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Nonselective cyclooxygenase (COX) inhibition during allergic sensitization with ovalbumin in a murine model leads to an increase in the Type 2 cytokines interleukin-5 (IL-5) and IL-13; however, the effect of selective COX-1 and COX-2 inhibitors on these cytokines is unknown. We found that COX-1 protein was constitutively expressed in lung tissue. Expression of COX-1 protein did not increase with ovalbumin sensitization, but expression of COX-2 protein did. Ovalbumin-sensitized mice treated with either selective COX-1 inhibitor SC58560 (OVA-COX-1 inhibitor) or selective COX-2 inhibitor SC58236 (OVA-COX-2 inhibitor) had significantly greater airway hyperresponsiveness (p < 0.05) and higher levels of IL-13 (p < 0.05) in lung supernatants than did untreated mice that were ovalbumin sensitized (OVA). Lung mRNA levels for the chemokine receptors CCR1 through CCR5 (expressed on eosinophils, basophils, lymphocytes, and dendritic cells) were increased in the OVA-COX-2 inhibitor and OVA-indomethacin groups. We conclude that in the BALB/c mouse, COX inhibition with either a COX-1 or COX-2 inhibitor during allergen sensitization augments production of IL-13 and increases airway hyperresponsiveness.
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Keywords: airway hyperresponsiveness; cyclooxygenase; IL-13; ovalbumin
There is an augmented Type 2 inflammation and allergen-induced airway hyperresponsiveness (AHR) when mice are treated with indomethacin, a nonselective cyclooxygenase (COX) inhibitor, during allergic sensitization (1). This amplified Type 2 inflammation is characterized by an increase in lung interleukin-5 (IL-5) and IL-13, as well as lung interstitial eosinophilia (1). There are two COX enzyme isoforms, COX-1 and COX-2, that catalyze the committed step in the synthesis of prostaglandins and thromboxane (2). Allergen-induced pulmonary inflammation and AHR have been investigated in COX-1- and COX-2-deficient mice (3). After ovalbumin sensitization, allergen-induced lung inflammation defined by histopathology, serum IgE, and bronchoalveolar lavage (BAL) cells was significantly increased in COX-1-deficient mice compared with either wild-type or COX-2-deficient mice. Only allergically sensitized COX-1-deficient mice had increased baseline resistance and responsiveness to methacholine (3). However, the effects of acute inhibition of COX-1 or COX-2 only during allergen sensitization are unknown.
COX-1 and COX-2 products are crucial factors in the development of T lymphocytes, but the isoenzymes differ in
their contribution to lymphocyte development (4). COX-1 is
widely expressed in lymphoid cells in the embryonic thymus
and COX-2 is predominant in a subset of medullary stromal
cells in 3- to 5-week-old mice (5). COX-1 critically affects the
transition from CD4
CD8 cells to CD4+CD8+ cells. On the
other hand, COX-2 has an important role in early thymocyte
proliferation and differentiation and subsequent maturation of the CD4+ T lymphocyte lineage (4). Therefore, the effect of acute COX inhibition with pharmacologic inhibitors of these enzymes during allergic sensitization may be different from
that seen in the COX-1- and COX-2-deficient mice that lack
these enzymes throughout the course of embryonic development. We therefore treated groups of mice with either a selective COX-1 inhibitor (SC58560), a selective COX-2 inhibitor
(SC58236), indomethacin, or vehicle during allergic sensitization
to determine the effect of acute COX inhibition on the development of the allergic phenotype. The selectivity of the COX-1
(SC58560) and COX-2 (SC58236) inhibitors has been previously established (6-8).
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Mice
Pathogen-free 8-week-old female BALB/c mice were purchased from Harlan (St. Louis, MO). In caring for animals, the investigators adhered to the Guide for the Care and Use of Laboratory Animals prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council (revised 1996).
