Introduction

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The pathogenesis of asthma involves a complex interplay between cells, chemical mediators, cytokines, chemokines, neurogenic mechanisms, and environmental influences. Inflammation plays a major pathogenic role in asthma development (1). Many inflammatory mediators such as histamine, platelet-activating factor, leukotrienes (LTs), nitric oxide (NO), and various cytokines, including tumor necrosis factor (TNF), interleukin (IL)-4, IL-5, IL-13, and chemokines such as macrophage inflammatory protein (MIP)-1, macrophage chemotactic protein 3, regulated on activation, normal T cells expressed and secreted, and eotaxin, are produced in increased amounts in the airways of patients with asthma (1). The cysteinyl LTs, namely LTC₄, LTD₄, and LTE₄, have been identified as the main constituents of the previously described slow-reacting substance of anaphylaxis. These LTs appear to play an especially important role in the pathogenesis of asthma by inducing tissue edema, increasing vascular permeability and mucus production, and promoting smooth-muscle proliferation and cellular infiltration (6). Further, LTC₄ and LTD₄ are the most potent bronchoconstrictors yet studied in human subjects (6). At least two distinct cysteinyl LT receptors, CysLT₁ and CysLT₂, have been discovered (7), but LTD₄ biologic effects result from CysLT₁ receptor activation (6). Interestingly, this receptor has been identified on human smooth-muscle cells and alveolar macrophages (AMs) (8, 9). However, there is limited information on the potential role of LTD₄ on AM functions.

Pulmonary macrophages, commonly called AMs, are the most abundant cells not only in the alveoli and distal air spaces but also in the conducting airways. They are the first line of defense against infectious agents and other immunologic insults, and one of their functions is to downregulate the immune response in the lung (10). However, there is increasing evidence suggesting that AMs participate in the production and maintenance of airway inflammation in asthma and allergic diseases (11). AMs are a potent sorce of mediators such as TNF, MIP-1, eotaxin, and NO, all known to be involved in inflammatory responses. Further, AMs contribute to eosinophil influx in asthma (11). Thus, modulation of AM cytokine production may play a significant role in the pathogenesis of asthma.

Given the presence of LTD₄-specific receptors on AMs, we hypothesized that LTD₄, which is released by mast cells during allergic reactions, activates or primes AMs to release inflammatory chemokines and/or cytokines that recruit and stimulate inflammatory cells potentiating the inflammatory process observed in asthma. We have previously demonstrated that modulation of cytokine production of rat AM cell line NR8383 was similar to freshly isolated normal human and rat AMs (12). Thus, NR8383 cells were used as the source of AMs for this study. When AMs were pretreated with LTD₄, they secreted significantly more MIP-1, TNF, and NO after stimulation with bacterial antigens, lipopolysaccharides (LPSs). Moreover, LTD₄ alone or followed by LPS stimulation increased messenger RNA (mRNA) levels of MIP-1 and TNF. These immunomodulatory effects of LTD₄ were mediated by Cys-LT₁ receptor as demonstrated by the inhibitory effect of the antagonist, Verlukast (MK-679). Thus, LTD₄ potentiated the release of proinflammatory mediators by AMs, suggesting a role of LTD₄ in modulating inflammation through its action on these cells.

