Leukotrienes are important mediators of the eosinophilic influx and mucus hypersecretion in the lungs in a murine model of asthma. We used in situ PCR in this model of human asthma to detect lung mRNA for 5-lipoxygenase (5-LO) and 5-LO-activating protein (FLAP), key proteins necessary for leukotriene synthesis. Lung tissue was obtained on day 28 from mice treated with i.p. (days 0 and 14) and intranasal (days 14, 25, 26, and 27) OVA or saline. After fixation, the tissue sections underwent protease- and RNase-free DNase digestion, before in situ RT-PCR using target-specific cDNA amplification. 5-LO and FLAP-specific mRNA was visualized by a digoxigenin detection system, and positive cells were analyzed by morphometry. 5-LO and FLAP-specific mRNA and protein were associated primarily with eosinophils and alveolar macrophages in the airways and pulmonary blood vessels in OVA-sensitized/challenged mice. 5-LO and FLAP protein expression increased on a per-cell basis in alveolar macrophages of OVA-treated mice compared with saline controls. Pulmonary blood vessel endothelial cells were also positive for 5-LO, FLAP mRNA, and protein. 5-LO inhibition significantly decreased 5-LO and FLAP-specific mRNA and protein expression in the lung inflammatory cells and endothelial cells. These studies demonstrate a marked increase in key 5-LO pathway proteins in the allergic lung inflammatory response and an important immunomodulatory effect of leukotriene blockade to decrease 5-LO and FLAP gene expression.

Airway eosinophilia, mucus hypersecretion, edema, and epithelial injury are key inflammatory features of asthma. Important in the mediation of this inflammatory process are the 5-lipoxygenase (5-LO)4 arachidonic acid products, leukotriene (LT) B4, and cysteinyl leukotrienes, LTC4, LTD4, and LTE4 (1). As a consequence of IgE-mediated mast cell degranulation after bronchial Ag challenge in allergic asthmatics, increased levels of leukotrienes are found in the bronchoalveolar lavage (BAL) fluid of these subjects (2, 3). Although LTB4, the major 5-LO product of human neutrophils, monocytes, and alveolar macrophages, may induce leukocyte trafficking into the lungs by stimulation of leukocyte chemokinesis/chemotaxis and adherence to vascular endothelium, its role in asthma is unclear. More closely linked to asthma pathogenesis are cysteinyl leukotrienes C4, D4, and E4, principal arachidonate products of eosinophils. The cysteinyl leukotrienes induce eosinophil chemotaxis, mucus hypersecretion, increased vasopermeability, and bronchial smooth muscle constriction. Metabolism of arachidonic acid to leukotrienes requires the action of both 5-LO, a 78-kDa protein (4), and 5-LO-activating protein (FLAP), an 18-kDa arachidonic acid-binding protein (5, 6). In bronchial mucosal biopsies from normal subjects, aspirin-intolerant asthma patients, and aspirin-tolerant asthma patients, 5-LO and FLAP colocalize by immunocytochemistry to monocyte-macrophages and eosinophils in the lung tissue (7). Alveolar macrophages also immunostain positive for 5-LO protein in lung tissue from patients with idiopathic pulmonary fibrosis (8).

Murine models have been developed that reproduce characteristic features of human asthma. We developed a murine asthma model using OVA as allergen in which OVA-treated mice demonstrate increased levels of total and OVA-specific IgE in the blood and LTB4 and LTC4 in BAL fluid, increased expression of Th cells with type 2 (Th2) cytokine (IL-4, IL-5, and IL-13) phenotype in peribronchial lymph nodes, and airway eosinophilia, mucus hypersecretion, and hyperreactivity to methacholine (9, 10). Using this in vivo model, we found that treatment with either a 5-LO or FLAP inhibitor blocks the allergen-induced airway inflammatory response. In particular, leukotrienes were found to be important mediators of the airway mucus hypersecretion and eosinophil infiltration. To characterize allergen-induced airway 5-LO pathway activation, we examined by in situ PCR and immunocytochemistry the expression of FLAP and 5-LO mRNA and protein in the lungs of allergic mice. In OVA-treated animals, we found a marked increased in 5-LO and FLAP-positive eosinophils and mononuclear leukocytes surrounding the pulmonary vasculature and airways. Pulmonary endothelial cell gene expression of both 5-LO and FLAP was also observed in the allergen-challenged mice. Further, leukotriene blockade in the OVA-sensitized/challenged mice inhibited both 5-LO and FLAP gene expression in the lungs.

A total of 500 μg/ml crystalline OVA (Pierce Chemical, Rockford, IL) in saline was mixed with equal volumes of 10% (w/v) aluminum potassium sulfate (alum; Sigma, St. Louis, MO) in distilled water and incubated for 60 min at room temperature after adjustment to pH 6.5 using 10 N NaOH. After centrifugation at 750 × g for 5 min, the OVA/alum pellet was resuspended to the original volume in distilled water.

Specific rabbit polyclonal anti-human leukocyte FLAP and 5-LO Abs, which have been extensively characterized (11, 12, 13) and recognize rodent FLAP and 5-LO, respectively (14, 15, 16), were generously provided by Dr. Jilly F. Evans (Merck Frosst Center for Therapeutic Research, Pointe Claire-Dorval, Québec, Canada). The antisera to FLAP (designated H4 TB4) was raised against the thyroglobulin conjugate of amino acid residues 41–52 of FLAP. The antisera recognizes FLAP in the membrane fraction of leukocytes at a dilution of 1:150. The 5-LO antisera (designated LO-32) identifies leukocyte 5-LO by Western immunoblot at a dilution of 1:200 in 1% gelatin: TBST and also immunoprecipitates leukocyte 5-LO (13).