Allergen Sensitization Protocol
Mice were injected intraperitoneally with 0.1 ml (10 µg) of ovalbumin (chicken OVA, Grade V; Sigma, St. Louis, MO) complexed with 20 mg of Al(OH)3 on Day 0 (Figure 1). On Days 14 through 21, the mice were placed in an acrylic box and exposed to aerosols of 1% ovalbumin diluted in sterile phosphate-buffered saline, using an ultrasonic nebulizer (Ultraneb 99; DeVilbiss, Somerset, PA) for 40 minutes each day. Methacholine challenges were performed on Day 22.
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COX Inhibitor Administration
Indomethacin (30 µg/ml) was administered in the drinking water
starting on Day 
2. An indomethacin stock was made by dissolving 150 mg of indomethacin in 50 ml of ethanol. Three times per week throughout the experimental protocol, 2 ml of the indomethacin stock
solution was added to 200 ml of water in the animals' water bottles.
The water of the control mice was also changed three times per week
and 2 ml of ethanol was added to 200 ml of water in the water bottles
of those mice. The COX-1 inhibitor (SC58560) and the COX-2 inhibitor (SC58236) were also started in the drinking water on Day 2.
Stock solutions were made of each agent by adding 150 mg of the
compound to 47.5 ml of polyethylene glycol 200 and 2.5 ml of Tween
20. The final dilution of the COX-1 inhibitor was made by adding 2 ml
of the stock solution to 200 ml of drinking water to give a final concentration of 30 µg/ml. The final dilution of the COX-2 inhibitor was
made by adding 0.4 ml of the stock solution to 200 ml of drinking water to give a final concentration of 6 µg/ml. The water of the OVA-
COX-1 inhibitor and OVA-COX-2 inhibitor mice was also changed weekly.
Western Blot
Lung tissue homogenates were prepared in 20 mM Tris-HCl (pH 8.0)-1 mM EGTA-1 mM EDTA with a protease inhibitor cocktail tablet (Roche Molecular Biochemicals, Mannheim, Germany). After a 10-minute centrifugation at 10,000 × g, the supernatant was centrifuged for 60 minutes at 100,000 × g to prepare the microsome. The microsomes were suspended in homogenizing buffer, mixed with an equal volume of 2× sodium dodecyl sulfate sample buffer, and boiled for 5 minutes. The extracted proteins were separated on a 10% sodium dodecyl sulfate gel under reducing conditions and transferred to Immobilon-P transfer membranes (Millipore, Bedford, MA). The membranes were blocked overnight with 20 mM Tris-HCl (pH 7.4)-500 mM NaCl-5% nonfat milk-0.05% Tween 20 and incubated for 3 hours at room temperature with either rabbit polyclonal antiserum against murine COX-2 (Cayman Chemicals, Ann Arbor, MI) or goat polyclonal IgG against murine COX-1 (Santa Cruz Biotechnology, Santa Cruz, CA). The primary antibodies were detected with goat anti-rabbit IgG-horseradish peroxidase for COX-2 (Santa Cruz Biotechnology) and donkey anti-goat IgG-horseradish peroxidase for COX-1 and exposed on film by using enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ).
Measurement of Arachidonic Acid Metabolites
Arachidonic acid metabolites were measured in BAL fluid harvested on Day 22. Prostaglandin E2 (PGE2) and thromboxane were measured by a modified stable isotope dilution assay that used gas chromatography-negative ion chemical ionization-mass spectrometry as previously described (9). Leukotriene C4 (LTC4), LTD4, and LTE4 were collectively quantified (LTC4/LTD4/LTE4) by enzyme immunoassay, employing a peptidoleukotriene polyclonal antiserum (Amersham Pharmacia Biotech).