	Materials and Methods

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Cell Culture

NR8383 (cell culture CRL-2192; ATCC, Rockville, MD), is an AM cell line initiated by lung lavage of a normal Sprague-Dawley rat (13). These cells represent a homogenous source of highly responsive AMs which were previously used in vitro to study macrophage-related activities (12, 14). NR8383 cells were maintained in Ham's F-12 media (GIBCO BRL, Burlington, ON, Canada) with 10% fetal bovine serum (FBS) (GIBCO), 1% N-2-hydroxyethylpiperazine- N'-ethane sulfonic acid (Hepes) buffer (GIBCO), 1% penicillin- streptavidin (GIBCO), and 0.2% garamycin (Schering Canada Inc., Pointe-Claire, PQ, Canada) in a humid incubator at 37°C with 5% CO₂. Cells were spun down at 250 × g and suspended at 1 × 10⁶ AMs/ml in RPMI-1640 medium (GIBCO) with 5% FBS, 1% Hepes buffer, and antibiotics as mentioned earlier. Cell viability (93 ± 2%) was determined by Trypan Blue exclusion. After 2 h adherence in 48- or 96-well plates (Falcon; Becton Dickinson Labware, Lincoln Park, NJ) at 37°C, cells were washed and different concentrations (10⁶ to 10¹⁴ M) of LTD₄ (Cayman Chemical, Ann Arbor, MI) or LTE₄ (gift from Dr. P. Bourgeat, Laval University, Sainte-Foy, PQ, Canada) were added for different periods of time as described later. Pretreatments were followed or not by 2 h activation with LPS (Salmonella enteritidis; Sigma Chemical Co., St. Louis, MO) at 1 ng/ml (this LPS concentration was chosen after a dose-response analysis and represents the lowest concentration needed to cause a significant release of TNF). At the end of the treatment, supernatants were recovered and stored at 70°C for future analysis. LTD₄ receptor antagonist Verlukast (MK-679), kindly provided by Merck Frosst Canada Inc. (Kirkland, PQ, Canada), was added 30 min before LTD₄ treatment as described later.

Enzyme-Linked Immunosorbent Assay for MIP-1 and TNF

MIP-1 content in cell-free supernatants was measured using a double-ligand method as previously described (15). Briefly, flat-bottomed 96-well microtiter plates (Costar, Cambridge, MA) were coated with goat antimouse-MIP-1 antibody (R&D Systems, Minneapolis, MN) for 24 h at 4°C. Plates were washed with phosphate-buffered saline solution containing 1% Tween-20 (Sigma) and nonspecific binding was blocked for 24 h at 4°C with 5% bovine serum albumin (ICN, Montreal, PQ, Canada). Plates were washed and samples were added for measurement. After 2 h at room temperature, plates were washed and biotinylated goat antimouse-MIP-1 antibody was added (1 h). After washes, streptavidin-peroxidase conjugate was added for 30 min and plates were washed before adding the substrate tetramethyl benzidine (Sigma). The reaction was stopped 30 min later by adding 1 M H₂SO₄. Plates were read at 450 nm with correction wavelength of 570 nm in a Vmax kinetic microplate reader (Thermo Max; Molecular Devices, Menlo Park, CA). A standard curve was done for each plate using different concentrations of recombinant rat MIP-1 (BioSource International, Camarillo, CA). This enzyme-linked immunosorbent assay consistently detected MIP-1 concentrations > 7 pg/ml, whereas the immunoassay kit for rat TNF (BioSource International) detected concentrations > 4 pg/ml.

Measurement of NO Production

Given that NO metabolite can be measured in supernatants 24 to 48 h after stimulation with LPS, AMs were incubated with LTD₄ (1 × 10¹⁰ M) for 24 h followed by 24 h stimulation with LPS (1 ng/ml). Cell-free supernatants were assayed for NO₂ content using Greiss reaction as previously described (16). NO₂ concentration, proportional to optical density at 540 nm, was determined using a Vmax kinetic microplate reader (Thermo Max; Molecular Devices) with reference to a standard curve (NaNO₂).

