Cyclooxygenase Inhibition Increases Interleukin 5 and Interleukin 13 Production and Airway Hyperresponsiveness in Allergic Mice

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1 Cyclooxygenase Inhibition Increases Interleukin 5 and Interleukin 13 Production and Airway Hyperresponsiveness in Allergic Mice R. STOKES PEEBLES, Jr., RYSZARD DWORSKI, ROBERT D. COLLINS, KASIA JARZECKA, DAPHNE B. MITCHELL, BARNEY S. GRAHAM, and JAMES R. SHELLER Departments of Medicine, Pathology, and Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee The immunomodulatory role of arachidonic acid metabolites in allergic sensitization is undefined. Prostaglandin E 2 (PGE 2 ), a product of arachidonic acid metabolism through the cyclooxygenase pathway, has been reported to favor Type 2-like cytokine secretion profiles in murine and human CD4 T cells by inhibiting the production of Type 1-associated cytokines. On the basis of these in vitro data, we hypothesized that indomethacin, a nonselective cyclooxygenase inhibitor, would diminish allergen-induced production of Type 2 cytokines in mice, and protect against airway hyperresponsiveness (AHR) to methacholine. We found that ovalbumin-sensitized mice that were treated with indomethacin (OVA indomethacin mice) had significantly greater AHR (p 0.05) and higher levels of IL-5 ( versus 66 4 pg/ml) and IL-13 (1, versus pg/ml) in lung supernatants than mice sensitized with ovalbumin alone (OVA mice), while levels of IL-4 and serum IgE were not different. Lung mrna expression of the C-C chemokine MCP-1 was increased in OVA indomethacin mice, while there was no difference between the two groups in lung mrna expression of eotaxin, MIP-1, MIP-1, or MIP-2. Histologic examination revealed greater pulmonary interstitial eosinophilia in OVA indomethacin mice as well. Contrary to our expectations, we conclude that in the BALB/c mouse, cyclooxygenase inhibition during allergen sensitization increases AHR, production of IL-5 and IL-13, and interstitial eosinophilia. Cyclooxygenase is the critical enzyme necessary for the formation of prostaglandins and thromboxane (1). Prostaglandin E 2 (PGE 2 ), one of the products of the cyclooxygenase pathway, may favor a Type 2-like cytokine secretion profile in murine and human CD4 T cells by inhibiting production of the Type 1-associated cytokines interleukin 2 (IL-2) and interferon (IFN- ) (2 5). In addition, in human monocytes PGE 2 inhibits production of IL-12, a potent inducer of T cell differentiation toward Type 1 cytokine production (6). PGE 2 in some reports does not increase T lymphocyte IL-4 production in vitro (2, 3, 7, 8), while others have shown that IL-4 and IL-5 are upregulated by PGE 2 in the presence of IL-2 (5). In summary, it is hypothesized that the upregulation of PGE 2 resulting from decreased aspirin use enhances allergic sensitization and asthma by amplifying the relative Type 2/Type 1 cytokine imbalance in genetically predisposed individuals (9). On the basis of these in vitro data, we hypothesized that mice in which cyclooxygenase activity was blocked by a nonsteroidal antiinflammatory drug (NSAID) during an allergen sensitization protocol would have a diminished production of the Type 2 cytokines IL-4, IL-5, and IL-13; decreased serum IgE production; and protection against allergen-induced airway hyperresponsiveness (AHR) to methacholine. To test this hypothesis, we used a well-characterized murine model of allergic sensitization and airway hyperresponsiveness, employing ovalbumin (OVA) as an antigen. To inhibit cyclooxygenase activity and prostaglandin synthesis, we treated the mice during the allergen sensitization protocol with indomethacin, a nonselective cyclooxygenase inhibitor. The dose of indomethacin used inhibits PGE 2 production and does not cause illness in experimental animals (10). Our results suggest that cyclooxygenase inhibition during allergen sensitization in mice actually augments induction of the Type 2 cytokines IL-5 and IL-13 and enhances allergen-induced AHR. METHODS Mice Pathogen-free, 8-wk-old female BALB/c mice were purchased from Harlan (St. Louis, MO). They were shipped in filtered crates and housed in a high-efficiency particulate air (HEPA)-filtered Duo-flo laminar flow unit. Cages, bedding, food, and water were sterilized before use. Room temperature was maintained between 24 and 27 C and a 12-h-on, 12-h-off light cycle was provided. 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 g) 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 22, the mice were placed in an acrylic box and exposed to aerosols of 1% ovalbumin diluted in sterile phosphate-buffered saline (PBS), using an ultrasonic nebulizer (Ultraneb 99; DeVilbiss, Somerset, PA), for 40 min each day. Opposite the aerosol orifice was a small exhaust orifice vented into a chemical hood to ensure continuous air flow. Nonsensitized mice were injected intraperitoneally with Al(OH) 3 on Day 0. Methacholine challenges were performed on Day 23. Indomethacin Administration Indomethacin (30 g/ml) was administered in the drinking water starting on Day 1. 