Cytokine and Chemokine Detection by RNase Protection Assay
Total lung RNA was isolated with guanidine thiocyanate. Probes for a
panel of cytokines (MCK-1; PharMingen, San Diego, CA) and chemokine receptors (macrophage chemotactic protein-1; PharMingen) were used according to the manufacturer's instructions. Briefly, RNA
was dissolved in 80% formamide-0.4 M NaCl-1 mM EDTA-40 mM piperazine-N,N'-bis(2-ethanesulfonic acid), heated to 90° C for 5 minutes, and hybridized for 12-16 hours with corresponding [
-32P]UTP-
labeled antisense probes at 56° C. The unhybridized RNA was digested
with 100 µl of RNase T1+A (250 U/µl; PharMingen) and 100 µl of
RNase A (80 ng/ml; PharMingen) for 45 minutes at 30° C. After phenol-chloroform extraction and ammonium acetate-ethanol precipitation, the protected hybridized RNA was denatured and electrophoresed on a 5% polyacrylamide gel. The gel was dried and exposed to film.
Quantitation of IL-4, IL-5, IL-13, and IL-6 in Lung Tissues
Levels of IL-4, IL-5, IL-13, and IL-6 in lung tissues of the four groups of mice were measured with commercially available enzyme-linked immunosorbent assay kits (IL-4, IL-5, and IL-6 from Endogen [Woburn, MA] and IL-13 from R&D Systems [Minneapolis, MN]) according to the manufacturer's protocols. On Days 16, 18, 20, and 22 the lungs from four mice in each group were analyzed for cytokine levels. Briefly, one lung from each mouse was ground, using a mortar and pestle and ground glass. The suspension of ground lung and ground glass was then centrifuged at 800 × g for 10 minutes. The supernatant was then either frozen for later use or added to precoated wells, and incubated for 2 hours. Dilutions of recombinant cytokine were included for generation of a standard curve. Peroxidase-labeled anti-cytokine antibody was added to detect bound cytokine, and the plates were developed by the addition of tetramethylbenzidene substrate. Concentrations of cytokines in the lung supernatants were calculated from the standard curve produced. The cytokine level from each lung was measured in duplicate.
Methacholine Challenge
Mice were anesthetized with intraperitoneal injections of pentobarbital sodium (85 mg/kg) and a tracheostomy tube was put in place. The internal jugular vein was cannulated and a microsyringe was attached to intravenous tubing for methacholine administration. The mice were then placed in a whole body plethysmography chamber and mechanically ventilated (10, 11). Methacholine dose-response curves were obtained by calculating the mean ± standard error for individual animals at each methacholine dose.
Protocol for Examining Lung Sections
The mice were killed by cervical dislocation on Day 22 and the lung block was removed. The lung tissue was stored in 4% paraformaldehyde, paraffin embedded, cut in 6-µm sections, mounted, and stained with hematoxylin and eosin for routine histology, with periodic acid- Schiff reagent to assess mucus, and with Wright-Giemsa. Four animals per treatment group were examined. Morphometric analysis was performed in the following fashion: (1) Representative areas of the right and left lung from each mouse were photographed separately at a magnification of ×40 with a 35-mm camera attached to an Olympus America (Melville, NY) BH-2 microscope; (2) the photograph was scanned into Adobe Photoshop (Adobe, San Jose, CA); (3) the images were adjusted to maximal contrast by setting the input level at 150; (4) the number of pixels was read from the histogram at both the blue and highlight levels; (5) the ratio of blue to highlight was calculated for each lung and the average of these two readings gave a value of lung infiltrate for each mouse; and (6) the results of all four mice in each treatment group were averaged to give a value of infiltrate for each group.
In addition, under oil immersion, the number of lymphocytes, eosinophils, and plasma cells within the bronchial walls (five consecutive high-power fields) and in the peribronchial vessel walls (five consecutive high-power fields) were counted. These morphometric and cell quantitations were each performed by two experienced pathologists who were blinded to the treatment groups.
