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Departments of
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Medicine and
Pathology, University of Washington, Seattle, WA 98195
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 x 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).
Animals
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).
Allergen immunization/challenge protocol
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.
Lung histology
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.
Immunocytochemistry
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).
RT-PCR for 5-LO and FLAP gene expression
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).
Southern blot analysis
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
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, 1x 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 1x 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 1x 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.
Morphometry
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 40x, 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).
Statistical analyses
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).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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).
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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. 2A). 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. 2B). 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. 3A). 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. 3B).
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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. 3B), 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.
Localization of 5-LO and FLAP mRNA and protein expression to lung inflammatory cells and endothelial cells
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. 4A). FLAP mRNA expression was
not seen in pulmonary endothelial cells from saline-treated control
mice (Fig. 4B); monocyte-macrophages in the control lung
tissue exhibited weak FLAP mRNA expression (Fig. 4B).
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. 4C). Distribution of 5-LO
(Fig. 5, A and B)
mRNA expression by in situ PCR was similar to that of FLAP (Fig. 4A) 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. 6A) and FLAP (Fig. 6B) 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. 7A) and 5-LO (not shown).
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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. 8B),
only 12.3% and 13.4% were ++ or greater for 5-LO and FLAP respected
in the saline-treated controls (Fig. 8A). The distribution
of staining intensities of alveolar macrophages in
OVA-sensitized/challenged mice treated with the 5-LO inhibitor zileuton
(Fig. 8C) was similar to that alveolar macrophages of the
saline-treated controls (Fig. 8A); 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).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-
B 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.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan.
3 Address correspondence and reprint requests to Dr. William R. Henderson, Jr., Department of Medicine, 1959 N.E. Pacific Street, Box 356523, University of Washington, Seattle, WA 98195-6523.
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.
Received for publication March 30, 2000. Accepted for publication July 19, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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on the 5-lipoxygenase pathway of rat lung macrophages. Am. J. Respir. Cell Mol. Biol. 6:93.
4 integrin) on intrapulmonary but not circulating leukocytes inhibits airway inflammation and hyperresponsiveness in a mouse model of asthma. J. Clin. Invest. 100:3083.[Medline]
B activation through CD28 requires reactive oxygen production by 5-lipoxygenase. EMBO J. 14:3731.[Medline]
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