LTB4-induced neutrophil survival.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells

Neutrophils were obtained from peripheral blood of healthy medication-free volunteers after informed consent in accordance with an Internal Review Board-approved protocol. Peripheral blood leukocytes were enriched by dextran sedimentation, layered over a Ficoll-Hypaque cushion, and centrifuged at 400 x g for 20 min. Mononuclear leukocytes were collected at the interface, whereas neutrophils were obtained from the pellet, depleted of erythrocytes by osmotic shock, washed twice with PBS, and resuspended in RPMI 1640 (Life Technologies, Burlington, Ontario, Canada) supplemented with 10% heat-inactivated FBS (Sigma-Aldrich, Oakville, Ontario, Canada), 100 U/ml penicillin, and 100 µg/ml streptomycin in a humidified atmosphere with 5% carbon dioxide at 37°C.

Reagents

All reagents were ourchased from Sigma-Aldrich, unless otherwise indicated. Nonpertinent monoclonal OKT3 Ab (hybridoma supernatant) was obtained from American Type Culture Collection (Manassas, VA); FITC-conjugated rabbit anti-mouse IgG was purchased from Bio/Can Scientific (Mississauga, Ontario, Canada); LTB4 and platelet-activating factor (PAF) were obtained from Cayman Chemicals (Ann Arbor, MI); U75302, AA861, and AACOF3 were purchased from Biomol (Plymouth Meeting, PA); and LY255283 was obtained from Eli Lilly (Indianapolis, IN).

Northern blot analysis

Total cellular RNA was extracted by the guanidium thiocyanate method (35), separated by electrophoresis on 1% agarose, and transferred onto a Hybond-N+ (Amersham Pharmacia Biotech, Baie d’Urfé, Quebec, Canada) membrane for Northern blot analysis. The cDNA corresponding to the open reading frame of human BLT1 was cloned from the genomic DNA of Raji cells by PCR using primers (forward, 5'-CGGATCCAACACTACATCTTCTGCAGCACCC-3'; and reverse, 5'-GCGAATTCTAGTTCAGTTTAACTTGAG-3') based on the published sequence (GenBank accession no. D89078). The cDNA sequence was verified by DNA sequencing (University of Calgary, Calgary, Alberta, Canada). The amplified BLT1 fragment contained 1058 bp and was used as a probe for Northern blot hybridization. Control hybridizations were performed with the human GAPDH cDNA probe obtained from the American Type Culture Collection or with 28S cDNA, a gift from Dr. E. Müller (Bern, Switzerland). The probes were labeled with a multiprime DNA labeling system (Amersham Pharmacia Biotech) using [{alpha}-32P]deoxycytidine 5'-triphosphated CTP (sp. act., 3000 Ci/mmol; Amersham Pharmacia Biotech). Membranes were prehybridized and hybridized as published previously (36), except with a hybridization temperature of 72°C. Membranes were then exposed to Hyperfilm MP (Amersham Pharmacia Biotech) with intensifying screens at -80°C.

Western blot analysis

Cells were lysed in radioimmunoprecipitation assay buffer containing protease inhibitors (leupeptin, soybean trypsin inhibitor, and aprotinin), and 40 µg of proteins was separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were incubated with the mAb (hybridoma supernatant, 1/5 dilution), followed by an HRP-conjugated sheep anti-mouse Ab, and the complexes were revealed with the ECL detection system (Amersham, Arlington Heights, IL).

Flow cytometry

The expression of BLT1 in neutrophils was assessed using an anti-BLT1 mAb generated by immunizing BALB/c mice with Chinese hamster ovary (CHO) cells stably transfected with BLT1. The Ab labeled BLT1-, but not CysLT1- or PAF receptor-transfected cells or cells transfected with the vector alone.

For flow cytometry studies, neutrophils were washed with PBS and fixed with 2% paraformaldehyde for 15 min at room temperature. Cells were resuspended with PBS-2% BSA and labeled for 30 min at room temperature with anti-BLT1 Ab (or with control nonpertinent Ab). Cells were then washed with PBS and incubated for 30 min with FITC-conjugated rabbit anti-mouse IgG (Bio/Can Scientific) diluted in PBS with 2% BSA. Finally, cells were washed again and resuspended in PBS before single-color immunofluorescence analysis of 5000 cells was performed on a FACScan flow cytometer (BD Biosciences, San Jose, CA).

