To investigate whether to assess the effect of dexamethasone on CysLTR1 expression in A549 cells, cells were incubated with fresh medium in presence or absence of various dexamathasone from 10-9M to10-6M (Fig. 11). Treatment of A549 cells with various concentration dexamethasone increased CysLTR1 mRNA expression compared to untreated cells. Maximum enhancement was observed between 10-6M and 10-5M dexamethasone.
DEX (10-8M) - + - - + -
IL-4 significantly increased CysLTR1 promoter activity in mutant genotype (p=0.008), which was significantly inhibited by the pretreatment of MK-571 in mutant genotype (p=0.004). In contrast, dexamethasone pretreatment increased promoter activity in both mutant and wild genotype (p< 0.001). ** P<0.001and
*P<0.01.
**
**
Fig. 11. Realtime PCR analysis effects on CysLTR1 mRNA with addition of dexamethasone in A549 cells.A549 cells were further incubated with fresh medium in the presence or absence of dexamethasone (10-5M~10-9M) for 6hr. RNA was extracted and realtime-PCR was performed as described methods.
A: A549 cell only; B: A549 cells + IL-4 (10ng/ml, 18hr); C: dexamethasone (10-9M);
D: dexamethasone (10-8M); E: dexamethasone (10-7M); F: dexamethasone (10-6M);
G: dexamethasone (10-5M)
IV. DISCUSSION
CysLTs such as LTC4, LTD4, and LTE4 are important proinflammatory lipid mediators in the development of asthma by mediating bronchoconstriction as well as increasing mucus secretion, vascular permeability, and cellular infiltration and airway hyperresponsiveness (Lewis and Soberman, 1990; Henderson WR, 1994).
CysLTs exert their biological action by binding to types of GPCRs such as CysLTR1 and CysLTR2 (Hui Y and Funk, 2002). Because CysLTR1 mRNA is abundantly expressed in inflammatory leukocytes such as eosinophils, monocytes and macrophages, it is thought that CysLTR1 may have an important role in these cells, main effectors in asthma, particularly in AIA. Although the pathogenesis of aspirin intolerance has not understood yet, overproduction of CysLTs and CysLT receptors may play major roles in pathogenesis mechanism of AIA.
In the present study, we are the first to demonstrate allele polymorphisms of CysLTR1 promoter gene in AIA patients. The 3 novel SNPs that we discovered (C-634T, A-475C, and A-336G) were associated with AIA risk in males; males with AIA had significantly higher frequencies of minor alleles (T-C-G) of three SNPs than male control subjects. Moreover, the two common 3-SNP haplotypes were associated with AIA in males. The ht1 [C-A-A] haplotype was associated with decreased disease risk and the ht2 [T-CG] haplotype with increased disease risk. Within the AIA group, the male patients were younger than female patients.
We therefore can speculate that male AIA patients carrying ht2 [T-C-G] may be at greater risk of developing AIA at an earlier age. In female subjects, there were no
significant associations of CysLTR1 genotype or 3-SNP haplotype with AIA risk.
These results suggest that the male patient with mutant allele of CysLTR1 has a higher risk genetic factor in AIA development. In fact, this result was consistent with other previous report that the male subjects showed higher level of CysLTR1 than female subjects in corectral adenocarcinoma (Ohd and Sjolander, 2003).
These results suggest that the observed differences in association between males and females may be related to allele-dependent dysregulation of the CysLTR1 gene in AIA patients.