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. (Received in original form November 15, 1999 and in revised form February 14, 2000) Supported by K08-HL-03730, GM 15431, and R01-AI Correspondence and requests for reprints should be addressed to James R. Sheller, M.D., Center for Lung Research, T-1217 MCN, Vanderbilt University Medical Center, Nashville, TN James.Sheller@mcmail.vanderbilt.edu Am J Respir Crit Care Med Vol 162. pp , 2000 Internet address: Measurement of Arachidonic Acid Metabolites Arachidonic acid metabolites were measured in bronchoalveolar lavage (BAL) fluid harvested on Day 23. PGE 2 was measured by a modified stable isotope dilution assay that used gas chromatographynegative ion chemical ionization-mass spectrometry as previously described (11). Leukotrienes C 4 /D 4 /E 4 (LTC 4 /D 4 /E 4 ) were quantified by enzyme immunoassay employing a peptido-leukotriene polyclonal

2 Peebles, Dworski, Collins, et al.: Cyclooxygenase Inhibition Increases IL-5, IL-13 Production and AHR 677 Total IgE Concentrations Before sacrifice on Day 23, sera were collected from sensitized mice; then analyzed by ELISA to determine levels of total IgE. To determine total IgE levels, 96-well Immunolon II plates (Nunc, Roskilde, Denmark) were coated with a 1:200 dilution of rat monoclonal antimurine IgE clone LO-ME-3 (Serotec, Oxford, UK). Plates were washed with PBS 0.5% Tween and blocked with PBS 1% bovine serum albumin for 1 h. The plates were then washed before adding 100 l of serum diluted 1:30 in PBS. Plates were incubated overnight and washed, and 100 l of rat anti-mouse IgE clone LO-ME-2 (Serotec) diluted 1:2,000 was added to each well. After 1 h of incubation at 37 C, the plates were again washed and horseradish peroxidase (HRP) activity was determined with a tetramethylbenzidine (TMB; Sigma) developing solution (1% TMB in dimethyl sulfoxide [DMSO], M sodium acetate, and 0.45% H 2 O 2 final concentration). Substrate development was stopped with 2.5 M H 2 SO 4 and optical density was measured at 450 nm (OD 450 ). Concentration was extrapolated by use of an IgE standard (Maine Biotech, Portland, ME). Figure 1. Time line of experimental protocol. antiserum (Cayman Chemical, Ann Arbor, MI) after prior purification on C 18 columns (Altech, Los Altos, CA). 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 chemokines (MCK-5; PharMingen) were used according to the manufacturer instructions. Briefly, RNA was dissolved in 80% formamide, 0.4 M NaCl, 1 mm EDTA, and 40 mm piperazine-n,n -bis(2-ethanesulfonic acid), heated to 90 C for 5 min, and hybridized for h with corresponding [ - 32 P]UTP-labeled antisense probes at 56 C. The unhybridized RNA was digested with 100 l of RNase T1 A (250 U/ l; PharMingen) and 100 l of RNase A (80 ng/ml; PharMingen) for 45 min 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 (ELISA) kits (IL-4, IL-5, and IL-6 [Endogen, Woburn, MA]; IL-13 [R&D Systems, Minneapolis, MN]) according to the manufacturer protocols. On Day 18, 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 solution of ground lung and ground glass was then centrifuged at 1,000 rpm for 10 min. The supernatant was then either frozen for later use or added to precoated wells, and incubated for 2 h. 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 placed. 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 (12). Transpulmonary pressure was measured as airway opening pressure referenced to pressure within the chamber. A four-way connector was attached to the tracheostomy tube. One port of the four-way connector was attached to the inspiratory side of a ventilator (rodent ventilator, model 683; Harvard, South Natick, MA) while another port was connected to the ventilator expiratory side port. Mice were ventilated at a rate of 200 breaths/min with a tidal volume of 5 6 ml/kg and a positive end-expiratory pressure of 2 cm H 2 O. Lung volume changes were measured by detecting pressure changes in the plethysmographic chamber (model MC ; Validyne, Northridge, CA). Flow was measured by differentiation of the volume signal. Pressure, flow, and volume changes were recorded. Lung resistance was continuously computed (LabVIEW Graphical Programming for Instruments; National Instruments, Austin, TX) by fitting flow, volume, and pressure to an equation of motion. Acetyl- -methacholine (Sigma) was dissolved in normal saline and administered intravenously at a starting dose of 5 g/kg. The average volume per methacholine dose was approximately 35 l. Threefold-increasing concentrations of methacholine were administered at 2-min intervals, and only after transpulmonary pressure and tidal volume returned to within 10% of baseline. Pulmonary variables were recorded for at least 10 breaths during the peak response, within 30 s after each intravenous methacholine dose. 