All animal use protocols were proved by the University of Washington Animal Care Committee. Female BALB/c mice 6–8 wk of age were purchased from B and K Universal (Kent, WA).

As previously described (9, 10), mice received an i.p. injection of 0.2 ml (100 μg) OVA complexed with alum on days 0 and 14. On days 14, 25, 26, and 27, mice were anesthetized i.p. with 0.2 ml 6.5 mg/ml ketamine/0.44 mg/ml xylazine in normal saline before receiving an intranasal (i.n.) dose of 100 μg OVA in 0.05 ml normal saline on day 14 and an i.n. dose of 50 μg OVA in 0.05 ml normal saline on days 25, 26, and 27. Lung tissue was obtained 24 h after the last i.n. challenge on day 28. The control group received normal saline with alum i.p. on days 0 and 14 and normal saline without alum i.n. on days 14, 25, 26, and 27. To assess the effect of 5-LO inhibition on 5-LO and FLAP mRNA and protein expression, in some studies 35 mg/kg of the 5-LO inhibitor zileuton (N-(1-benzo[b]thien-2-ylethyl]-N-hydroxyurea; kindly provided by Drs. Randy L. Bell and George W. Carter, Abbott Laboratories, Abbott Park, IL) was given i.p. 30 min before each i.n. challenge on days 25, 26, and 27.

The left lung tissue was fixed in 10% buffered formalin. After embedding in paraffin, the tissues were cut into 5-μm sections. Eosinophils were stained with Discombe’s solution (0.05% aqueous eosin and 5% (v/v) acetone in distilled water) for 5 min, rinsed with distilled water, and counterstained with 0.07% methylene blue.

For immunocytochemistry, left lung tissue was dissected and fixed immediately in Carnoy’s fixative for 12 h at 4°C. Tissues were dehydrated with graded alcohol, infiltrated with xylene, embedded in paraffin at 56°C, and 5-μm sections were cut. The sections were deparaffinized in xylene and hydrated by sequential immersions into absolute ethanol, 70% ethanol, and 0.05 M PBS (pH 7.2). To block nonspecific binding of the secondary Ab and reduce background staining, a 5% solution of nonfat, dry milk in PBS was applied to the sections for 30 min at room temperature. After rinsing in PBS, the sections were overlaid with rabbit polyclonal Abs against 5-LO or FLAP. The polyclonal Abs were diluted in the 5% milk solution in PBS and used at a dilution of 1:80 to detect FLAP and 1:50 to detect 5-LO. These Abs were applied individually in the amount of 200 μl per slide for 60 min in a moist chamber at room temperature followed by 16 h at 4°C. Two controls were employed substituting the primary Ab incubation with either PBS or normal rabbit IgG (Vector Laboratories, Burlingame, CA) diluted 1:1000 in a 5% solution of nonfat dry milk in PBS, under the same incubation conditions. The avidin-biotin complex alkaline phosphatase procedure was used for 5-LO and FLAP localization. After rinsing in PBS, the sections were incubated for 60 min with biotinylated goat anti-rabbit IgG (Zymed, San Francisco, CA) diluted 1:80 in a 5% solution of nonfat, dry milk in PBS containing 1% normal goat serum (Vector Laboratories) and then in an avidin-biotinylated alkaline phosphatase complex (Vector Laboratories) at a dilution of 1:100 for 30 min according to the manufacturer’s instructions. Sections were treated with alkaline phosphatase-substrate solution to which levamisole was added to block endogenous alkaline phosphatase activity. Sections were counterstained with either 2% methyl green or 0.5% methylene blue for 3 min. Colocalization of 5-LO and FLAP to inflammatory cells was performed as previously described (17).

The right lung of each mouse was frozen on dry ice slabs and homogenized in TRI REAGENT solution (Molecular Research Center, Cincinnati, OH; 1 ml/50–100 mg tissue) using a 5-ml grinder with a Teflon pestle. Total RNA was isolated according to the manufacturer’s instructions. As previously described (9), 3 μg of total RNA in diethyl pyrocarbonate-water were transcribed in a final 20-μl reaction mixture containing 0.5 mM dNTP (Boehringer Mannheim, Indianapolis, IN), 25 ng/μl random hexamer primers (Pharmacia Biotech, Piscataway, NJ), 10 U/μl Moloney murine leukemia virus transcriptase (Life Technologies, Gaithersburg, MD), 1 U/μl placental RNase (RNasin) inhibitor (Promega, Madison, WI), 5 mM DTT, 3 mM MgCI2, 75 mM KCl, and 50 mM Tris-HCl (pH 9.3; Life Technologies). The mixture was incubated at 37°C for 60 min for RT and then 95°C for 5 min to inactivate the transcriptase. The RT-cDNA temple (1–5 μl) underwent PCR in a 50-μl reaction mixture containing 200 μM dNTPs, 0.03 U/μl Taq polymerase (Amersham, Arlington Heights, IL), 1 μM sense-stand primers, 1 μl anti-sense-strand primer, 1.5 mM MgCl2, 20 mM ammonium sulfate, and 50 mM Tris-HCl (pH 9.0; Amersham). The samples underwent denaturation at 94°C for 1 min, annealing at 59°C for 1 min, and extension at 72°C for 2 min. To verify that equal amounts of cDNA were added in each PCR within an experiment and a uniform amplification process, the housekeeping gene hypoxanthine-guanine phosphoribosyl transferase (HPRT) was also reversed transcribed and amplified in each assay. PCR cycles used for each primer pair were as followed 5-LO (18), FLAP (19), and HPRT (5). The cycle number had been limited to ensure the PCR did not reach the plateau of amplification. The primer pair for 5-LO, FLAP, and HPRT were as follows: 5′-TTCACATCCTCAAGCAGCAC and 3′GTAGCCAAACATGAGGTCTFCC (5-LO), 5′-GGCCMGTCACCCTCATCAGCG and 3′-CCCGGCGAAGGACATGAGGAACAGG (FLAP), and 5′-GTTGGATACAGGCCAGACT′UGTTC and 3′-GAGGGTAGGCTGGCCTATAGGCT (HPRT; Ref. 20). The PCR products were electrophoresed and visualized on 1.8% agarose gel (Life Technologies, Grand Island, NY) containing 0.5 μg/ml ethidium bromide. For estimation of the relative levels of gene expression, type 55 film (Polaroid, Cambridge, MA) was used to photograph the ethidium bromide-stained gels. The photographic negatives were scanned on a flat-bed scanner (Arcus II, Agfa-Gevaert N.V., Mortsel, Belgium), and the digital data of the band intensities of each amplified product were analyzed using public domain National Institutes of Health Image software (http://zippy.nimh.nih.gov). The sum of pixel values of each single band for 5-LO, FLAP, and HPRT was calculated with the gel background absorbance subtracted to determine the ratio of 5-LO or FLAP to HPRT amplified products (21).