LTB4 production

Production of LTB4 by neutrophils was measured using a specific LTB4 enzyme immunoassay kit from Cayman Chemicals according to the manufacturer’s directions. The lower limit of detection was 4 pg/ml. Neutrophils (5 x 106/ml) were exposed to DEX (100 nM) or its vehicle, ethanol, for 1, 2, or 18 h, then pretreated with either medium or GM-CSF (700 pM) and TNF-{alpha} (1.2 nM) for 30 min at 37°C and treated for 5 min with adenosine deaminase (0.1 U/ml), to relieve the natural suppressive effect of endogenous adenosine (37), before stimulation with either thapsigargin (100 nM) or the neutrophil agonists fMLP and PAF (each at 300 nM at 5-min intervals) and further incubation for 10 min at 37°C.

Intracellular calcium mobilization

For Ca2+ mobilization assays, 5 x 106 cells were loaded in HBSS (Life Technologies) containing 350 mg/L NaHCO3 and 10 mM HEPES (pH 7.0), with the calcium indicator fura-2-acetoxymethyl ester (Molecular Probes, Eugene, OR) for 30 min at room temperature. Loaded cells were washed twice, suspended in fresh loading buffer, and added to a constantly stirred cuvette at 37°C in an SLM/Aminco spectrofluorometer (SLM Instruments, Urbana, IL). The concentration of extracellular Ca2+ was brought to 2 mM by addition of a solution of CaCl2 into the cuvette 10 min before recordings. Maximal cell fluorescence was obtained by adding Triton X-100 to a final concentration of 0.5%. Minimal fluorescence was determined by subsequent addition of the chelator EGTA in Tris-HCl buffer (100 mM, pH 9.0) at 125 mM. Stimulus consisted of LTB4 or PAF.

Chemotaxis assay

Neutrophil chemotactic activity was performed with Boyden chambers using a modified Boyden chamber chemotaxis assay. A volume of 200 µl of cells (6 x 105) in Gey’s balanced salt solution (Life Technologies) supplemented with 20 mg/ml BSA was added to the upper chamber. The lower chamber contained graded concentrations of LTB4 or its vehicle, diluted in Gey’s balanced salt solution with 2% BSA. The two chambers were separated by a 5-µm pore size polycarbonate filter (Osmonics, Westborough, MA). After incubation for 2 h at 37°C in 5% CO2, the chambers were disassembled and the upper side of the filter was scraped free of cells. Cells on the lower side were removed with 5 mM EDTA and centrifuged before counting in the FACScan.

Assessment of apoptosis

Cell apoptosis was assessed by direct examination of nuclear morphology by light microscopy and by cell surface binding of phophatidylserine by annexin V. For the latter, neutrophils were stained with annexin V and propidium iodide for dual labeling to detect apoptosis and necrosis, respectively. In selected experiments, neutrophils were labeled with anti-caspase 3 Ab (BD PharMingen, Mississauga, Ontario, Canada). Stained cells were examined with a FACScan flow cytometer (BD Biosciences) using CellQuest Pro software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
When PMNs were exposed for 4 h to graded concentrations of DEX, their steady-state expression of BLT1 mRNA was markedly enhanced (Fig. 1GoA). The effect was concentration dependent, being observed at the lowest concentration of 0.1 nM with a plateau at 100 nM DEX. The effect was prevented by the glucocorticoid receptor (GR) antagonist mifepristone (RU486) (Fig. 1GoB). The effect of DEX was also time dependent, being quite evident by 2 h, with maximal BLT1 up-regulation at 4 h of exposure (Fig. 1GoC).