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 23 and the lung block was removed. The lung tissue was stored in 4% paraformaldehyde, paraffin embedded, cut in 6- m sections, mounted, and stained with hematoxylin and eosin for routine histology, periodic acid Schiff (PAS) to assess mucus, and Luna stain to specifically evaluate eosinophils (13). Slides were examined by one observer in a blinded fashion as previously described (14). The following compartments of the lung were assessed: alveolar spaces, airways at all levels, interstitium, and vessels (both arteries and veins). Inflammatory infiltrates were evaluated for location, severity, and composition (cell types: small mononuclear cells, transformed lymphocytes, histiocytes, neutrophils, and eosinophils). The degrees of inflammation were graded as follows: 0, no infiltrate; 1, most vessels have an infiltrate up to four cells thick; 2, most vessels have an infiltrate five to seven cells thick; 3, most vessels have an infiltrate greater than seven cells thick. Interstitial alveolar cellularity was graded as follows: 0, no infiltrate; 1, minimal increased cellularity without widening of septa; 2, obvious increased cellularity with widening of septa; and 3, markedly increased cellularity with thickened septa; this score also includes blood or edema fluid in the tissue space. Statistical Analysis Results are expressed as means standard error of the mean (SEM). Dose response curves to methacholine were compared by repeated measures analysis of variance (ANOVA) with the Fisher least significant difference performed as a post hoc analysis. Measurements of PGE 2, LTC 4 /D 4 /E 4, cytokines by ELISA, chemokines by RNase protection assay (RPA), and total IgE were analyzed by ANOVA with the Fisher least significant difference performed as a post hoc analysis. Differences were considered to be significant if p 0.05.

3 678 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL Figure 2. The concentrations of LTC 4 /D 4 /E 4 and PGE 2 in BAL fluid harvested on Day 23 (n 4). The data shown are representative of three separate experiments. *p RESULTS Indomethacin Administration during Allergen Sensitization Decreases PGE 2 Levels and Increases Cysteinyl Leukotriene Levels in BAL Fluid The PGE 2 level measured in the BAL fluid on Day 18 was pg/ml in the OVA group and pg/ml in the OVA indomethacin group (p 0.05) as shown in Figure 2. In unsensitized mice not treated with indomethacin (NS), the PGE 2 level in the BAL fluid was pg/ml. PGE 2 was not measured in unsensitized mice treated with indomethacin (NS indomethacin). In contrast, the level of LTC 4 /D 4 /E 4 was 18 pg/ ml in the OVA indomethacin group and 3 pg/ml in the OVA group (n 4 in each group, results representative of three separate experiments). LTC 4 /D 4 /E 4 was not measured in the BAL fluid of unsensitized mice. Thus, indomethacin treatment during allergen sensitization decreased the concentration of PGE 2, yet increased the cysteinyl leukotrienes. IL-5 and IL-13 Are Increased in Indomethacin-treated OVA-sensitized Mice, while IL-4 and IL-6 Are Unchanged Cytokines were measured in the ground lung supernatants on Day 18, the fifth day of 8 d of ovalbumin aerosol treatment. We have previously found that cytokine levels peak on this day even though aerosol treatments continued for three more days (data not shown). Levels of IL-4, IL-5, or IL-13 were not detected in the lung supernatants of the NS and NS indomethacin groups. Indomethacin treatment had no significant effect on IL-4 protein levels in lung supernatants of the allergically sensitized mice (Figure 3). However, indomethacin treatment of ovalbumin-sensitized BALB/c mice significantly increased IL-5 Figure 3. The concentrations of IL-4, IL-5, IL-13, and IL-6 in the lung supernatants from mice on Day 18 (n 4). The data shown are representative of three separate experiments. *p ( versus 66 4 pg/ml) and IL-13 (1, versus pg/ml) protein levels in lung supernatants (p 0.05; n 4 in each group, results representative of three separate experiments). Thus, prostaglandin inhibition increased, rather than decreased, cytokines that are considered to be central in allergen-induced AHR. IL-6 was also measured in the ground lung supernatants on Day 18. There was a trend for the OVA indomethacin group to have a decrease in IL-6 (146 5 versus pg/ml) protein in lung supernatants (p 0.1; n 4). Levels of IFN- were undetectable in the ground lung supernatants from any of