RT-PCR products for 5-LO and FLAP were resolved on 1.8% agarose gel and transferred to Hybond-N+ Nylon membrane (Amersham) by capillary flow transfer overnight. The membrane was baked at 80°C for 1 h and placed in hybridization buffer. The membranes were hybridized with oligonucleotide probe labeled with digoxigenin (DIG)-dUTP by using DIG oligonucleotide 3′ tailing kit (Boehringer Mannheim). The oligonucleotide probe was 5′-TGAACAGGTTCTCCATCGCT for 5-LO and 5′-CAGTbTCTGGTTGGCAGTGTA for FLAP. The hybridization procedure was performed according to the manufacturer’s recommendations. The DIG probe, which hybridized with PCR products, was detected with the DIG detection system (Boehringer Mannheim).

In situ RT-PCR was performed as described previously (22, 23). Left lung tissue was fixed in 10% neutral buffered formalin for 6–24 h, embedded in paraffin, and 4-μm-thick tissue sections mounted to silane-coated glass slides (Perkin-Elmer, Foster City, CA). After deparaffinization, the tissue was digested with 2 mg/ml pepsin (Sigma) and exposed to 10 IU/tissue section RNase-free DNase overnight (Boehringer Mannheim) to avoid nonspecific amplification of DNA. The DNase-treated tissue sections were incubated at room temperature for 15 min and then at 42°C for 60 min in a humid chamber with 20 μl of solution containing 5 mM MgCl2, 1× PCR buffer II (50 mM KCl, 10 mM Tris-HCl (pH 8.3)), 1 mM dNTP, 2.5 μl random primer, 1 U/μl RNase inhibitor, 2.5 U/μl reverse transcriptase (GeneAmp RNA PCR kit; Perkin-Elmer). The sections were washed with TBS (0.1 M Tris (pH 7.5), 0.1 M NaCl) and dehydrated with 100% ethanol. In situ RT-PCR was performed using the Perkin-Elmer GeneAmp in situ PCR System 1000 instrument. The solution for amplification of the cDNA contained 1× PCR buffer II (GeneAmp kit), 4.5 mM MgCl2, 200 μM dNTP, 100 μM BSA (20 mg/ml, molecular biology grade; Sigma), 10 μM DIG 11-dUTP (Boehringer Mannheim), 1 μM sense and antisense primers, and 5 U/50 μl AmpliTaq DNA polymerase (GeneAmp kit). The sequences of primers for 5-LO and FLAP were the same as for solution RT-PCR. Slides were overlaid with 50 μl PCR solution and covered with GeneAmp AmpliCover Discs. The slides were placed in the GeneAmp in situ PCR system 1000 instrument and thermal cycling was performed for 25–35 cycles as follows: denaturation at 95°C for 1 min, annealing 59°C for 1 min, and extension 72°C for 2 min. Unincorporated DIG-dUTP was removed by washing with 0.2% BSA in 1× SSC at 52°C for 10 min. A DIG detection kit (Boehringer Mannheim) was used to detect the amplified products. The slides were incubated in a humid chamber with anti-DIG alkaline phosphatase conjugate (1:200 dilution in TBS and 0.2% BSA) at 37°C for 30 min. Free conjugates were removed by washing with a solution containing 0.1 M Tris (pH 9.5), 0.1 M NaCl, and 0.1 M MgCl2 at room temperature for 2 min. Sections were incubated with chromogen nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate at 37°C for 10–20 min. Following appropriate color development, the slides were washed in distilled water for 1 min, followed by counterstaining with 0.1% nuclear fast red for 5 min. The positive control for in situ RT-PCR was to eliminate DNase digestion, an intensive nuclear signal was generated from DNA repair and mispriming. The negative control was the tissue treated with DNase, and the reverse transcription step was not conducted.

The number of inflammatory cells in the lung interstitium expressing FLAP mRNA and protein per unit airway area (2200 μm2) was determined by morphometry as described (17, 19, 24); atelectatic areas were not examined. A minimum of 10 fields were randomly examined by light microscopy by a blinded observer. Slides were viewed using an Olympus Vanox microscope at a magnification of 40×, and all cells in each field were counted in a grid of 64 squares representing 2200 μm2; the error of repeat counting was <2%. Intensity of 5-LO and FLAP immunostaining of alveolar macrophages was assessed on a semiquantitative scale ranging from 0 to 3+. Alveolar macrophages were assigned a score for intensity of immunostaining for either 5-LO or FLAP based on the following criteria: 0 (no intensity), 1 (low intensity), 2 (medium intensity), and 3 (high intensity).