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  		FIGURE 1. DEX up-regulates BLT1 gene expression in a concentration- and time-dependent manner. A, Neutrophils were treated for 4 h with graded concentrations of DEX. B, Neutrophils were treated for 4 h with 100 nM DEX in the absence or presence of the glucocorticoid receptor antagonist RU486. C, Neutrophils were cultured in the presence of DEX (100 nM) for indicated times. After each of the above treatments, cells were lysed and total RNA was extracted, separated by electrophoresis, and analyzed by Northern blot for BLT1 and GAPDH gene expression. Controls contained the highest concentration of diluent, ethanol, needed for 10-6 M DEX. The illustrations are representative of three to four independent experiments.

 
BLT1 mRNA half-life was assessed in PMNs by blocking transcription of new mRNA with actinomycin D (ActD) and subsequently measuring residual BLT1 mRNA. Untreated cells had a basal BLT1 mRNA half-life of ~2 h (data not shown), and pretreatment of PMNs with DEX had no effect on BLT1 mRNA stability. In contrast, when PMNs were pretreated with ActD before exposure to DEX, the up-regulation of BLT1 mRNA expression by DEX was completely prevented, suggesting that transcriptional activity was essential for DEX-induced BLT1 gene expression (Fig. 2Go).



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  		FIGURE 2. DEX-induced up-regulation of BLT1 mRNA involves a transcriptional mechanism. Neutrophils were pretreated, or not, with ActD (5 µg/ml) for 20 min and then stimulated for 3 h with DEX (100 nM) or the diluent for DEX (ethanol (EtOH)). Cells were then lysed and total RNA was extracted, separated by electrophoresis, and analyzed by Northern blot for BLT1 gene expression. The experiment illustrated is representative of three performed.

 
DEX-induced up-regulation of BLT1 expression was also abrogated to a large extent by pretreatment of neutrophils with the LTB4 antagonist LY255283, suggesting that endogenous LTB4 participated in the DEX effect (Fig. 3Go). The LTB4 receptor antagonist U75302 had a similar effect (data not shown). Although endogenous production of LTB4 by neutrophils was measurable under a variety of stimulatory conditions, only a small increase in basal production of LTB4 was observed after an 18-h treatment with DEX (vehicle = 7.2 ± 2 pg/ml; DEX = 17 ± 4 pg/ml). Exogenously added LTB4 was also capable of up-regulating BLT1 mRNA expression (Fig. 3Go).



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  		FIGURE 3. The LTB4 antagonist LY255283 abrogates DEX-induced BLT1 up-regulation, whereas LTB4 enhances BLT1 expression. Neutrophils were pretreated, or not, with 1 µM LY255283 and were further incubated in the absence or presence of 100 nM DEX (upper left panel) or 100 nM LTB4 (upper right panel). Cells were then lysed and total RNA was extracted, separated by electrophoresis, and analyzed by Northern blot for BLT1 gene expression. Lower panel, Corresponding normalized BLT1:28S ratios of densitometry values. Representative illustrations of two independent experiments.

 
To measure the expression of BLT1 protein in human PMNs by flow cytometry, we generated anti-BLT1 mAb as indicated in Materials and Methods. The Ab revealed a main protein band of ~52 kDa on Western blots of lysates from BLT1-transfected, but not mock-transfected, CHO cells (Fig. 4Go). A minor protein band was also revealed at ~70 kDa, corresponding to differential glycosylation of BLT1 (R. Gaudreau, J. Harris, M. Rola-Pleszczynski, and J. Stankova, manuscript in preparation). As expected, the basal level of BLT1 expression on PMNs was quite evident (Fig. 5Go). Nevertheless, pretreatment of PMNs with DEX further increased BLT1 expression (~3-fold) at 18 h. Pretreatment of PMNs with LTB4 for 18 h also resulted in augmented BLT1 expression, albeit to a lesser extent than with DEX. BLT1 expression returned to basal values by 48 h (data not shown).In contrast, pretreatment of PMNs with GM-CSF for 18 h did not enhance BLT1 expression (data not shown).