Statistical Analysis
Results are expressed as means ± standard error of the mean. Dose- response curves to methacholine were compared by repeated measures analysis of variance with Fisher's least significant difference performed as a post hoc analysis. Measurements of PGE2, cytokines (by enzyme-linked immunosorbent assay), chemokines (by RNase protection assay), and AHR were analyzed by analysis of variance with Fisher's least significant difference performed as a post hoc analysis. Differences were considered to be significant if p < 0.05.
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COX-2 Protein Is Increased in the Lungs of Ovalbumin-sensitized Mice
To determine the protein expression of COX-1 and COX-2 in the lungs of allergically sensitized mice, a time course experiment was performed. The lungs from mice were harvested on Days 14 (immediately before the first exposure to aerosolized ovalbumin), 16, 18, and 22, and Western blot analysis for COX-1 and COX-2 was performed (Figure 2). COX-1 was constitutively expressed, with the same protein levels before ovalbumin aerosol (Day 14) as at the three time points measured after ovalbumin aerosols were initiated (Days 16, 18, and 22). In contrast, expression of COX-2 was induced when the mice were subjected to treatments with aerosolized ovalbumin. There was an increase in COX-2 expression after the initiation of ovalbumin aerosol that continued through the period of aerosol treatments.
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COX-2 Inhibitor Has Selective COX-2 Inhibitory Activity
To confirm the selectivity of our COX-2 inhibitor, we measured serum thromboxane from our four groups of mice after incubating the serum with ionophore to maximally stimulate platelet thromboxane production. The specific COX-2 inhibitor did not decrease plasma thromboxane levels after stimulation with ionophore, whereas the COX-1 inhibitor and indomethacin did significantly inhibit thromboxane generation (data not shown).
COX-1 and COX-2 Inhibitor Administration during Allergen Sensitization Decreases PGE2 Levels in BAL Fluid and Nonsignificantly Increases BAL Fluid Levels of LTC4/LTD4/LTE4
The PGE2 level measured in the BAL fluid on Day 22 was 175 ± 16 pg/ml in the OVA group, 46 ± 11 pg/ml in the OVA-COX-1 inhibitor group, 27 ± 5 pg/ml in the OVA-COX-2 inhibitor group, below the limit of assay detection (4 pg/ml) in the OVA- indomethacin group, and 6 ± 2 pg/ml in the nonsensitized group (Figure 3). The PGE2 in the BAL fluid of the OVA group was significantly greater than in any other group. There was no significant difference in the PGE2 levels in the BAL fluid between the OVA-COX-1 inhibitor group and the OVA-COX-2 inhibitor group. The OVA-indomethacin group had significantly lower BAL fluid PGE2 levels compared with the OVA-COX-1 inhibitor group and there was a trend for lower BAL PGE2 levels in the OVA-indomethacin group compared with the OVA- COX-2 inhibitor group. Thus, specific COX-1 and COX-2 inhibitors, as well as the nonspecific COX inhibitor indomethacin, all significantly decreased PGE2 levels in BAL when administered throughout the duration of the allergen sensitization protocol. The LTC4/LTD4/LTE4 level measured in the BAL fluid on Day 22 was 75 ± 12 pg/ml in the OVA group, and was nonsignificantly increased in the OVA-COX-1 inhibitor group (119 ± 12 pg/ml), OVA-COX-2 inhibitor group (101 ± 7 pg/ml), and OVA-indomethacin group (114 ± 21 pg/ml) (Figure 4).