The data are reported as the mean ± SE of the combined experiments. Student’s two-tailed t test for independent means was employed to determine significant differences (p < 0.05).

Airway mucus hypersecretion and a marked leukocytic infiltration of the pulmonary interstitium were observed in mice sensitized and challenged with OVA compared with saline-treated controls (Fig. 1, B vs A). Eosinophils were the predominant cell in both the lung parenchymal infiltrates and the airway mucus plugs of the OVA-treated mice (Fig. 1, C and D).

FIGURE 1.

Airway inflammation in OVA-treated mice. Lung sections from saline-treated mice (A) and OVA-sensitized/challenged mice (B–D) were stained in Discombe’s solution and counterstained with methylene blue. A, In control animals, inflammatory cells are not present in the pulmonary tissue. Mucus release is not observed in the airway (AW) lumen. B, OVA-treated mice exhibit airway (AW) mucus hypersecretion and extensive infiltration of the pulmonary interstitium by inflammatory cells (arrows). C, Infiltration of the lung interstitium by eosinophils (arrowheads) is observed in OVA-sensitized/challenged mice. Numerous eosinophils (arrows) are located in the airway (AW) mucus plugs. D, In the lung interstitium, eosinophils (arrowheads) constitute the predominant cell of the intense leukocytic infiltrate in the OVA-treated mice. Magnification, ×200 (A and B) and ×400 (C and D).

FIGURE 1.

Airway inflammation in OVA-treated mice. Lung sections from saline-treated mice (A) and OVA-sensitized/challenged mice (B–D) were stained in Discombe’s solution and counterstained with methylene blue. A, In control animals, inflammatory cells are not present in the pulmonary tissue. Mucus release is not observed in the airway (AW) lumen. B, OVA-treated mice exhibit airway (AW) mucus hypersecretion and extensive infiltration of the pulmonary interstitium by inflammatory cells (arrows). C, Infiltration of the lung interstitium by eosinophils (arrowheads) is observed in OVA-sensitized/challenged mice. Numerous eosinophils (arrows) are located in the airway (AW) mucus plugs. D, In the lung interstitium, eosinophils (arrowheads) constitute the predominant cell of the intense leukocytic infiltrate in the OVA-treated mice. Magnification, ×200 (A and B) and ×400 (C and D).

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5-LO and FLAP mRNA expression were examined by RT-PCR of RNA extracted from lung tissue of OVA-treated mice. Only one band each of predicted size for 5-LO (332 bp) and FLAP (352 bp) was found in ethidium bromide-stained agarose gels (Fig. 2,A). Southern blot analysis confirmed specific binding of the 5-LO probe to the 332-bp product of the 5-LO cDNA and the FLAP probe to the 352-bp product of FLAP cDNA (Fig. 2,B). The expression of 5-LO and FLAP mRNA relative to HPRT increased 3.6-fold (p < 0.001, OVA vs saline) and 2.1-fold (p < 0.001, OVA vs saline), respectively, in OVA-sensitized/challenged mice compared with saline-treated control mice (Fig. 3,A). Expression of 5-LO and FLAP mRNA was also examined in the OVA-treated mouse lung tissue by in situ RT-PCR. Morphometric analysis indicated a 6.0-fold (p < 0.005, OVA vs saline) and 4.2-fold (p < 0.001) increase in total cells positive for 5-LO and FLAP mRNA expression, respectively, in lung tissue from OVA-treated mice compared with controls (Fig. 3 B).

FIGURE 2.

RT-PCR and Southern blot analysis of lung 5-LO and FLAP mRNA from OVA-treated mice. A, mRNA, after extraction from lung tissue of OVA-sensitized/challenged mice, underwent RT-PCR for detection of 5-LO and FLAP cDNA products. The amplified PCR products for the primer pairs specific for 5-LO (332 bp) and FLAP (352 bp) cDNA were visualized in ethidium bromide-stained 1.8% agarose gel. B, Southern blot analysis was performed as described in Materials and Methods. DIG-labeled 5-LO and FLAP probes were used for colorimetric detection of blotted cDNA.

FIGURE 2.

RT-PCR and Southern blot analysis of lung 5-LO and FLAP mRNA from OVA-treated mice. A, mRNA, after extraction from lung tissue of OVA-sensitized/challenged mice, underwent RT-PCR for detection of 5-LO and FLAP cDNA products. The amplified PCR products for the primer pairs specific for 5-LO (332 bp) and FLAP (352 bp) cDNA were visualized in ethidium bromide-stained 1.8% agarose gel. B, Southern blot analysis was performed as described in Materials and Methods. DIG-labeled 5-LO and FLAP probes were used for colorimetric detection of blotted cDNA.

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FIGURE 3.

5-LO inhibition blocks 5-LO and FLAP mRNA expression in lung tissue from OVA-treated mice. Lung tissue was obtained from saline-treated mice (saline) and OVA-sensitized/challenged mice in the absence (OVA) or presence of treatment with the 5-LO inhibitor zileuton (zileuton/OVA). A, RT-PCR was performed for detection of 5-LO and FLAP mRNA in the tissue. The ratio of 5-LO and FLAP transcripts relative to HPRT was calculated for each condition as described in Materials and Methods. B, The number of 5-LO or FLAP mRNA-positive inflammatory cells per unit area (2200 μm2 was determined in the airway interstitial tissue by morphometry; 10 lung sections per mouse were examined. The number of mice analyzed is indicated; probability values are shown for zileuton/OVA vs OVA.

FIGURE 3.