Reverse Transcription/Polymerase Chain Reaction

Given that mRNA is expressed before the release of the protein, AMs (10⁶ cells/ml) were pretreated with LTD₄ (10¹¹ M) for only 2 h and stimulated or not with LPS (2 h). Cells were collected (3 × 10⁶ cells) and total RNA was extracted using TRIzol reagent (GIBCO). Total RNA was quantified using RiboGreen RNA quantification reagent (Molecular Probes, Inc., Eugene, OR) and read on a Fluoroskan Ascent FL (Labsystems, Franklin, MA). For complementary DNA synthesis, 1 µg of total RNA was reverse transcribed by Moloney murine leukemia virus reverse transcription (RT) enzyme (GIBCO) using Peltier Thermal Cycler 200 (MJ Research, Inc., Watertown, MA) according to the manufacturer's protocol. Polymerase chain reaction (PCR) was performed using Qiagen Taq DNA polymerase protocol and reaction was done in 20 µl final volume. The primers used were: (1) rat -actin sense: 5'-ATG CCA TCC TGC GTC TGG ACC TGG C-3', and antisense: 5'-AGC ATT TGC GGT GCA CGA TGG C-3' (607 base pair [bp]); (2) murine TNF sense: 5'-TTC TGT CTA CTG AAC TTC GGG GTG ATC GGT CC-3', antisense: 5'-GTA TGA GAT AGC AAA TCG GCT GAC GGT GTG GG-3' (354 bp); (3) rat MIP-1 sense: 5'-ATG AAG GTC TCC ACC ACT-3', and antisense: 5'-TCA GGC ATT CAG TTC CAG-3' (279 bp); and (4) rat inducible NO synthase (iNOS) sense: 5'-ACA ACA GGA ACC TAC CAG CTC A-3', antisense: 5'-GAT GTT GTA GCG CTG TGT GTC A-3' (651 bp). Rat eotaxin sense and antisense primers were purchased from BioSource International. Products were run on a 2% agarose gel and stained with ethidium bromide (5 mg/ml).

Densitometric Analysis

Relative mRNA expression was quantified by densitometric scanning analysis using NIH Image 1.61 and normalized against -actin. Pictures of the gels were taken using Alphamager 2000 version 3.2 (Alpha Innotech Corp., San Leandro, CA) and the image was imported to an NIH image analysis program to determine band density using surface under the curve. The ratio of the band density of each treatment on its -actin was calculated.

Statistical Analysis

Analysis of variance combined with Fisher's protected least significant difference test or Student's t test for paired data were used to compare treatments. Differences were considered significant when P < 0.05.

	Results

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Production and Expression of MIP-1

To investigate the modulatory effect of LTD₄ on AMs, NR8383 cells were treated with different concentrations of LTD₄ (10⁶ to 10¹⁴ M) for 6 h followed or not by 2 h stimulation with a low concentration of LPS (1 ng/ml). Cell-free supernatants were tested for the presence of MIP-1, a chemotactic factor for eosinophils. LTD₄ alone did not significantly modulate the release of MIP-1 (Figure 1). However, LTD₄ (10¹¹ to 10⁸ M) significantly increased MIP-1 release when AMs were further stimulated with LPS. Maximum stimulation of MIP-1 release (47% increase) was observed at 10¹¹ M LTD₄. Different time periods of pretreatment (0, 0.5, 2, 4, and 6 h) with numerous concentrations of LTD₄ (10⁶ to 10¹⁴ M) were investigated and showed that a minimum of 6 h pretreatment with LTD₄ was required to significantly increase LPS-stimulated MIP-1 release whereas LTD₄ alone had no effect (data not shown). Interestingly, treatment of AMs with LTE₄ (10¹¹ M) also significantly increased the release of MIP-1 (49.7 ± 6.5%).

View larger version (47K): [in this window] [in a new window]   Figure 1.   Stimulation of MIP-1 release by LTD4. AMs were pretreated for 6 h with different concentrations of LTD4 (1014 to 106 M), stimulated or not with LPS (1 ng/ml) for 2 h and cell-free supernatants were tested for MIP-1 content. Pretreatment with LTD4 (1011 to 108 M) significantly increased MIP-1 released (*P < 0.02 and **P < 0.01) when AMs were stimulated with LPS. Means ± standard error of the mean (SEM) of nine experiments are shown.

To further explore the specificity of LTD₄ stimulation, a specific LTD₄ receptor antagonist, Verlukast (MK-679), was used. AMs were pretreated with Verlukast (10¹¹ M) for 30 min before the addition of LTD₄. Verlukast did not modify MIP-1 release by LPS-stimulated AMs (LPS, 0.78 ± 0.09 ng/10⁶ AMs; Verlukast plus LPS, 0.88 ± 0.13 ng/10⁶ AMs) (Figure 2). However, the potentiation of MIP-1 release by LTD₄ was significantly inhibited by the presence of Verlukast (LTD₄ plus LPS, 1.15 ± 0.13 ng/10⁶ AMs; Verlukast plus LTD₄ plus LPS, 0.85 ± 0.11 ng/10⁶ AMs, P < 0.002).

