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Published ahead of print on May 19, 2004, doi:10.1164/rccm.200311-1595OC
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American Journal of Respiratory and Critical Care Medicine Vol 170. pp. 485-491, (2004)
© 2004 American Thoracic Society
doi: 10.1164/rccm.200311-1595OC

Original Article

Higher Prostaglandin E2 Production by Dendritic Cells from Subjects with Asthma Compared with Normal Subjects

Julie A. Long, Mirjana Fogel-Petrovic, Darryl A. Knight, Philip J. Thompson and John W. Upham

Asthma and Allergy Research Institute, Sir Charles Gairdner Hospital; School of Medicine and Pharmacology, University of Western Australia; and Institute for Child Health Research, Perth, Western Australia, Australia

Correspondence and requests for reprints should be addressed to John Upham, M.B., B.S., F.R.A.C.P., Ph.D., Institute for Child Health Research, P.O. Box 855, West Perth, WA 6872, Australia. E-mail: johnu{at}ichr.uwa.edu.au


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dendritic cells (DCs) are thought to play an important role in the pathogenesis of allergic disorders through their ability to interact with T cells to initiate and amplify helper T cell Type 2 immune responses. The mechanisms by which this occurs are not completely understood, nor is it clear whether DC function differs between normal individuals and individuals with asthma. We compared the function of DCs from 10 subjects with allergic asthma and 10 normal individuals, focusing on the production of prostaglandin E (PGE) 2, interleukin (IL)-10, and IL-12 p70, mediators that play an important role in helper T cell Type 1/Type 2 polarization. Monocyte-derived DCs were established by culturing monocytes with granulocyte-macrophage colony-stimulating factor and IL-4 for 7 days, and then stimulated with LPS plus IFN-{gamma}. PGE2, IL-10, and IL-12 production was evaluated by ELISA, whereas cyclooxygenase-1, and -2 messenger RNA expression was analyzed using reverse transcription-polymerase chain reaction. LPS-stimulated monocyte-derived DCs from individuals with asthma exhibited increased PGE2 and IL-10 production, but equivalent IL-12 p70 synthesis, when compared with DCs from normal subjects. Increased PGE2 synthesis by DCs from subjects with asthma was associated with an increase in cyclooxygenase-2 messenger RNA expression. These findings support the notion that DC function is significantly altered in allergic asthma.

Key Words: asthma • cyclooxygenase • dendritic cells • interleukin-10 • prostaglandin E 2

Allergic asthma is widely regarded as a T cell-mediated disorder in which allergen-specific T cells secrete helper T cell type 2 (Th2) cytokines such as interleukin (IL)-4, IL-5, and IL-13 that orchestrate key features of asthma including IgE production, airway eosinophilia, and mucus secretion. However, T cells are unable to respond to antigen independently of antigen-presenting cells, and there is now increasing evidence that antigen-presenting cells, particularly dendritic cells (DCs), are highly relevant to both allergic sensitization and to the continuing reinforcement of Th2 immunity that occurs with repeated allergen exposure (1).

In animal models of allergy, DCs play a central role in priming Th2 immune responses to inhaled antigens and the initiation of allergic airway inflammation (2, 3). DCs are also critical to the maintenance of chronic eosinophilic airway inflammation after sensitization (4–6).

Studies in humans also suggest that DCs play a central role in the development of immune responses to inhaled antigens. DC precursors disappear from the circulation after allergen inhalation and are rapidly recruited into the bronchial mucosa (7, 8). In addition, increased numbers of DCs expressing the high-affinity IgE receptor (Fc{epsilon}R1) are present within the airway mucosa of subjects with asthma (9, 10).

These differences in DC function between atopic and healthy control subjects are seemingly not confined to the airways, but may also exist within precursor populations in the bone marrow and/or circulation. In asthma, monocyte-derived DCs reportedly have an increased capacity to present antigen to T cells (11), and DCs from allergic subjects appear to secrete an altered pattern of inflammatory mediators, compared with DCs from nonatopic individuals. Thus, DCs from allergic subjects synthesize greater amounts of IL-1ß, IL-6, and tumor necrosis factor-{alpha} when compared with DCs from healthy individuals (12–14). In contrast, studies investigating the production of polarizing cytokines IL-10 and IL-12 by DCs have yielded conflicting results, with various reports suggesting that DCs from atopic individuals secrete either more (13) or less (14) IL-10, and either less (14) or equivalent amounts (13, 15) of IL-12, compared with DCs from normal individuals.