5-LO inhibition blocks 5-LO and FLAP mRNA expression in lung tissue from OVA-treated mice. Lung tissue was obtained from saline-treated mice (saline) and OVA-sensitized/challenged mice in the absence (OVA) or presence of treatment with the 5-LO inhibitor zileuton (zileuton/OVA). A, RT-PCR was performed for detection of 5-LO and FLAP mRNA in the tissue. The ratio of 5-LO and FLAP transcripts relative to HPRT was calculated for each condition as described in Materials and Methods. B, The number of 5-LO or FLAP mRNA-positive inflammatory cells per unit area (2200 μm2 was determined in the airway interstitial tissue by morphometry; 10 lung sections per mouse were examined. The number of mice analyzed is indicated; probability values are shown for zileuton/OVA vs OVA.

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As determined by RT-PCR of lung RNA (Fig. 3,A), the selective 5-LO inhibitor zileuton significantly inhibited both 5-LO gene expression (p = 0.01, zileuton/OVA vs OVA) and FLAP (p = 0.01, zileuton/OVA vs OVA) in OVA-sensitized/challenged mice. By morphometric analysis (Fig. 3 B), zileuton significantly reduced the number of 5-LO and FLAP mRNA-positive cells in lung tissue of OVA-treated mice by 2.5-fold (p = 0.01, zileuton/OVA vs OVA) and 2.4-fold (p = 0.01, zileuton/OVA vs OVA), respectively.

In OVA-sensitized/challenged mice, FLAP mRNA expression localized to endothelial cells of blood vessels and inflammatory cells in the lung interstitium surrounding the airways (Fig. 4,A). FLAP mRNA expression was not seen in pulmonary endothelial cells from saline-treated control mice (Fig. 4,B); monocyte-macrophages in the control lung tissue exhibited weak FLAP mRNA expression (Fig. 4,B). Compared with the OVA-treated group, a marked decrease in lung FLAP mRNA expression was seen in OVA-sensitized/challenged mice treated with the 5-LO inhibitor zileuton (Fig. 4,C). Distribution of 5-LO (Fig. 5, A and B) mRNA expression by in situ PCR was similar to that of FLAP (Fig. 4,A) in the lung tissue from the OVA-sensitized/challenged mice with 5-LO mRNA seen in leukocytes infiltrating the airways and pulmonary endothelial cells. By immunocytochemistry, 5-LO (Fig. 6,A) and FLAP (Fig. 6,B) protein localized to the pulmonary blood vessel endothelial cells and leukocytes in the interstitium and blood vessels in the OVA-treated mice. The proportion of FLAP-positive cells that were monocyte-macrophages or eosinophils in the lung interstitium of the saline, OVA, and zileuton/OVA groups were as follows: 99.4% monocyte-macrophages/0.6% eosinophils (saline group, n = 10), 60.3% monocyte-macrophages/39.7% eosinophils (OVA group, p < 0.0001, OVA vs saline for both monocyte-macrophages and eosinophils), n = 10), and 74.9% monocyte-macrophages/25.1% eosinophils (zileuton/OVA vs OVA for both monocyte-macrophages and eosinophils). On a per cell basis, FLAP (Fig. 7, A vs C; and Fig. 8) and 5-LO (Fig. 8) protein expression were greatly increased in the alveolar macrophages in the OVA-treated mice compared with the saline controls. Heterogeneity in the intensity of immunostaining of the leukocytes in the OVA-treated mice was seen for FLAP (Fig. 7 A) and 5-LO (not shown).

FIGURE 4.

5-LO inhibition decreases FLAP expression in lungs of OVA-treated mice. In situ RT-PCR was performed for detection of FLAP mRNA in lung tissue from OVA-sensitized/challenged mice in the absence (A) or presence of zileuton (C) and saline-treated mice (B). A, In the OVA-sensitized/challenged mice, inflammatory cells (arrows) with FLAP mRNA expression are distributed in the lung interstitium around the airways (AW). The blood vessel (BV) endothelial cells (arrowheads) are positive for FLAP mRNA. B, In control mice, as seen by the lack of DIG reaction products, FLAP mRNA expression is not observed in the blood vessel (BV) endothelial cells. Monocyte-macrophages (arrows) in the lung interstitium of the saline-treated mice have less intense reaction products for FLAP than in the OVA-treated mice. C, The 5-LO inhibitor zileuton markedly decreased FLAP mRNA expression (arrows) in the airway (AW) interstitium and blood vessel (BV) endothelial cells in the OVA-treated mice. Magnification, ×160.

FIGURE 4.

5-LO inhibition decreases FLAP expression in lungs of OVA-treated mice. In situ RT-PCR was performed for detection of FLAP mRNA in lung tissue from OVA-sensitized/challenged mice in the absence (A) or presence of zileuton (C) and saline-treated mice (B). A, In the OVA-sensitized/challenged mice, inflammatory cells (arrows) with FLAP mRNA expression are distributed in the lung interstitium around the airways (AW). The blood vessel (BV) endothelial cells (arrowheads) are positive for FLAP mRNA. B, In control mice, as seen by the lack of DIG reaction products, FLAP mRNA expression is not observed in the blood vessel (BV) endothelial cells. Monocyte-macrophages (arrows) in the lung interstitium of the saline-treated mice have less intense reaction products for FLAP than in the OVA-treated mice. C, The 5-LO inhibitor zileuton markedly decreased FLAP mRNA expression (arrows) in the airway (AW) interstitium and blood vessel (BV) endothelial cells in the OVA-treated mice. Magnification, ×160.

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FIGURE 5.

Localization of 5-LO mRNA expression in lung tissue of OVA-treated mice by in situ RT-PCR. Lung tissue of OVA-sensitized/challenged mice underwent in situ RT-PCR for detection of 5-LO as described in Materials and Methods. A, Intense 5-LO mRNA expression is observed in inflammatory cells (arrows) surrounding the airways (AW) in the OVA-sensitized/challenged mouse lung. B, 5-LO mRNA expression (arrowheads) is noted in the endothelium of the pulmonary blood vessel (BV) adjacent to the airway (AW). Magnification, ×300.