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IL-13 Protein and Expression of mRNA for IL-5 Are Increased in the Lungs of OVA-sensitized Mice Treated with COX-1 and COX-2 Inhibitors and Indomethacin, whereas IL-4 Levels Are Not Different
Cytokines were measured in the ground lung supernatants on Days 16, 18, 20, and 22. There was no significant difference in IL-4 concentrations in the lung supernatants between the four groups at any of the four time points (data not shown). Indomethacin-treated mice had detectable levels of IL-5 in the lung supernatants on Day 16 (384 ± 23 pg/ml) and Day 18 (177 ± 26 pg/ml), whereas none of the other groups had detectable levels at any time point. The time course of IL-13 production in lung supernatants is shown in Figure 5. On Day 18, the OVA-sensitized mice that were treated with the COX-1 inhibitor, the COX-2 inhibitor, and indomethacin had significantly greater levels of IL-13 in lung supernatants compared with the untreated group. On Day 16, the OVA-indomethacin group had increased IL-13 compared with all groups. The levels of IL-13 in all four groups were significantly greater on Day 18 compared with any other day, despite the fact that exposure to OVA aerosol continued through Day 21. IL-6 was measured in the lung supernatants on all 4 days, but the concentrations were not above the detection level of the assay at any time point.
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On the basis of the findings that peak IL-5 production was found on Day 16 in the OVA-indomethacin group, we performed an RNase protection assay to determine whether there were differences between the four groups in the expression of cytokine lung mRNA on that day. The results are shown in Figures 6A and 6B. There was a significant increase in IL-5 and IL-13 mRNA in the OVA-COX-1 inhibitor group, OVA-COX-2 inhibitor group, and OVA-indomethacin group compared with the untreated OVA group, with the OVA- COX-2 inhibitor group and OVA-indomethacin groups showing greater expression of these cytokines than the OVA- COX-1 inhibitor group. There was no significant difference between the groups in terms of the lung mRNA for any of the other cytokines measured.
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COX-1 and COX-2 Inhibitors Increase Chemokine Receptor Expression mRNA in OVA-sensitized Mice
RNase protection assays were performed on the lungs of mice harvested on Day 16 of the protocol. There was a significant increase in the expression of CCR1, CCR2, CCR3, CCR4, and CCR5 in the OVA-COX-2 inhibitor and OVA-indomethacin groups compared with the OVA-COX-1 inhibitor and OVA groups (p < 0.05) as shown in Figures 7A and 7B. There was a trend toward an increase in the expression of CCR1 and CCR5 in the OVA-COX-1 inhibitor group compared with the OVA group on Day 16 (p = 0.08 and p = 0.1, respectively). Thus, COX inhibition with a COX-2 inhibitor upregulated the expression of chemokine receptors important in the chemotaxis of inflammatory cells in the OVA challenge model.
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COX-1 and COX-2 Inhibition Does Not Change Allergen-induced Lung Pathology in the Lung
By both morphometric analysis and quantitation of lymphocytes, eosinophils, and plasma cells, there were no significant differences in the degree of inflammation of the lungs of the four groups by histopathologic analysis (data not shown). A large amount of mucus was present in the airways of all four groups.
COX-1 and COX-2 Inhibitors Increase Allergen-induced AHR
Methacholine challenge was performed on Day 22, 1 day after the last OVA aerosol. The AHR was significantly greater in the OVA-COX-1 inhibitor, OVA-COX-2 inhibitor, and OVA- indomethacin groups compared with the OVA group (Figure 8). At the highest dose of methacholine (411 µg/kg), the OVA- COX-1 inhibitor group had a lung resistance of 12.6 ± 1.2 cm H2O/ml per second, the OVA-COX-2 inhibitor group had a lung resistance of 14.3 ± 3.9 cm H2O/ml per second, and the OVA-indomethacin mice had a lung resistance of 14.7 ± 1.8 cm H2O/ml per second, whereas the lung resistance of the OVA mice was 5.8 ± 0.6 cm H2O/ml per second. Thus, COX inhibition with selective COX inhibitors increased AHR in the OVA challenge model.