the groups. RNase protection assays (RPAs) were performed on the lungs from mice harvested on Day 18 of the protocol (Figures 4A and 4B). There were no differences in the levels of cytokine mrna in the lung tissue for IL-4 and IL-5 between the OVA indomethacin and OVA groups; however, the level of IL-13 mrna was significantly increased in the OVA indomethacin group. There was a trend for the IL-6 mrna to be increased in the OVA group compared with the OVA indomethacin group (p 0.08). There was no detectable mrna for IFN- in either the OVA or OVA indomethacin group. Indomethacin Treatment Increased Lung MCP-1 mrna in OVA-sensitized Mice RNase protection assays (RPAs) were performed on the lungs from mice harvested on Day 18 of the protocol. There was no difference in mrna levels for eotaxin, MIP-1, MIP-1, or MIP-2 between the OVA indomethacin and OVA groups (Fig- Figure 4. (A) Data from an RNase protection assay showing cytokine mrna present in lungs harvested on Day 18. The data are expressed as mrna for each chemokine as a percentage of L32 (n 3). *p (B) RNase protection assay showing cytokine mrna present in lungs harvested on Day 18.

4 Peebles, Dworski, Collins, et al.: Cyclooxygenase Inhibition Increases IL-5, IL-13 Production and AHR 679 Figure 5. (A) Data from an RNase protection assay showing chemokine mrna present in lungs harvested on Day 18. The data are expressed as mrna for each chemokine as a percentage of L32 (n 3). *p (B) RNase protection assay showing cytokine mrna present in lungs harvested on Day 18. ures 5A and 5B), although the lung mrna for these chemokines was significantly greater in the OVA indomethacin and OVA groups compared with the two groups of mice that were not OVA sensitized. However, the lung mrna for MCP-1 was significantly greater in the OVA indomethacin group compared with the OVA group (p 0.05). In the nonsensitized mice there was no difference in MCP-1 lung mrna expression between the mice treated with indomethacin and those that were not. Thus indomethacin treatment increased MCP-1 mrna expression only in those mice that were OVA sensitized. Indomethacin Did Not Increase Total IgE Levels in the Sera of OVA-sensitized Mice On Day 23, the day after the last OVA aerosol treatment, blood was drawn after the methacholine challenge for measurement of total IgE levels. The average total IgE level was g/ml in the sera of the OVA indomethacin mice and g/ml in the OVA group (n 4 in each group, results representative of two separate experiments). This represents a trend for an increase in total IgE in the OVA indomethacin mice compared with the OVA group (p 0.08) (Figure 6). Mice that are not sensitized have no detectable IgE in their sera (data not shown). Indomethacin Increased Allergen-induced Lung Pathology in the Lung Interstitium The OVA indomethacin group had increased interstitial inflammation compared with the OVA group (Figure 7). This inflammation was composed of a large number of eosinophils (2 ), macrophages (2 ), and small lymphocytes (2 ), while there were only a few macrophages (1 ) in the interstitium of the OVA group. There was no difference in the inflammation in the bronchovascular or perivenous spaces between the two groups. Both the OVA and OVA indomethacin groups had a significant inflammation in the bronchovascular and perivenous compartments with extensive infiltration of eosinophils, small lymphocytes, and plasma cells. There was also a large amount of mucus in the airways of both the OVA and OVA indomethacin groups. There was essentially no inflammation in the bronchovascular, perivenous, and interstitial compartments of the two groups of nonsensitized mice, nor was there any mucus in the airways of these two groups. Indomethacin Increased Allergen-induced AHR Methacholine challenge was performed on Day 23, 1 d after the last OVA aerosol treatment. The AHR was significantly greater in the OVA indomethacin group compared with the OVA group (Figure 8). At the highest dose of methacholine, the OVA indomethacin mice had a lung resistance of cm H 2 O/ml/s, while the lung resistance of the OVA mice was cm H 2 O/ml/s. Indomethacin treatment did not increase AHR in either the NS or NS indomethacin group. DISCUSSION Prostaglandins influence many important inflammatory processes (15). These mediators are produced by the phospholipase A 2 conversion of membrane phospholipids to arachi- Figure 6. Total IgE levels in serum (p 0.08) harvested on Day 23 (n 6). The data shown are representative of two separate experiments. Figure 7. Tabulation of histologic analysis showing number of eosinophils and lymphocytes and their distribution in the bronchovascular (BV), perivenous (PV), and interstitial (IS) locations in all four groups on Day 23. *Histologic scoring is as outlined in METHODS.