FIGURE 5.

Localization of 5-LO mRNA expression in lung tissue of OVA-treated mice by in situ RT-PCR. Lung tissue of OVA-sensitized/challenged mice underwent in situ RT-PCR for detection of 5-LO as described in Materials and Methods. A, Intense 5-LO mRNA expression is observed in inflammatory cells (arrows) surrounding the airways (AW) in the OVA-sensitized/challenged mouse lung. B, 5-LO mRNA expression (arrowheads) is noted in the endothelium of the pulmonary blood vessel (BV) adjacent to the airway (AW). Magnification, ×300.

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FIGURE 6.

Expression of 5-LO and FLAP in endothelial cells of OVA-treated mice. Lung tissue from OVA-sensitized/challenged mice (A and B) and saline-treated mice (C and D) underwent immunocytochemistry for localization of 5-LO (A and C) and FLAP (B and D) protein as described in Materials and Methods. A, The pulmonary blood vessel (BV) endothelial cells stain positive for 5-LO (arrows) by immunocytochemistry. 5-LO-positive leukocytes are seen in the lung interstitium. Magnification, ×300. B, Localization of FLAP on the endothelial cell surface (arrows) is seen in the pulmonary blood vessels (BV) adjacent to the airways (AW) in the OVA-treated mice. Intravascular and lung tissue leukocytes are also positive for FLAP. Magnification, ×300. C, The blood vessel (BV) endothelial cells in the control lungs are negative for 5-LO immunostaining. Occasional monocyte-macrophages (arrows) in the lung interstitium around the airways (AW) are weakly positive for 5-LO. Magnification, ×160. D, Blood vessel (BV) endothelial cells also lack immunocytochemical reaction products for FLAP in the lungs of saline-treated mice. Few cells are seen in the alveoli (A) of the controls. Magnification, ×160.

FIGURE 6.

Expression of 5-LO and FLAP in endothelial cells of OVA-treated mice. Lung tissue from OVA-sensitized/challenged mice (A and B) and saline-treated mice (C and D) underwent immunocytochemistry for localization of 5-LO (A and C) and FLAP (B and D) protein as described in Materials and Methods. A, The pulmonary blood vessel (BV) endothelial cells stain positive for 5-LO (arrows) by immunocytochemistry. 5-LO-positive leukocytes are seen in the lung interstitium. Magnification, ×300. B, Localization of FLAP on the endothelial cell surface (arrows) is seen in the pulmonary blood vessels (BV) adjacent to the airways (AW) in the OVA-treated mice. Intravascular and lung tissue leukocytes are also positive for FLAP. Magnification, ×300. C, The blood vessel (BV) endothelial cells in the control lungs are negative for 5-LO immunostaining. Occasional monocyte-macrophages (arrows) in the lung interstitium around the airways (AW) are weakly positive for 5-LO. Magnification, ×160. D, Blood vessel (BV) endothelial cells also lack immunocytochemical reaction products for FLAP in the lungs of saline-treated mice. Few cells are seen in the alveoli (A) of the controls. Magnification, ×160.

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FIGURE 7.

Increased FLAP protein in alveolar macrophages in OVA-treated mice. Lung sections were obtained from OVA-sensitized/challenged mice in the absence (A and D) or presence (B) of zileuton, and saline-treated controls (C). Rabbit polyclonal Ab H4 TB4 against FLAP was used to detect FLAP in A–C by immunocytochemistry as described in Materials and Methods. D, Nonimmunized rabbit IgG was used as a control instead of anti-FLAP Ab. Methylene blue nuclear counterstaining of sections was performed. A, Immunostaining for FLAP protein is seen in alveolar macrophages (arrows) and eosinophils (arrowhead) within the alveoli (A) of the OVA-treated mice. Heterogeneity of the intensity of the FLAP staining is observed in the inflammatory cells. B, Alveolar macrophage (arrow) and eosinophil (arrowhead) staining for FLAP is reduced by zileuton in the OVA-sensitized/challenged mice. C, Little FLAP expression is observed in alveolar macrophages (arrows) in lungs of saline-treated mice. D, When nonimmunized rabbit IgG was employed as a control, the alveolar macrophages do not exhibit immunostaining for FLAP. Magnification, ×850.

FIGURE 7.

Increased FLAP protein in alveolar macrophages in OVA-treated mice. Lung sections were obtained from OVA-sensitized/challenged mice in the absence (A and D) or presence (B) of zileuton, and saline-treated controls (C). Rabbit polyclonal Ab H4 TB4 against FLAP was used to detect FLAP in A–C by immunocytochemistry as described in Materials and Methods. D, Nonimmunized rabbit IgG was used as a control instead of anti-FLAP Ab. Methylene blue nuclear counterstaining of sections was performed. A, Immunostaining for FLAP protein is seen in alveolar macrophages (arrows) and eosinophils (arrowhead) within the alveoli (A) of the OVA-treated mice. Heterogeneity of the intensity of the FLAP staining is observed in the inflammatory cells. B, Alveolar macrophage (arrow) and eosinophil (arrowhead) staining for FLAP is reduced by zileuton in the OVA-sensitized/challenged mice. C, Little FLAP expression is observed in alveolar macrophages (arrows) in lungs of saline-treated mice. D, When nonimmunized rabbit IgG was employed as a control, the alveolar macrophages do not exhibit immunostaining for FLAP. Magnification, ×850.

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FIGURE 8.