Prostaglandin E (PGE) 2 is an immunoregulatory molecule that is known to enhance Th2 differentiation (16). However, PGE2 has received little attention in relation to DC function in allergic disease. Although DCs are known to be responsive to exogenous PGE2, it was previously thought that DCs were unable to synthesize PGE2 themselves (17). We and others have shown that human DCs can indeed synthesize PGE2 and express cyclooxygenase (COX)-1 and COX-2 (18, 19).

Accordingly, the aim of this study was to investigate the function of DCs obtained from subjects with allergic asthma and healthy subjects, focusing on the production of PGE2 and the secreted cytokines IL-12 p70 and IL-10, which are thought to play an important role in Th1/Th2 polarization. Because there is considerable interest in the relationship between environmental endotoxin exposure, immune function, and the pathogenesis of allergic disease (20, 21), we examined DCs after in vitro activation with LPS and IFN-{gamma}.

Our results show that LPS plus IFN-{gamma}–stimulated monocyte-derived DCs from individuals with atopic asthma exhibit increased production of PGE2 and IL-10, but equivalent IL-12 p70 synthesis, compared with DCs from healthy subjects. Furthermore, the increased PGE2 levels in subjects with asthma correlated with changes in expression of COX-1 and COX-2, the key enzymes involved in the regulation of PGE2 synthesis. Some of the results of this study have been previously reported in abstract form (22).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
See the online supplement for further details concerning methods.

Subjects
Subjects aged between 18 and 50 years were recruited from an asthma clinic within the Asthma and Allergy Research Institute (Perth, Australia). All subjects underwent skin-prick testing to five common aeroallergens (house dust mite, grass pollen, mold, cat dander, and dog hair). Subjects with asthma were all symptomatic and atopic (4-mm or greater reaction to at least one allergen), whereas the control group consisted of healthy subjects without any respiratory symptoms, and negative skin-prick tests. All the patients stopped using inhaled steroids for 24 hours before blood sample collection. Exclusion criteria included the following: use of oral corticosteroids within the last month, symptoms of upper or lower respiratory tract infection within the last month, and smoking within the last 12 months. All subjects gave their informed consent to participate in the study, which had been approved by the Human Ethics Committee of the University of Western Australia (Perth, Australia). The characteristics of the subjects are described in Table 1.


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  		TABLE 1. Characteristics of patients

 
Generation of DCs
Monocyte-derived DCs were prepared from adherent peripheral blood mononuclear cells cultured for 7 days in the presence of granulocyte-macrophage colony-stimulating factor (50 ng/ml; Schering Plough, Sydney, Australia) and IL-4 (10 ng/ml; R&D Systems, Minneapolis, MN), using methods described previously (23). Polymyxin B (10 µg/ml; Sigma, Sydney, Australia) was added during DC differentiation, to avoid cellular activation caused by any trace amounts of endotoxin.

Stimulation of DCs
After 7 days of culture, nonadherent cells corresponding to the DC-enriched fraction were counted, washed, and resuspended in medium without polymyxin B. DCs (1 x 106 cells/ml) were added to 24-well tissue culture plates, primed with IFN-{gamma} (20 ng/ml) for 3 hours, and stimulated with LPS (1 µg/ml) for 6 or 24 hours.

Flow Cytometry
Cells were stained with the following monoclonal antibodies purchased from BD Pharmingen (San Diego, CA): CD1a–FITC (fluorescein isothiocyanate), CD14–FITC, CD80–PE (phycoerythrin), CD86–PE, and HLA-DR–CyChrome and appropriate mouse IgG controls.

Cytokine and PGE2 Assays
IL-12 p70 and IL-10 were measured using commercially available ELISA kits (BD Pharmingen). PGE2 levels were determined by enzyme immunoassay (Cayman Chemicals, Ann Arbor, MI).