View larger version (46K): [in this window] [in a new window]   Figure 2.   Inhibition of LTD4 effect on MIP-1 release by AMs with the receptor antagonist Verlukast (MK). Verlukast was added 30 min before pretreatment of AMs with LTD4 (6 h, 1011 M) followed by LPS stimulation (2 h, 1 ng/ml). LTD4 significantly increased (**P < 0.01) MIP-1 release. This effect of LTD4 was significantly inhibited (P < 0.002) by the presence of Verlukast. Treatment with Verlukast alone had no significant effect on the release of MIP-1. Means ± SEM of five to nine experiments are shown.

To determine whether the potentiation of MIP-1 release reflected an increase in steady-state levels of mRNA for MIP-1, RT-PCR was performed on RNA isolated from sham-treated cells and cells treated with or without LTD₄ (10¹¹ M for 2 h) followed by LPS stimulation (2 h) in the presence or absence of Verlukast (Figure 3). Densitometric analysis of PCR bands was performed and the ratio of the MIP-1 band to the -actin band of the same RT was calculated. LTD₄ treatment alone increased (21%) MIP-1 mRNA expression in AMs (Figure 4). Although LPS stimulation markedly enhanced MIP-1 mRNA expression (81%), LTD₄ further increased this expression (93%) whereas the receptor antagonist Verlukast abrogated the effect of LTD₄ (Figures 3 and 4).

View larger version (64K): [in this window] [in a new window]   Figure 3.   Modulation of MIP-1, TNF, and iNOS mRNA by LTD4. AMs were pretreated with LTD4 (2 h, 1011 M) and stimulated with LPS (2 h, 1 ng/ml). Verlukast (MK) was added 30 min (1011 M) before LTD4 treatment. RT-PCR was performed for MIP-1, TNF, iNOS, and -actin. Lane 1, sham-treated AMs; lane 2, AMs treated with LTD4 alone; lane 3, AMs treated with LPS alone; lane 4, AMs treated with LTD4 plus LPS; lane 5, AMs treated with MK plus LPS; lane 6, AMs treated with MK plus LTD4 plus LPS. A representative experiment of three different experiments is shown. Densitometric analysis was used to quantify the increase in PCR products.

View larger version (18K): [in this window] [in a new window]   Figure 4.   Percent increase of MIP-1 (A) and TNF (B) mRNA levels by LTD4. AMs were pretreated with LTD4 (2 h, 1011 M) and stimulated with LPS (2 h, 1 ng/ml). Verlukast (MK) was added 30 min before LTD4 treatment. The ratio of the band density of each treatment on the density of its -actin band was calculated and expressed in percent compared with sham-treated cells. Means of three different experiments are shown.

The modulation of eotaxin, another chemotactic factor for eosinophils, was also investigated. However, eotaxin mRNA was not detected in unstimulated or LTD₄ and LPS-stimulated AMs; whereas the positive control, pulmonary cells isolated from allergen-challenged rats, highly expressed eotaxin mRNA (data not shown).

TNF Modulation

Given that TNF may be important in amplifying asthmatic inflammation, the modulation of this proinflammatory cytokine by LTD₄ was investigated. AMs were pretreated with LTD₄ (10¹¹ M) for 6 h followed or not by LPS stimulation (2 h) and TNF was measured in cell-free supernatants. LTD₄ pretreatment significantly increased TNF release (21%) when AMs were stimulated with LPS (Figure 5). However, there was no significant increase of LTD₄-stimulated TNF release in the presence of Verlukast (10¹¹ M) (Figure 5). Further, the time-course analysis (2, 4, and 6 h) showed that LTD₄ alone did not stimulate the release of TNF and that at least 6 h pretreatment were needed to potentiate TNF release as for MIP-1 release (data not shown).