Effect of 5-LO inhibition on distribution of 5-LO and FLAP protein staining intensities in alveolar macrophages in OVA-treated mice. Lung tissue was obtained from (A) saline-treated mice (saline), and OVA-sensitized/challenged mice in the (B) absence (OVA) or (C) presence (zileuton/OVA) of zileuton. The percentage of alveolar macrophages expressing different staining intensities of 5-LO and FLAP protein by immunocytochemistry (0 to +++ staining intensity scale) is shown.

FIGURE 8.

Effect of 5-LO inhibition on distribution of 5-LO and FLAP protein staining intensities in alveolar macrophages in OVA-treated mice. Lung tissue was obtained from (A) saline-treated mice (saline), and OVA-sensitized/challenged mice in the (B) absence (OVA) or (C) presence (zileuton/OVA) of zileuton. The percentage of alveolar macrophages expressing different staining intensities of 5-LO and FLAP protein by immunocytochemistry (0 to +++ staining intensity scale) is shown.

Close modal

5-LO inhibition by zileuton reduced FLAP (Fig. 7, B vs A; Fig. 8, C vs B; and Fig. 9) and 5-LO (Fig. 8, C vs B; and Fig. 9) protein expression in the alveolar macrophages. Although 73.1% and 64.5% of the alveolar macrophages stained ++ or greater for 5-LO and FLAP protein by immunocytochemistry, respectively, in the lungs of OVA-treated mice (Fig. 8,B), only 12.3% and 13.4% were ++ or greater for 5-LO and FLAP respected in the saline-treated controls (Fig. 8,A). The distribution of staining intensities of alveolar macrophages in OVA-sensitized/challenged mice treated with the 5-LO inhibitor zileuton (Fig. 8,C) was similar to that alveolar macrophages of the saline-treated controls (Fig. 8,A); a marked reduction in cells staining either ++ or +++ for 5-LO and FLAP was seen in the zileuton/OVA group compared with the OVA group (Fig. 8, C vs B). An averaged staining intensity score (0–3 scale) for the alveolar macrophages in the lungs was determined for each study group (Fig. 9). The intensity of immunostaining for 5-LO and FLAP of the alveolar macrophages increased 3.5-fold (p < 0.0001, OVA vs saline) and 2.7-fold (p < 0.0001, OVA vs saline), respectively, in the OVA-sensitized/challenged mice compared with saline-treated controls. The 5-LO inhibitor zileuton significantly reduced alveolar macrophage protein expression of 5-LO (p < 0.0001, zileuton/OVA vs OVA) and FLAP (p < 0.0001, zileuton/OVA vs OVA) in the OVA-sensitized/challenged mice (Fig. 9).

FIGURE 9.

5-LO inhibition reduces 5-LO and FLAP staining intensities in alveolar macrophages in OVA-treated mice. As described in Materials and Methods, alveolar macrophages were assigned an immunostaining intensity score (0–3) for 5-LO and FLAP. An averaged staining intensity score was calculated for the total number of alveolar macrophages counted in the lung preparations from saline-treated mice (saline) and OVA-sensitized/challenged mice in the absence (OVA) or presence (zileuton/OVA) of zileuton treatment (n = 1016 and 758 alveolar macrophages counted, respectively, for assessment of 5-LO and FLAP staining intensity). Probability values are shown for Zileuton/OVA vs OVA.

FIGURE 9.

5-LO inhibition reduces 5-LO and FLAP staining intensities in alveolar macrophages in OVA-treated mice. As described in Materials and Methods, alveolar macrophages were assigned an immunostaining intensity score (0–3) for 5-LO and FLAP. An averaged staining intensity score was calculated for the total number of alveolar macrophages counted in the lung preparations from saline-treated mice (saline) and OVA-sensitized/challenged mice in the absence (OVA) or presence (zileuton/OVA) of zileuton treatment (n = 1016 and 758 alveolar macrophages counted, respectively, for assessment of 5-LO and FLAP staining intensity). Probability values are shown for Zileuton/OVA vs OVA.

Close modal

By in situ RT-PCR and immunocytochemistry, we examined the expression and cellular distribution of 5-LO and FLAP mRNA and protein in lung tissue of mice after allergen challenge. We found a marked increase in 5-LO and FLAP-positive cells in the allergic lung tissue. 5-LO and FLAP gene expression was found in interstitial and alveolar inflammatory cells (i.e., eosinophils and alveolar macrophages), and pulmonary blood vessel endothelial cells. This influx of 5-LO and FLAP-positive inflammatory cells accounts in part for the marked increase in 5-LO and FLAP gene expression in the mouse lungs after allergen challenge. The data also suggest that FLAP and 5-LO gene expression increased on an individual cell basis in the leukocytes of the OVA-treated mice.

Increased expression of 5-LO and FLAP in lung inflammatory cells in the allergen-immunized/challenged mice may result from cytokines released in the allergic airways. In this murine asthma model, there is increased expression of Th cell type 2 (Th2) cytokines (IL-4, IL-5, IL-13) in bronchial lymph nodes (10) and in BAL fluid (17). Allergen challenge induces FLAP mRNA expression in human peripheral blood leukocytes (25). Activated lymphocytes increase 5-LO and FLAP protein expression in the monocyte-like cell THP-1 (14) and human blood monocytes (26) in vitro, effects which are reproduced by IL-3 or GM-CSF. Similarly, GM-CSF increases protein levels of both 5-LO (27) and FLAP (28) in human blood neutrophils, and IL-5 increases expression of FLAP in human blood eosinophils (29). The expression of 5-LO and FLAP in blood monocytes is also increased by alveolar lining fluid, suggesting a possible mechanism for augmented 5-LO synthetic capacity of the 5-LO pathway in alveolar macrophages (30).