Isolation of RNA and Reverse Transcription-Polymerase Chain Reaction
Total RNA was isolated from unstimulated DCs and DCs stimulated with LPS plus IFN-{gamma} (for 6 hours), using the RNeasy kit (Qiagen, Valencia, CA). Total RNA was reverse transcribed to cDNA and amplified with the One-Step reverse transcription-polymerase chain reaction (RT-PCR) kit (also from Qiagen). See the online supplement for additional details on the PCR cycling conditions. Ten-microliter samples of PCR product from six subjects with asthma and six normal individuals were run simultaneously on a single gel for each set of primers (COX-1, COX-2, and glyceraldehyde-3-phosphate dehydrogenase) and stained with ethidium bromide. An image was taken of the gel, using Kodak ID image analysis software (Eastman Kodak, Rochester, NY), and analyzed with ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Relative amounts of target messenger RNA (mRNA) were determined relative to glyceraldehyde-3-phosphate dehydrogenase mRNA in the corresponding sample and data were expressed as normalized arbitrary values.

Statistical Analysis
Except where indicated, data sets were normally distributed and data are expressed as means ± SEM. Significance of differences between patient groups was evaluated by unpaired t test analysis whereas differences between treatment and control samples were evaluated by paired t test. A p value of less than 0.05 was deemed significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
DCs from Subjects with Asthma Release Higher Levels of PGE2 in Response to LPS
DCs obtained from subjects with allergic asthma (n = 10) exhibited significantly higher PGE2 release than DCs from normal subjects (n = 10) after LPS plus IFN-{gamma} stimulation (116.3 ± 26.0 vs. 51.8 ± 18.9 pg/ml; p = 0.01) (Figure 1). In contrast, baseline production of PGE2 by unstimulated DCs did not differ between subjects with asthma and normal subjects (38.9 ± 10.7 vs. 18.0 ± 7.0 pg/ml; p = 0.11).



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  		Figure 1. Higher production of prostaglandin E (PGE) 2 by dendritic cells (DCs) from patients with asthma. Monocyte-derived (Mo-) DCs were obtained from 10 subjects with asthma and 10 healthy subjects, unstimulated (Control) or primed with 20 ng/ml IFN-{gamma} and then stimulated with LPS (1 µg/ml) for 24 hours. Mo-DCs were cultured from circulating monocytes as described in METHODS. Levels of PGE2 were determined by enzyme immunoassay. The dashed line represents the lower limit of detection of the assay.

 
DCs from Subjects with Asthma Have Higher Expression of COX-2 and COX-1 mRNAs
Because COX is the rate-limiting enzyme in the formation of PGE2, we next examined expression of both genes (COX-1 and COX-2) in a randomly selected subset of individuals with asthma (n = 6) and healthy individuals (n = 6). In unstimulated DCs, constitutively expressed COX-1 mRNA was higher at baseline in subjects with asthma compared with normal subjects (48,240 ± 2,008 vs. 33,850 ± 6,244 arbitrary units; p = 0.05), whereas COX-2 mRNA was barely detectable in DCs derived from both subjects with asthma and normal subjects (2,308 ± 971.8 vs. 2,269 ± 749.4 units) (Figures 2a and 2d).




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  		Figure 2. Expression of COX-2 and COX-1 messenger RNA (mRNA) in DCs from patients with asthma. Mo-DCs were unstimulated (–) or stimulated (+) with LPS plus IFN-{gamma} for 6 hours and one-step RT-PCR was performed with specific primers as described under METHODS. (a–c) Representative experiments from three individuals with asthma (A) and three healthy individuals (N). In (d) and (e), the densitometric values for COX-2 and COX-1 mRNA expression are shown for Mo-DCs from six subjects with asthma and six normal subjects. Densitometric values were normalized to RNA input relative to corresponding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression. Each densitometric value is represented as a fold increase (relative to lane 1), as indicated beneath gels (a and b).

 
After 6 hours of stimulation with LPS plus IFN-{gamma}, COX-2 mRNA was markedly upregulated in DCs from both normal subjects and subjects with asthma. The mean fold increase in COX-2 expression was 15-fold (p < 0.003) in normal subjects and 21-fold (p < 0.001) in subjects with asthma. Although the magnitude of COX-2 mRNA upregulation tended to be higher in DCs obtained from subjects with asthma compared with normal subjects (49,080 ± 6,286 vs. 31,840 ± 5,714 units), this did not reach statistical significance (p = 0.07; Figure 2d). However, the magnitude of COX-2 upregulation appeared to correlate with the greater increase in PGE2 production observed in DCs from subjects with asthma.