View larger version (51K): [in this window] [in a new window]   Figure 5.   Stimulation of TNF release by LTD4. AMs were pretreated with LTD4 (6 h, 1011 M) stimulated or not with LPS (2 h, 1 ng/ml); sham-treated AMs (C). LTD4 significantly (*P < 0.02) increased TNF release when AMs were stimulated with LPS. No significant increase was observed in the presence of Verlukast (MK, 1011 M). Means ± SEM of four to six experiments are shown.

The modulation of TNF production by LTD₄ was investigated at the mRNA levels using RT-PCR (Figure 3). LTD₄ treatment alone stimulated the expression of TNF mRNA (32%) in AMs (Figure 4). Moreover, LTD₄ further increased TNF expression (319%) when cells were stimulated with LPS (242%). However, Verlukast abrogated LTD₄ effect without affecting TNF mRNA levels stimulated by LPS (Figures 3 and 4).

NO

To investigate the role of LTD₄ in modulating NO production by AMs, cells were treated with LTD₄ (10¹⁰ M) for 24 h and stimulated or not with LPS (1 ng/ml) for 24 h. No significant modulation of NO production was observed in the absence of LPS stimulation (data not shown). However, a significant increase (P < 0.01) in NO release was observed when AMs were pretreated with 10¹⁰ M LTD₄ for 24 h followed by LPS stimulation (Figure 6). Moreover, the presence of Verlukast abrogated LTD₄ potentiation of NO release. AMs spontaneously released small amounts of NO (1.7 µM/10⁶ AMs) but this production was not modulated by 10¹² to 10⁶ M LTD₄ treatment (data not shown). Although NO synthesis is mainly catalyzed by iNOS, in macrophages the mRNA levels of iNOS were not modulated by LTD₄ treatment (Figure 3).

View larger version (54K): [in this window] [in a new window]   Figure 6.   Stimulation of NO release by LTD4. AMs were pretreated with LTD4 (24 h, 1010 M) followed by LPS stimulation (24 h, 1 ng/ ml). LTD4 significantly (*P < 0.02) increased NO release when AMs were stimulated with LPS. In the presence of Verlukast (MK, 1010 M) no significant increase was observed. Means ± SEM of six to 12 experiments are shown.

	Discussion

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The cysteinyl LTs LTC₄, LTD₄, and LTE₄ have been known for many years as potent bronchoconstrictors (6). The demonstration of Cys-LT₁ receptor on smooth-muscle cells has initiated the development of antiasthma agents that interfere with cysteinyl LTs which all bind to this receptor (17). Oral administration of LT receptor antagonists to atopic patients hours before allergen challenge significantly attenuated both early and late allergen-induced bronchoconstriction (6). Moreover, these antagonists are efficient in exercise-induced bronchoconstriction, in chronic asthma, and in decreasing needs for corticosteroids and ₂-agonist (6, 18). Interestingly, decreased levels of eosinophils in peripheral blood and airways (18, 19) and reduced postchallenge increase of bronchial hyperresponsiveness (6) have been observed with LTD₄ receptor antagonist treatment, suggesting some anti-inflammatory activities. Given the presence of LTD₄ receptor on AMs (9) and the role of these cells in inflammation (11), it was tempting to speculate that LTD₄ may modulate the production of inflammatory mediators by AMs.