Augmentation of the 5-LO pathway in lung cells in the OVA-treated mice may lead to prolonged release of leukotrienes in the airways of these animals. We have previously found 3.4- and 5-fold increases in levels of LTB4 and LTC4, respectively, 24 h after the third OVA challenge of OVA-immunized in this murine asthma model (9). We report here that the selective 5-LO inhibitor zileuton blocked the gene expression of 5-LO and FLAP significantly in the lungs of OVA-sensitized/challenged mice. The decreased expression of 5-LO and FLAP mRNA and protein in lung tissue from 5-LO inhibited mice is likely the result of a reduction in 1) the number of leukocytes infiltrating the lungs and 2) 5-LO and FLAP mRNA and protein expression in the infiltrating cells. Leukotrienes may be important in activation of some genes. For example, IL-2 gene expression and NF-κB by ligation of CD28 receptors on T cells requires 5-LO activation (31). In human monocytes, LTB4 stimulate c-fos and c-jun gene transcription and AP-1 binding activity (32). Thus, inhibition of leukotriene synthesis by the 5-LO inhibitor zileuton may decrease production of transcription factors important for 5-LO and FLAP gene induction.

Heterogeneity in FLAP and 5-LO protein expression by immunocytochemistry was observed in the inflammatory cells infiltrating the airways with some cells intensely stained and others nonreactive for FLAP and 5-LO in the OVA-treated mice. The lung interstitium is an intermediate stage for the maturation of blood monocytes to alveolar macrophages. In the mouse, ∼15% of the monocytes exiting the peripheral circulation become pulmonary macrophages trafficking first through the pulmonary interstitium before entering the alveolar spaces (33); 93% of these lung macrophages are alveolar macrophages, and 7% are tissue macrophages (33). An increase in leukotriene synthetic capability is observed in the maturational changes of blood monocytes to alveolar macrophages. In cells from normal human subjects, BAL fluid macrophages have >10-fold greater release of LTB4 than blood monocytes (34). In rats, as macrophages differentiate in the lungs from peripheral blood monocytes, there is an increase in 5-LO metabolism; interstitial macrophages are more similar to peripheral precursors in that they have less 5-LO synthetic capability than alveolar cells (35). When rat alveolar macrophages are separated on the basis of density, the least dense cells correspond to the most mature macrophage population and the most dense cells correspond to the youngest; LTB4 release increases with the degree of maturation and correlates with increased 5-LO protein content in the cells (36). This heterogeneity of alveolar macrophages has also been observed for proteins other than 5-LO synthetic pathway constituents. The largest alveolar macrophages in BAL fluid from rats have higher expression of Fc-receptor for IgG than smaller alveolar macrophages; the smaller cells are similar to blood monocytes, which have lower Fc-receptor activity (37). We found that FLAP and 5-LO protein expression was also heterogeneous in the inflammatory cells in the interstitial and alveolar compartments of the lungs.

By in situ RT-PCR, we also noted 5-LO and FLAP mRNA expression in blood vessel endothelial cells from OVA-treated mice but not saline-treated controls. Piper et al. (38) first noted release of leukotriene-like material from porcine pulmonary arteries and other blood vessels. Voelkel et al. (16) demonstrated immunocytochemical localization of 5-LO and FLAP protein in vascular endothelial cells from hypoxic rat lungs. Overexpression of 5-LO and FLAP in small and medium-sized arteries is also observed in lung tissue of patients with primary pulmonary hypertension (39). Activation of 5-LO and FLAP in pulmonary endothelium by allergen challenge indicates a potential important role of the lung vasculature in mediation of airway inflammation by release of 5-LO arachidonate products. For example, endothelial cell production of leukotrienes could amplify inflammatory cell trafficking from the peripheral blood to and around the pulmonary blood vessels. LTB4 stimulates leukocyte chemotaxis, chemokinesis, and adherence to vascular endothelium. LTB4-induced endothelial cell hyperadhesiveness for neutrophils is dependent on increased CD11/CD18 expression on the neutrophil surface and a specific domain of the adhesion molecule CD54 found on endothelial cells (40, 41). Cysteinyl leukotrienes are also involved in the adherence of leukocytes to vascular endothelium. LTC4/LTD4 induce translocation of P-selectin (GMP-140, PADGEM, CD62) to the luminal surface of endothelial cells from subcellular sites in Weibel-Palade bodies (42); LTE4 leads to an influx of eosinophils into the airway lamina propria (43).

In summary, allergen challenge in a murine model of asthma induces a marked increase in 5-LO and FLAP gene and protein expression in inflammatory cells infiltrating the airways and in pulmonary endothelial cells. Increased release of biologically potent 5-LO pathway arachidonic acid products as a consequence of this activation likely significantly contributes to the pathophysiology of the allergic pulmonary inflammatory response. These data indicate that leukotriene modifiers, recently introduced for the pharmacotherapy of asthma, may also exert important anti-inflammatory actions by inhibiting both the influx of 5-LO and FLAP positive inflammatory cells into the airways after allergen challenge and gene expression of 5-LO and FLAP in allergic lung inflammation.

We thank Gertrude K. S. Chiang, Mechthild Jonas, Falaah Jones, and Y.-Z. Tien for excellent technical assistance and Rachel Norris for typing the manuscript.

1

This work was supported by National Institutes of Health Grants Al42989, HL61756, and HL30542.

4

Abbreviations used in this paper: 5-LO, 5-lipoxygenase; alum, aluminum potassium sulfate; BAL, bronchoalveolar lavage; FLAP, 5-LO-activating protein; HPRT, hypoxanthine-guanine phosphoribosyltransferase; i.n. intranasal; DIG, digoxigenin; LT, leukotriene.

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