In contrast, COX-1 mRNA expression was downregulated 1.5-fold (p < 0.01) and 1.7-fold (p < 0.01) in both subjects with asthma and normal individuals, respectively, after stimulation with LPS plus IFN-{gamma} (Figures 2b and 2e). However, expression of COX-1 was significantly higher in DCs from subjects with asthma compared with DCs from normal individuals (32,450 ± 2,451 vs. 20,420 ± 4,807 units; p < 0.05).

DCs from Subjects with Asthma Produce Higher Levels of IL-10
Production of IL-10 by DCs at baseline was comparable in subjects with asthma (n = 10) and healthy subjects (38.8 ± 12.4 vs. 17.4.0 ± 8.1 pg/ml, n = 10; p = 0.17) (Figure 3). Although LPS plus IFN{gamma} stimulation induced a profound increase in IL-10 in both subjects with asthma and normal individuals (p = 0.05 and p < 0.01, respectively), the magnitude of this increase was significantly higher in subjects with asthma when compared with healthy subjects (1,200 ± 482.4 vs. 179.9 ± 54.4 pg/ml, respectively; p < 0.05). The mean increase from IL-10 levels at baseline was 31-fold in subjects with asthma compared with 10-fold in normal individuals.



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  		Figure 3. Higher production of interleukin (IL)-10 by both resting and stimulated DCs from patients with asthma. Mo-DCs derived from 10 subjects with asthma (A) and 10 healthy (N) subjects were seeded at 1 x 106 cells in 1-ml cultures. Cultures were either left unstimulated (Control) or primed with 20 ng/ml IFN-{gamma} and stimulated with 1 ug/ml LPS for 24 hours. IL-10 levels were determined by ELISA. The dashed line represents the lower limit of detection of the assay.

 
Production of IL-12 by DCs from Subjects with Asthma and Normal Subjects
In contrast to IL-10 and PGE2, bioactive IL-12 p70 was undetectable in unstimulated DCs. Production of IL-12 p70 in response to LPS plus IFN-{gamma} stimulation was not significantly different in DCs obtained from subjects with asthma compared with normal subjects (1,201 ± 529.9 vs. 552.0 ± 184.7 pg/ml, n = 10; p = 0.26) (Figure 4).



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  		Figure 4. Similar levels of IL-12 production by DCs from patients with asthma and healthy individuals. Mo-DCs derived from 10 subjects with asthma (A) and 10 healthy (N) subjects were seeded at 1 x 106 cells in 1-ml cultures. Cultures were either left unstimulated (Control) or primed with 20 ng/ml IFN-{gamma} and stimulated with 1 ug/ml LPS for 24 hours. IL-12 was undetectable in control (unstimulated) cultures. IL-12 levels were determined by ELISA. The dashed line represents the lower limit of detection of the assay and the unbroken lines represent mean values.

 
Expression of CD86 by DCs from Subjects with Asthma and Normal Subjects
Having shown that DCs from subjects with asthma synthesize more PGE2 and IL-10 than do DCs from control subjects, it was important to consider whether these changes could be attributed to variations in DC activation. However, flow cytometric analysis of unstimulated DCs showed that expression of the costimulatory molecule CD86 did not differ between DCs from subjects with asthma and normal individuals (mean fluorescence intensity, 91.5 ± 26.4 vs. 48.4 ± 13.0; p = 0.41) (Figure 5). Upregulation of CD86 expression was seen in both subject groups after stimulation with LPS, but this did not differ between DCs from subjects with asthma and normal individuals (mean fluorescence intensity, 218.6 ± 37.3 vs. 204.6 ± 52.4; p = 0.93). Similar results were obtained for CD80 and HLA-DR (data not shown).



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  		Figure 5. CD86 expression on DCs from patients with asthma and healthy individuals. Expression of CD86 was examined by flow cytometry on Mo-DCs derived from patients with asthma (hatched bars, n = 7) and healthy individuals (solid bars, n = 4). Mo-DCs were unstimulated (Control) or treated with 1 ug/ml LPS for 24 hours and preprimed with 20 ng/ml IFN-{gamma}. Results are shown as the average mean fluorescence intensity (MFI) as determined by flow cytometry. There was no significant difference in CD86 expression between subjects with asthma and healthy subjects, using the Mann–Whitney statistical test.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This is the first report demonstrating that DCs from subjects with allergic asthma produce significantly higher levels of PGE2 and IL-10 in response to LPS, compared with DCs from healthy subjects. The observed increase in PGE2 was associated with alterations in the expression of both COX-1 and COX-2 in the asthmatic group, despite a concomitant reduction in COX-1 gene expression after stimulation in both subject groups. In contrast, we observed no difference in IL-12 p70 production or expression of costimulatory molecules.