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  		FIGURE 4. Anti-BLT1 mAb identifies BLT1 protein in transfected cells. A, CHO cells were transiently transfected either with the empty vector (CHO) or with BLT1 cDNA-containing vector (CHO-BLT1). Western blot analysis of whole cell lysates revealed a main band at ~52 kDa and a minor band at ~70 kDa, which were not revealed in mock-transfected CHO cells. B, Anti-BLT1 mAb labeling of BLT1-transfected, but not platelet-activatinf factor receptor-transfected, CHO cells as assessed by flow cytometry.

 


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  		FIGURE 5. DEX and LTB4 augment the expression of BLT1 protein in neutrophils. Neutrophils were incubated with 100 nM DEX (line) or its diluent (shaded histogram, upper panels) or with 100 nM LTB4 (line) or its diluent (shaded histogram, lower panels) for 18 h. Cells were then stained with an isotype control Ab (left panels) or anti-BLT1 mAb (right panels), followed by a secondary FITC-conjugated anti-mouse Ab. Representative illustrations of three to five independent experiments.

 
Up-regulation of BLT1 expression by DEX was accompanied by enhanced functional responses of PMNs to LTB4. Pretreatment of PMNs with DEX for 18 h resulted in a markedly augmented responsiveness of the cells to LTB4 in terms of intracellular Ca2+ mobilization (Fig. 6Go). The LTB4 antagonist LY255283 completely prevented Ca2+ transients in response to LTB4 in both control and DEX-treated PMNs. Responsiveness to PAF, as expected, was not affected by LY255283. Increased responsiveness of DEX-treated cells to LTB4 was also evident in the threshold concentration needed to trigger the calcium transient. Whereas untreated PMNs responded to 1 nM or greater concentrations of LTB4, DEX-treated cells responded to 10-fold lower concentrations (Fig. 6Go, E and F). Moreover, pretreatment of PMNs with LTB4 for 18 h also resulted in a markedly augmented responsiveness of the cells to LTB4 in terms of intracellular Ca2+ mobilization (Fig. 6Go, G and H).



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  		FIGURE 6. DEX and LTB4 augment LTB4-induced Ca2+ mobilization. Neutrophils were incubated for 18 h with diluent ethanol (A, C, E, and G), DEX 100 nM (B, D, and F), or LTB4 100 nM (H). The cells were loaded with fura 2-acetoxymethyl ester and stimulated with 100 nM LTB4 (A–D, G, and H) or PAF (C and D). C and D, Cells were preincubated with the LTB4 antagonist LY255283 (50 µM) for 2 min before the indicated stimulation with agonists. E and F, Cells from the same donor were stimulated with graded concentrations of LTB4 after overnight treatment with ethanol (E) or DEX (F). The illustrated experiments are representative of three performed.

 
Chemotactic responses to LTB4 were also up-regulated by pretreatment of cells for 18 h with DEX (Fig. 7Go), but no significant change in chemokinetic activity was observed (data not shown).



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  		FIGURE 7. DEX enhances LTB4-stimulated chemotaxis. Neutrophils were cultured in the presence of diluent ethanol (medium) or DEX (100 nM) for 18 h. Chemotactic activity was measured in response to LTB4 as described in Materials and Methods. This figure illustrates the mean ± SEM of six independent experiments. *, p < 0.05; **, p < 0.01; DEX-treated vs diluent-treated cells.

 
PMN survival was next assessed after exposure to DEX or LTB4. Both substances effectively reduced PMN apoptosis by ~50% (Fig. 8Go). Increased PMN survival, at 24 h after DEX treatment, was further enhanced to >90% by concomitant treatment with LTB4. At 48 h, however, all responses were attenuated, with concomitant LTB4 and DEX treatment inducing <50% PMN survival. GM-CSF also inhibited neutrophil apotosis, as assessed by measuring activated caspase 3-containing cells, at a level similar to that of LTB4 (Fig. 9Go). Concomitant treatment of cells with GM-CSF and LTB4 resulted in an additive effect on PMN survival.



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  		FIGURE 8. DEX and LTB4 effects are additive in preventing neutrophil apoptosis. Neutrophils were cultured in the presence of diluent (medium), DEX (100 nM), LTB4 (100 nM), or combination of DEX and LTB4 for 24 or 48 h. The cells were stained with FITC-coupled annexin V and propidium iodide and analyzed on a FACS. Results are illustrated as annexin V-positive, propidium iodide-negative cells and represent the mean ± SEM of three independent experiments.