Chemotactic substances such as MIP-1 and eotaxin are important in eosinophil recruitment. Interestingly, 6 h pretreatment of AMs with concentrations of LTD₄ (10¹⁰ to 10¹¹ M) found in sputum of patients with asthma (20) potentiated LPS-stimulated MIP-1 production at the protein and mRNA levels. Although LTE₄, the metabolite of LTD₄, has been shown to be a less potent bronchoconstrictor than LTD₄ (21), no significant difference was observed between LTD₄ and LTE₄ on the increase of MIP-1 release. Interestingly, LTD₄ alone did not stimulate MIP-1 release, but did increase its mRNA level suggesting that LTD₄ may prime AMs to further stimulation as seen with interferons (15). Further, although LTD₄ exhibits negligible chemotactic activity for eosinophils (22), inhalation of LTD₄ and LTE₄ induces eosinophil infiltration in the lung (23, 24), suggesting an indirect role of these LTs in eosinophil recruitment. Our data suggest that AMs may participate in eosinophil recruitment by secreting MIP-1. However, LTD₄ and LPS did not modulate the production of eotaxin, a chemokine that has selective chemotactic activity for eosinophils (25). Although AMs have been identified as an important source of eotaxin in atopic asthmatic patients (5), our results suggest that LPS and LTD₄ are not good stimuli for the production of this chemokine by AMs.

AMs are a source of an important proinflammatory cytokine, namely TNF (26). Among its many effects, TNF increases bronchial hyperresponsiveness (27) and mediates recruitment of inflammatory cells such as neutrophils and eosinophils in the lung (28). Further, TNF can stimulate these inflammatory cells and the production of many inflammatory mediators (28). Interestingly, LTD₄ increased TNF mRNA expression (32%) and potentiated TNF production by AMs after LPS stimulation both at the protein and mRNA levels, suggesting a role of LTD₄ in eosinophil recruitment and stimulation through TNF production. Thus, LTD₄ may prime AMs to produce MIP-1 and TNF which are chemotactic for eosinophils, thereby contributing to the inflammatory process seen in asthma.

The importance of the highly reactive molecule NO, which acts as an intracellular messenger in many biologic processes, has been widely recognized. In the respiratory system, NO is known as an inflammatory mediator, a vasodilatator, and a nonadrenergic neurotransmitter (29). In response to immunologic stimulation or inflammation, NO is synthesized by iNOS in several cell types within the respiratory tract, including macrophages. Interestingly, exhaled NO has been shown to correlate with airway hyperresponsiveness (30). Further, LTD₄ receptor antagonist treatment has been shown to reduce the increased exhaled NO by about 20% (31). Our data support this immunomodulatory effect of LTD₄ on AMs (Figure 6). However, LTD₄ did not modulate iNOS mRNA expression. Interestingly, NO synthesis from L-arginine can also be catalyzed by a constitutive form of NO synthase (cNOS). Although AMs are well known to produce NO through iNOS, unstimulated AMs can produce NO via cNOS (32). Given the small increase in NO production by LTD₄ (16%), this augmentation may be mediated by cNOS instead of iNOS in AM, explaining the unchanged level of iNOS mRNA. However, further investigations are needed to confirm this hypothesis.

Respiratory viral infection is well known to induce airway hyperresponsiveness in patients with asthma (33). However, bacterial respiratory tract infection has also been associated with bronchial hyperresponsiveness (34). Bacterial endotoxins, LPSs, possess potent proinflammatory activities (35) contributing to airway inflammatory process observed in asthma. Our data suggest that LTD₄, released during asthmatic reaction, participates in the inflammation by priming AMs to release more inflammatory mediators after immunologic stimuli. Although the potentiation of MIP-1, TNF, and NO release by LTD₄ was small (47, 21, and 16%, respectively), an increase in these mediators will amplify the inflammatory response, delaying its resorption. Persistence of airway inflammation plays an important role in the development of asthma symptoms, and corticosteroids are currently the therapy of choice for the inflammatory component of asthma.

A new class of medication, the anti-LT drugs, has emerged as potential therapeutic agents for asthma. These drugs were developed to inhibit the effects of LTD₄ on airway smooth-muscle cells (17) and have been shown to be effective in asthma treatment (6, 18). Unexpectedly, decreased eosinophils in the airways and the sputum (19) have been observed in asthmatic patients treated with LT receptor antagonists, suggesting a role for these drugs in modulating lung inflammation. Our results may explain in part these anti-inflammatory LTD₄ receptor antagonist effects. However, further investigations are needed to fully understand the role of LTD₄ in the inflammatory process in asthma.