Other studies have reported increased PGE2 levels in bronchoalveolar lavage fluid and plasma of subjects with asthma (24, 25), and the concentration of PGE2 in plasma may correlate with the severity of airway obstruction (26). Similarly, increased expression of both COX-1 and COX-2 has been reported in the airways of subjects with asthma, using RT-PCR (27) and immunohistochemistry (25, 28). Although the cells responsible for COX expression were not fully identified in these studies, our findings suggest that DCs are likely to contribute to increased PGE2 production in asthma.

This is the first study to examine prostaglandin synthesis by DCs from subjects with asthma. Our findings confirm and extend reports (including from this laboratory) that human monocyte-derived DCs are capable of producing PGE2 and have increased COX-2 expression after stimulation with LPS (18, 19). Indeed, DCs express both COX-1 and COX-2. In most cell types, COX-1 is described as a constitutively expressed enzyme, whereas COX-2 is inducible and is upregulated in response to inflammatory stimuli. However, we have also shown that LPS can differentially regulate both isoforms of COX in dendritic cells (Figure 2). The simultaneous downregulation of COX-1 and upregulation of COX-2 after LPS plus IFN-{gamma} administration highlights the complex regulation of prostaglandin within DCs. Aside from a previous report by Liu and coworkers on the downregulation of COX-1 mRNA by LPS in cells from rat lungs (29), we are unaware of any other reports describing this phenomenon in human cells and thus future studies will address this.

We also detected an increase in IL-10 production in DCs obtained from individuals with allergic asthma compared with DCs from healthy individuals. Other investigators have shown that circulating monocytes from subjects with asthma and atopic dermatitis also synthesize increased amounts of PGE2 and IL-10 (30, 31), suggesting that enhanced PGE2 production and COX-2 upregulation characterize the monocyte/macrophage/DC lineage in allergic inflammatory disorders. However, it is important to highlight that our findings are not due to residual monocytes in the DC cultures, given that CD14 was undetectable (less than 1% of cells) by flow cytometry (data not shown).

We were not able to demonstrate a significant difference in IL-12 production between DCs from both groups of subjects. Similar findings have also been reported by others (15), although Reider and coworkers reported that DCs from atopic subjects are deficient in IL-12 synthesis when activated with CD40 ligand (14). It is possible that these variations in results reflect differences in culture conditions, particularly the nature of the stimuli by which DCs are activated.

At this stage the functional significance of the changes in DC function that we have observed are unknown. Although DCs are not the only cell type responsible for the increased levels of PGE2 production observed in asthma, DC-derived PGE2 is likely to have profound effects on T cell function during antigen presentation, that is, within the microenvironment of the "immunologic synapse" that forms between DCs and T cells (32). In particular, PGE2 may be involved in the induction and reinforcement of Th2 responses, either by directly affecting T cell polarization or indirectly by augmenting IL-10 release (33). We have observed that the COX-2 inhibitor nimesulide inhibits both PGE2 and IL-10 synthesis by DCs from normal subjects, but has no effect on IL-12 production (19). However, others have shown that both PGE2 and IL-10 selectively inhibit release of IL-12 and IFN-{gamma}, and expression of IL-12 receptor (16, 34). IL-10 has been reported to enhance Th2 responses, but may also play a role in tolerance to inhaled antigens (35).

Reports have also suggested that PGE2 plays a crucial role in DC migration (36, 37), and future studies are needed to determine the extent to which PGE2 production by DCs contributes to the accelerated trafficking and turnover of DCs in mouse models of asthma (38).