 


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  		FIGURE 9. GM-CSF and LTB4 effects are additive in preventing neutrophil apoptosis. Neutrophils were cultured in the presence of diluent, GM-CSF (5 or 10 ng/ml), LTB4 (100 nM), or combinations of GM-CSF and LTB4 for 24 h. Cells were then stained with PE-conjugated Ab to activated caspase 3 and were analyzed by flow cytometry. Results are illustrated as caspase-3-positive cells and represent the mean ± SEM of three independent experiments.

 
As expected, the BLT1 antagonist U75302 effectively blocked the LTB4-induced increased PMN survival at 24 h (Fig. 10GoA), as did LY255283 (data not shown). Interestingly, U75302 also abolished the effect of DEX and its additive effect with LTB4 in terms of PMN survival. Moreover, when endogenous LT synthesis was inhibited using the 5-LOX inhibitor AA861 or the cytosolic phospholipase A2 (cPLA2) inhibitor AACOF3, basal neutrophil apoptosis was increased (Fig. 10GoB). The antiapoptotic effect of DEX was blunted, whereas the effect of exogenous LTB4 was preserved.



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  		FIGURE 10. BLT1 antagonist U75302 blocks both LTB4- and DEX-induced neutrophil survival, whereas inhibition of LT synthesis mainly reduces the effect of DEX. Neutrophils were cultured with diluent (Control), DEX (100 nM), LTB4 (100 nM), or combination of DEX and LTB4 for 24 h, in the absence or presence of the BLT1 antagonist U75302 (1 µM) (A), the 5-LOX inhibitor AA861 (50 µM), or the cPLA2 inhibitor AACOF3 (25 µM) (B). Cells were then stained with FITC-coupled annexin V and propidium iodide and analyzed on a FACS. Results are illustrated as annexin V-positive, propidium iodide-negative cells and represent the mean ± SEM of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates a novel action of the glucocorticoid DEX which, prima facie, appears counterintuitive, namely the up-regulation of the high-affinity receptor for an inflammatory mediator. Although glucocorticoids are known to exert a number of anti-inflammatory activities through inhibition, for instance, of PGE2 synthesis (38), NF-{kappa}B-dependent cytokine gene expression (39), or adhesion molecule expression (40), they have also been found to have what appear to be proinflammatory properties. For instance, DEX has been shown to prolong survival of PMNs (31), which can be prevented by inhibition of the 5-LOX pathway (34), suggesting that endogenous 5-LOX products may be involved in DEX-mediated prevention of apoptosis. LTB4 has also been found to prolong PMN survival (10), and DEX has been shown to enhance 5-LOX-activating protein (41) and 5-LOX (42, 43, 44) gene expression and LT production. In this study, we present evidence that prevention of PMN apoptosis by DEX is associated with up-regulation of the high-affinity LTB4 receptor BLT1. Moreover, we show that DEX can have an additive effect with LTB4 in increasing PMN survival to >90% at 18 h, when DEX-induced up-regulation of BLT1 expression is evident. Finally, LTB4 antagonists prevent both DEX-induced BLT1 expression and DEX-induced PMN survival, suggesting a role for endogenous LTs in up-regulation of BLT1 expression. Interestingly, LTB4 itself can augment BLT1 expression and function. These findings suggest a possible mechanism for DEX-induced PMN survival, namely up-regulation of BLT1 with increased sensitivity to LTB4-dependent PMN survival.

DEX has been shown to modulate the expression of a number of other receptors, in both a positive and a negative sense. It can up-regulate some receptors, such as the GPCRs {beta}2-adrenergic receptor (45) and chemokine receptor CXCR4 (46) and the nuclear androstane receptor (47), whereas it down-regulates the chemokine receptor CCR3 (46), the endothelin A and B receptors (48), and the human glucocorticoid receptors {alpha} and {beta} (49).