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COX-1 and COX-2 (also known as prostaglandin endoperoxidase H synthases-1 and -2, respectively) are the critical enzymes in the conversion of arachidonic acid to the prostanoids
(2, 12, 13). COX-1 is constitutively expressed at high levels in
endothelium, monocytes, platelets, renal collecting tubules,
and seminal vesicles. In contrast, COX-2 expression is induced, rather than expressed constitutively. COX-2 expression is increased by growth factors and inflammatory mediators such as IL-1, tumor necrosis factor-, lipopolysaccharide,
and 12-O-tetradecanoylphorbol 13-acetate, whereas COX-2
expression can be suppressed by glucocorticoids. COX-2 has
important functions in reproduction, immunity, renal physiology, neurotransmission, bone reabsorption, and pancreatic secretion. COX-2 has been identified in epithelial cells, macrophages, fibroblasts, smooth muscle cells, and mast cells. COX-1
and COX-2 are structurally distinct enzymes that both enzymatically convert arachidonic acid into PGG2, and then reduce PGG2 to PGH2. PGH2 may then be converted by specific
synthases or isomerases to either PGD2, PGF2
, PGE2, PGI2,
or thromboxane. These products leave the cell via a carrier-mediated process and can activate G protein-linked prostanoid receptors (13).
Indomethacin is an inhibitor of both COX-1 and COX-2, and we have previously reported that its administration during allergic sensitization increased IL-5 and IL-13 production. Further, the increase in these Type 2 cytokines is associated with an increase in allergen-induced AHR (1). In the present article, we show that selective COX-1 and COX-2 inhibitors both increase IL-13 and AHR. One of the most interesting findings in this study is that the peak of IL-13 protein expression in the lung for all groups occurred on Day 18, despite the fact that the OVA aerosols were continued through Day 21. To our knowledge, this is the first report of the kinetics of IL-13 protein expression in the OVA system. Our results suggest that there is a mechanism by which cytokine production, and perhaps allergic inflammation, is downregulated to counteract an ongoing inflammatory stimulus such as allergen exposure.
Previously, we showed no difference between the OVA-
indomethacin and OVA groups in lung mRNA expression of
RANTES, eotaxin, and macrophage inflammatory protein-1,
chemokines thought to be responsible for eosinophil chemotaxis; however, lung expression of macrophage chemotactic
protein-1 was significantly greater in the OVA-indomethacin
group compared with the OVA mice. Macrophage chemotactic protein-1 is a member of the C-C chemokine family and
binds to the CCR2 receptor (14). CCR2 receptors are present on basophils, monocytes, activated T cells, dendritic cells, and natural killer cells, thus allowing for macrophage chemotactic protein-1-mediated biologic effects (14). In our previous
study, the increased interstitial eosinophilia was apparently
not due to increased chemokines currently believed to cause
eosinophil chemotaxis, as there was no increase in lung
mRNA expression of these chemokines (1). Alternatively,
lung eosinophilia might be due to increased expression of chemokine receptor expression from COX inhibition. Therefore,
we performed RNase protection assays on the lungs from
mice harvested on Day 16 of the protocol to examine the expression of the chemokine receptors. We found that the
OVA-COX-2 inhibitor and OVA-indomethacin mice had a
significant increase in the expression of CCR1, CCR2, CCR3,
CCR4, and CCR5 mRNA in the lung. Lymphocytes and dendritic cells express CCR1 through CCR5 (15). Basophils express CCR1 through CCR3, whereas eosinophils express CCR1
and CCR3. Macrophages express CCR1, CCR2, and CCR5
(15). Thus, upregulation of CCR-1 through CCR5 by COX-2 inhibition could affect many of the cell types involved in allergic inflammatory responses. By changing the expression of
chemokines, cytokines, and their receptors, COX inhibition
may influence the balance, composition, and regulation of the
immune response.