5 680 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL Figure 8. Results from methacholine challenge performed on Day 15. The data shown are a combination of two experiments (n 13 for OVA indomethacin and OVA; n 6 for NS indomethacin and NS). Data points represent the mean SEM measuring lung resistance. *p 0.05 OVA indomethacin versus all other groups. donic acid, followed by the cyclooxygenase conversion of arachidonic acid to prostaglandin H 2 (PGH 2 ). PGH 2 may then be converted by specific synthases or isomerases to either prostaglandin D 2 (PGD 2 ), prostaglandins F 2 (PGF 2 ), prostaglandin E 2 (PGE 2 ), prostaglandin I 2 (PGI 2 ), or thromboxane (15). The prostaglandins and thromboxane have diverse effects on the inflammatory cascade and are presumed to have a pathogenic role in asthma (16, 17). PGD 2 is increased in the BAL fluid of subjects with asthma (18) while both PGD 2 and PGF 2 contract human airway smooth muscle in vitro and are potent bronchoconstrictors in vivo (19). Thromboxane also causes human airway smooth muscle contraction in vitro (20). On the other hand, PGI 2 causes relaxation of isolated precontracted human bronchi (19), but has minimal effects on airway function in vivo (21). PGE 2 has a bronchoprotective effect in humans (22). Many groups have reported that PGE 2 in vitro inhibits lymphocyte production of the Type 1 cytokines IL-2 and interferon, thus promoting T cell differentiation toward a Type 2 cytokine profile (2 5). In our study, we found that cyclooxygenase inhibition by indomethacin treatment during allergen sensitization decreased PGE 2 and increased LTC 4 /D 4 /E 4 in BAL fluid, and increased the lung supernatant protein levels of the Type 2 cytokines IL-5 and IL-13. To our knowledge, this is the first time that prostaglandin synthesis inhibition has been reported to selectively increase specific Type 2 cytokines in the setting of allergic sensitization. We found that indomethacin treatment during allergen sensitization caused a trend for an increase in IL-4 protein levels in lung supernatants and on serum total IgE production, although these were not significant changes. These data corroborate the in vitro and in vivo demonstrations that IL-4 is required for the production of IgE and is the principal cytokine that stimulates switching of B cells to the IgE heavy chain (23). Others have reported that indomethacin treatment for 60 d decreased IL-6 production in a model of mineral oil-induced chronic intraperitoneal inflammation (10). In our model of allergic inflammation, we found that indomethacin treatment caused a trend toward decreased IL-6 production (p 0.1) in mice that were exposed to aerosolized ovalbumin for 8 d. It is possible that the duration of the inflammatory stimulus in our model was too short for indomethacin to have exerted a significant change in IL-6 concentrations. Indomethacin treatment during ovalbumin sensitization decreased PGE 2 while increasing the cysteinyl leukotrienes. The increase in the cysteinyl leukotrienes could result from shunting of arachidonate to the 5-lipoxygenase pathway (11). Alternatively, inhibition of PGE 2 by indomethacin is known to augment leukotriene-c 4 synthase activity, thereby increasing the production of the cysteinyl leukotrienes (24). Similarly, the increase in the cysteinyl leukotriene products of the 5-lipoxygenase pathway could result from an IL-5-mediated activity (25). IL-5 increases the expression of the 5-lipoxygenase-activating protein and translocates 5-lipoxygenase to the nucleus in normal blood eosinophils in vitro. This is associated with an increased capacity for cysteinyl leukotriene expression and is similar to the increase in 5-lipoxygenase expression seen in the eosinophils from subjects with allergic asthma (25). This increase in the cysteinyl leukotrienes may be important in the increased lung interstitial eosinophilic inflammation seen in the OVA indomethacin-treated mice, as these mediators are