The mechanisms underlying the increase in PMN survival by DEX are not understood. Endogenous LTs and, in particular, LTB4 may provide a basic aspect of PMN survival after stimulation by DEX, GM-CSF, or LPS (34). However, except for DEX, the other stimuli of PMN survival, and in particular GM-CSF, did not up-regulate BLT1. Only DEX induced an increase of BLT1 gene expression, suggesting that the mechanism of its antiapoptotic effect on PMNs may be distinct from that of other PMN stimuli. It also suggests a mechanism for the observed additive effect of combined DEX and LTB4 treatment on PMN survival. Interestingly, although endogenous production of LTB4 by neutrophils was measurable under a variety of stimulatory conditions, only a modest increase in basal LTB4 production was observed after treatment with DEX. In contrast, inhibition of LT synthesis by 5-LOX or cPLA2 inhibitors enhanced apoptosis and blunted the antiapoptotic effect of DEX, whereas the antiapoptotic effect of LTB4 was retained. The DEX effect may require not only basal LTB4 production but also up-regulated BLT1 expression, and basal endogenous LTB4 production may be necessary, but not sufficient, for the observed DEX-induced effect.

DEX has also been found to prolong survival in nonleukocytic cells, such as rat hepatoma epithelial cells (50) and human mammary epithelial cells (51). In the latter, DEX inhibited apoptosis by inducing the survival kinase gene sgk-1 (52), a known serum- and glucocorticoid-regulated kinase with catalytic domain homology to the antiapoptotic kinase akt (53). Recently, Strickland et al. (54) suggested that expression of GR{beta} by neutrophils renders them insensitive to glucocorticoid-induced cell death. In contrast, our findings suggest that neutrophils are sensitive to the actions of glucocorticoids, but that they respond with enhanced BLT1 expression and increased survival. If anything, GR{beta} would serve to dampen this effect by interfering with GR{alpha} signaling. Our findings indicate that DEX-induced augmentation of BLT1 expression is mediated by GR{alpha}, because the GR{alpha} antagonist RU486 can block this effect of DEX.

The mechanism underlying DEX-induced BLT1 gene expression remains to be elucidated. Although ActD pretreatment prevented the induction, we could not directly prove that DEX induced BLT1 gene transcription because nuclear run-on experiments on PMNs are prohibitively difficult. The promoter sequence of BLT1 published to date does not contain any consensus glucocorticoid response elements (26). However, it remains to be shown whether such elements are present in farther regions of the promoter or whether other elements may be involved.

In conclusion, we have shown for the first time to our knowledge that DEX can up-regulate BLT1 expression in human neutrophils, with consequently enhanced responses of the cells to LTB4. Moreover, LTB4 antagonists prevent both DEX-induced BLT1 expression and DEX-induced PMN survival. In contrast, exogenous LTB4 augments the expression of its own receptor, BLT1, and increases neutrophil survival, even in the presence of 5-LOX inhibition. Finally, concomitant exposure of PMNs to DEX and LTB4 results in an additive increase in cell survival, suggesting that the observed DEX effect on BLT1 expression may contribute to its antiapoptotic effect on neutrophils and may help preserve some of the host defense activities of these cells.


   Footnotes
 
1 This work was supported by funding from the Canadian Institutes for Health Research. Back

2 Address correspondence and reprint requests to Dr. Marek Rola-Pleszczynski, Department of Pediatrics, Immunology Division, Faculty of Medicine, Université de Sherbrooke 3001, North 12th Avenue, Sherbrooke, Quebec J1H 5N4, Canada. E-mail address: mrolaple{at}courrier.usherb.ca Back

3 Abbreviations used in this paper: LTB4, leukotriene B4; PMN, polymorphonuclear leukocyte; 5-LOX, 5-lipoxygenase; BLT, LTB4 receptor; GPCR, G protein-coupled receptor; DEX, dexamethasone; PAF, platelet-activating factor; CHO, Chinese hamster ovary; ActD, actinomycin D; cPLA2, cytosolic phopholipase A2; GR, glucocorticoid receptor. Back

Received for publication August 31, 2001. Accepted for publication February 1, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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