These COX-1 and COX-2 inhibitors have previously been shown to be selective for their COX enzymatic isoforms (6). We confirmed the selectivity of the COX-2 inhibitor by measuring plasma thromboxane after stimulation with ionophore and found that there was not a decrease in serum thromboxane levels in the COX-2 inhibitor-treated group, whereas there was a significant inhibition in the COX-1 inhibitor- and indomethacin-treated mice. Our results differ from those of Gavett and colleagues, who studied allergically sensitized mice lacking either COX-1 or COX-2 (3). In comparison with OVA-sensitized wild-type mice, they found a heightened degree of eosinophilic inflammation and higher levels of IgE in BAL fluid in the mice lacking COX-1 and COX-2. However, they found that AHR was increased only in the allergically sensitized mice lacking COX-1 and not in the group lacking COX-2 (3). We, on the other hand, found that AHR was increased in the OVA-indomethacin and OVA-COX-1 inhibitor group, but also in the OVA-COX-2 inhibitor group. This increase in AHR in the OVA-COX-2 inhibitor group compared with the OVA group may be related to the increase in IL-13 found in the lung supernatants of the OVA-COX-2 inhibitor group, as IL-13 has been identified as a central mediator of AHR (16, 17). The differences between our studies and those of Gavett and colleagues may be explained by the fact that their mice lacked the genes to produce either COX-1 or COX-2 during development, whereas we inhibited COX-1 and COX-2 only at the time of allergen sensitization. Thus, the lack of the COX isoforms during development may have had a profound effect on the development of T lymphocytes and possibly other cells in the immune system responsible for the development of the allergic response. Another key difference between our report and that by Gavett and coworkers is the strain of mice used. Gavett and colleagues used C57/B6 mice, whereas we used BALB/c mice (3). Substantial differences exist between strains of mice regarding their cytokine response to allergic sensitization and the development of AHR (18).
Our findings with the COX-1 and COX-2 inhibitors are
consistent with an inhibition of a prostanoid that may restrain
allergic inflammation. Studies suggest that PGD2 and thromboxane may be necessary for the production of the allergic response (19, 20). Mice in which the DP receptor is knocked out
have decreased allergic inflammation compared with wild-type control mice (19). CRTH2, a seven-transmembrane receptor preferentially expressed in Type 2 cells, eosinophils,
and basophils is also a receptor for PGD2 (21). In response to
PGD2, CRTH2 induced chemotaxis in Type 2 cells, and mediated PGD2-dependent blood eosinophil and basophil migration. Thus, PGD2 has significant proallergic inflammatory effects via signaling through either the DP or CRTH2 receptors. Thromboxane synthase inhibitors and thromboxane prostanoid
receptor antagonists used in a murine ovalbumin model of allergic airway inflammation are capable of inhibiting the production of Type 2 cytokines, in turn inhibiting airway eosinophilic
inflammation (20). Thus, PGD2 and thromboxane seem to be
necessary to generate allergic inflammation because inhibiting
the activity of these COX products downregulates the allergic
response. On the other hand, we have shown that COX inhibition actually heightens the allergic response (1). These studies
suggest that other COX products inhibited by COX-1 inhibitors, COX-2 inhibitors, and indomethacin, such as PGE2, may
have effects that downregulate allergic inflammation or that
promote Type 1 or interferon-
-mediated events. In vitro experiments suggest that PGE2 may augment allergic inflammation by inhibiting production of the Type 1-associated cytokines IL-2 and interferon- (22-25). In some reports, PGE2 increases IL-4 and IL-5 production in vitro in the presence of IL-2 (24), whereas others have not shown an increase in these cytokines (22, 23, 26, 27).
In human studies, PGE2 inhalation inhibits the pulmonary immediate- and late-phase responses to inhaled allergen (28, 29). Compared with vehicle inhalation, inhaled PGE2 also decreases the change in methacholine airway reactivity and reduces the number of eosinophils after inhaled allergen challenge (28). In addition, PGE2 blunts both exercise-induced and aspirin-induced bronchoconstriction in patients sensitive to these challenges (30, 31). Interestingly, although PGE2 has significant effects on pulmonary function in challenge models, it has no effect on baseline FEV1 or methacholine reactivity (29). The results from these studies suggest that PGE2 has a greater immunomodulatory effect than direct effect on airway caliber. This notion is supported by the fact that PGE2 inhalation before segmental allergen challenge significantly reduced the BAL levels of PGD2, an important product of mast cell degranulation, and the cysteinyl-leukotrienes (32).