described as being involved in eosinophil recruitment (26). In addition, indomethacin caused an increase in IL-5, a cytokine that increases eosinophil accumulation (27, 28) and is an important mediator causing eosinophil growth, recruitment, and survival (23). However, none of the chemokines thought to be responsible for eosinophil chemotaxis were elevated in the OVA indomethacin group compared with the OVA mice. The lung mrna expression of eotaxin, MIP-1, and MIP1 were not different between the OVA indomethacin and OVA groups. MCP-1 is a member of the C-C chemokine family and binds to the CCR2 receptor (29). CCR2 receptors are present on basophils, monocytes, activated T cells, dendritic cells, and natural killer cells, thus allowing for MCP-1-mediated biologic effects (29). Although the upregulation of MCP-1 as a result of indomethacin treatment may have recruited an increased number of activated T cells expressing IL-5 to the lung in the OVA indomethacin group, this possibility remains unexplored as we did not perform immunohistochemistry to delineate activated from nonactivated T cells. Our results are in agreement with those of Gavett and colleagues, who reported their findings in mice lacking either prostaglandin H synthase 1 (PGHS-1) or PGHS-2 (30). In comparison with wild-type mice, they found a heightened degree of eosinophilic inflammation, higher levels of IgE, decreased PGE 2 and increased leukotriene B 4 in BAL fluid, and increased airway responsiveness in the mice lacking PGHS-1. Our findings with indomethacin are consistent with an inhibition of a prostanoid, e.g., PGE 2, which may restrain allergic inflammation. Our results extend theirs to show that this effect is not the result of alterations in immune responses in transgenic mice that have never been exposed to PGHS-1 products, and by our finding that the Type 2 cytokines IL-5 and IL-13 are elevated as well, and may play a role in the increased inflammation and AHR we found in indomethacin-treated mice. IL-5 and IL-13 are reported to be critical for the development of allergic airway inflammation and hyperresponsiveness (31 37). The mechanism by which prostaglandin inhibition leads to the increase in IL-5 and IL-13 is unknown, but several possibilities exist. Perhaps in an in vivo system, PGE 2 does not increase a Type 2 cytokine profile as suggested by in vitro studies, and instead may downregulate Type 2 cytokine production. Another possibility is that the cysteinyl leukotrienes may have an effect on T cell function and could perhaps regulate IL-5 and IL-13 production. The cysteinyl leukotriene 1 receptor has been cloned and the highest expression of the mrna for this receptor is in spleen and peripheral blood leukocytes (38). To our knowledge, there have been no reports on the role of the cysteinyl leukotrienes in T cell development or on the distribution of the cysteinyl leukotriene 1 receptor on T cell subsets. We have previously shown that mice lacking

6 Peebles, Dworski, Collins, et al.: Cyclooxygenase Inhibition Increases IL-5, IL-13 Production and AHR 681 the 5-lipoxygenase gene produce less IgE in response to OVA than do wild-type mice, suggesting that the leukotrienes could be involved in allergic inflammation (12). Our report is the first to describe that cyclooxygenase inhibition during allergic sensitization increases IL-5 and IL-13 production and that this increase in these Type 2 cytokines is associated with an increase in allergen-induced AHR. Our findings suggest that the products of arachidonic acid metabolism have important immunomodulatory effects on allergic sensitization in vivo. References 1. Vane, J. R The mode of action of aspirin and similar compounds. J. Allergy Clin. Immunol. 58: Betz, M., and B. S. Fox Prostaglandin E2 inhibits production of Th1 lymphokines but not of Th2 lymphokines. J. Immunol. 146: Snijdewint, F. G., P. Kalinski, E. A. Wierenga, J. D. Bos, and M. L. 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