We have not been able to directly evaluate airway DC function in this study, as obtaining sufficient numbers of DCs from bronchoscopic biopsies or lavage for functional studies is technically difficult. Nonetheless, our data do show that specific functional differences exist between blood-derived DCs from subjects with atopic asthma and normal individuals, despite an identical 7-day period of in vitro culture. Although these differences in DC function might potentially be attributed to altered responsiveness of monocytes to granulocyte macrophage colony-stimulating factor or IL-4, the lack of any difference in IL-12 p70 synthesis, or expression of maturation markers such as CD80, CD86, or HLA-DR, between the two subject groups argues that the differences in PGE2 and IL-10 between the subjects with asthma and normal subjects are not due to nonspecific differences in DC maturation or activation. Previous studies have also shown that these parameters of dendritic cell function are readily suppressed by in vitro steroid exposure, usually at concentrations of 10–7 to 10–8 M of dexamethasone (39). It therefore appears unlikely that our finding of higher production of PGE2 and IL-10 by dendritic cells from subjects with asthma can be attributed to the residual effects of inhaled steroids, especially as the patients had not used inhaled steroids for 24 hours before the time of blood collection.

It is increasingly accepted that monocyte-derived DCs are phenotypically and functionally similar to those isolated from fresh human lung specimens (40), and our findings suggest that in the atopic individual, blood DCs and their monocyte precursors display distinct functional characteristics, even before they migrate into the lung. Similar observations have been made in relation to eosinophils in asthma, where it is well accepted that asthma is associated with specific changes in eosinophils and their progenitors, not only in the airway but also in the circulation and bone marrow (41). Equally at this stage it is not clear whether these differences are secondary to signals from the lung or whether they reflect a primary abnormality in DC precursor populations. Our findings do not allow us to determine the extent to which increased PGE2 and IL-10 production by DCs is involved in the initiation of allergic sensitization, or whether these changes have developed after sensitization and allergic inflammation.

It is interesting to speculate on what is causing the increases in PGE2 and IL-10 levels produced by DCs in subjects with asthma. These differences may relate to polymorphisms in either COX-1 or COX-2 or to an upstream regulatory mechanism such as NF-{kappa}B or IL-1ß. IL-1ß has been shown to induce COX-2 mRNA expression and increased levels of this cytokine have been found in bronchoalveolar lavage fluid of subjects with asthma, but it is also produced in larger amounts by DCs from allergic patients (13, 42). Although polymorphisms associated with asthma are yet to be identified in the COX genes, one has been identified in the IL-1ß gene (43). Increased NF-{kappa}B activity has also been observed in bronchial biopsies from subjects with asthma (44) and putative response elements for NF-{kappa}B have been identified in the promoter regions of both COX-1 and COX-2 genes (45, 46).

The stimuli employed in this study, LPS and IFN-{gamma}, are traditionally thought to have strong Th1-inducing properties when used to activate DCs (47). However, it is increasingly recognized that atopy and asthma are characterized not only by a Th2 response to allergens, but also by a reduced or modified Th1 response to microbial stimuli (48). The findings of the current study support this notion. Future studies will examine the extent to which allergens are also able to induce COX and PGE2 synthesis.

The contribution of PGE2 to the pathogenesis of allergic airway inflammation appears complex. In some instances PGE2 appears to have a bronchoprotective role in the lung, and is produced in normal human bronchial tissue (49, 50). In contrast, PGE2 can prolong eosinophil survival (51) and synergize with IL-4 to induce switching to IgE and IgG1 production in murine B cells (52). Although COX-deficient mice show exaggerated airway inflammation after challenge with antigen, suggesting that COX products may actually dampen airway inflammation, the extent to which this involves PGE2, as compared with other PGs, has yet to be determined (53).

An increasing body of evidence suggests that DCs function differently in the context of allergic airway inflammation (9–11, 38, 54). We propose that endogenous PGE2 and IL-10 are likely to be important mediators by which DCs regulate their own function and the function of allergen-specific T cells. Our findings also highlight the importance of understanding the regulation of prostaglandin production and regulation of both COX-1 and COX-2 activity in asthma and allergic disease, and this will need to be examined in future studies employing specific COX inhibitors or PG receptor antagonists.


   Acknowledgments
 
The authors thank Stephanie Phelps, Rachel Beard, Catherine Haig, Michelle Blackwood, and Alex Tregonning from the Clinical Trials Unit for their assistance with recruitment of subjects, and Neil Misso for his helpful advice.


   FOOTNOTES
 
Supported by grants from the Sir Charles Gairdner Hospital Research Fund and the National Health and Medical Research of Australia (no. 254524).

This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Conflict of Interest Statement: J.A.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; M.F.-P. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; D.A.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; P.J.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; J.W.U. does not have a financial relationship with a commercial entity that has an interest in the subject of this article.

Received in original form November 24, 2003; accepted in final form May 14, 2004


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 DISCUSSION
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