The in vivo effect of PGE2 in the early development of the allergic disease is currently undefined. However, we have demonstrated that PGE2 can cause bronchodilation of murine airways, and it is possible that part of the increase in AHR seen after COX inhibition could be a result of the loss of PGE2-mediated bronchodilation (33). Our data suggest that a COX product (not PGD2 or thromboxane, as mentioned above) restrains the development of allergic disease. Other COX products, such as PGI2 or 15d-PGJ2, could be candidates for mediating the COX-dependent suppression of allergic inflammation and AHR. The discovery of which prostanoid product is exerting this restraining effect would be the first step in developing a therapy that might involve a novel immunomodulatory agent that downregulates allergic disease. Another possibility is that the increase in leukotrienes present in the BAL, although not statistically significant, could contribute to the augmented Type 2 cytokine production and AHR that is present with COX inhibitor treatment.
We do not believe that this model, in which COX inhibition leads to an augmentation of allergic inflammation and AHR, is a model of aspirin-sensitive asthma. Previously, we have shown that indomethacin-treated, nonsensitized mice had no difference in AHR compared with vehicle-treated, nonsensitized mice. If this were a model of aspirin-sensitive asthma, then we would have suspected that there would have been a difference in AHR between the indomethacin-treated and vehicle-treated nonsensitized mice. Our model asks a much more fundamental question: What is the role of COX products in the development of allergic disease, from its inception to the development of the full allergic phenotype? Although extrapolation of murine results to the human system is hazardous, our present results suggest that COX inhibitors may have deleterious effects on the chain of events that results in the development of allergic response. These results are particularly significant, as COX-2 inhibitors are currently extremely popular pharmacologic agents for the treatment of inflammatory joint disease and perhaps will soon be used for prophylaxis against colon cancer.
Our group has previously shown that oral administration of prednisone to patients with atopic asthma increased COX-2 mRNA and protein expression significantly in alveolar macrophages and blood monocytes, whereas ex vivo, the alveolar macrophages and blood monocytes from the same atopic donors had a decrease in stimulated COX-2 with glucocorticoids (34). This shows a discordance between the in vivo and in vitro models and suggests that one possible in vivo antiinflammatory action of glucocorticoids is to increase COX-2 expression, as this study has found that a COX-2 inhibitor increases allergic inflammation.
In summary, allergic inflammation in the lung increases the expression of COX-2 and has no significant effect on constitutive COX-1 expression. In addition, treatment of mice with selective COX-1 and COX-2 inhibitors during allergen exposure increases allergic inflammation and AHR.
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Correspondence and requests for reprints should be addressed to Stokes Peebles, M.D., Center for Lung Research, T-1217 MCN, Vanderbilt University Medical Center, Nashville, TN 37215. E-mail: Stokes.Peebles{at}mcmail.vanderbilt.edu
(Received in original form June 8, 2001 and accepted in revised form January 30, 2002).
Acknowledgments: Supported by the American Lung Association of Tennessee (K08-HL-03730); an American Academy of Allergy, Asthma, and Immunology ERT Award; and by GM 15431, DK 48831, DK 26657, CA 77839, and R01-AI-45512. J.D.M. is the recipient of a Burroughs Wellcome Clinical Scientist Award in Translational Research.
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References |
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METHODS
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REFERENCES
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1.
Peebles RS Jr,,
Dworski R,
Collins RD,
Jarzecka AK,
Mitchell DB,
Graham BS,
Sheller JR.
Cyclooxygenase inhibition increases interleukin 5 and interleukin 13 production and airway hyperresponsiveness in allergic mice.
Am J Respir Crit Care Med
2000;
162:
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