In females, the CysLTR1 C-634T polymorphism was associated with AIA-related quantitative phenotype, total serum IgE level. Where no association was found in case of males. AIA was not through allergic mechanism but through a pharmacological mechanism, there has been no evidence of an IgE response to aspirin. (Cowburn et al, 1998; Lam and Frank Austen, 2000; Pierzchalska and Szczeklik 2003). In addition, CysLTs promoted generation of Th2 cytokines such as IL-4, IL-5, IL-13 and those cytokines also enhanced generation of CysLTs in marchrophages, eosinophils, and monocytes by inducing the expression of CysLTR1 (Thivierge and Pleszczynski, 2000; Thivierge and Rola-Pleszczynski, 2001; Chibana and Fukuda, 2003; Peters-Golden and Sampson, 2003;). IL-4 may also promote tissue eosinophilia by VCAM-1 (Schleimer et al, 1992), and it is an essential cofactor for IgE swiching in B lymphocyte (Del Prete, et al, 1988). There were extensive interactions between CysLTs and other mediators such as macrophage inflammatory protein (MIP)-1β (Frieri et al., 1999),
TNF-α (Elizabeth AM et al., 2002), NF-kB, and RANTES (Kawano T et al., 2003), which are relevant to asthmatic inflammation via a CysLTR1 mediated mechanism.
These previous reports show that the expression of CysLTR1 mRNA can be increased by Th2 inflammatory cytokines, implying a positive feedback by endogenously produced CysLTs. The role of CysLTR1 in total IgE production has a lack of supporting biological data. However, it may be possible that Th2 cytokine-rich environment can up-regulate the expression of CysLTR1 and therefore production of IgE in human with the C-634T polymorphism in CysLTR1.
To investigate the association of the promoter 3 SNPs with CysLTR1 expression, we performed in vitro functional studies using Jurkat cells, U937 cells, and A549 cells and these results suggested that the genotype, ht2 [T-C-G], may be associated with increased expression of the CysLTR1 gene. These results suggest that these genetic variants of CysLTR1 promoter may appear to code for transcription factor binding sites, unknown transcription factor might be capable of binding this region of CysLTR1 promoter, to contribute of CysLTs production. In gel shift assay, a specific band was noted on -475A probe among six probes, -634C, -634T,-475A, -475C, -366A, and -366G. These results suggest that the -475A SNP in the CysLTR1 gene was unknown nuclear protein binding sites, leading to modulated transcriptional activity and lead to expression of CysLTs. Although there was no transcription factor binding site matched on ‘Signal Scan’ and TRANSFAC online program, further studies will be needed to identify the transcription factor bound to these probes (-475A).
Human peripheral blood monocytes (Heise CE, 2000; Figueroa DJ, 2001), eosinophils (Thivierge M, 2000), and lung macrophages (Lynch KR, 1999, Heise CE, 2000) all express both CysLTR1 mRNA and protein. In another study, expression of CysLTR1 was up-regulated on CD45 leukocytes in nasal biopsy specimens obtained from subjects with aspirin sensitive chronic rhihinitis and polyposis, compared with non-aspirin sensitive patients with rhihinitis and polyposis (Sousa AR, 2002). Priming of either human monocytes or monocytes-derived macrophage with IL-4 increased their level of CysLTR1 mRNA and protein expression, and enhanced chemotactic response to LTD4 in vitro (Thivierge and Rola-Pleszczynski, 2001). Similary, CysLTR1 expression and chemotaxis to LTD4 in an eosinophils subline of human granulocytic leukemia cell line, HL-60, were both enhanced by priming with IL-5 (Thivierge M, 2000). In our study, A549 cells demonstrated a 2-fold increase of CysLTR1 mRNA expression when stimulated with IL-4, followed by a significant change in the surface CysLTR1 protein expression, consistent with data by Thivierge et al (2001). In other in vitro study, CysLTs served as eosinophil chemoattractants through a CysLTR1 dependent mechanism (Fregonese L, 2002). Taken together, these observations support the hyphothesis that CysLTs may serve as chemotactic mediators and (or) activating ligands for human effectors leukocyte, and that CysLTR1 expression of certain leukocytes or other tissue cells are modified by Th2 cytokines. In our study results that there were no significant changes observed in the promoter activities when the ht1 [C-A-A] construct were treated with IL-4 (10ng/ml). However, in mutant genotype, ht2 [T-C-G] construct, promoter activity was significantly
increased by IL-4 treatment (p=0.008), which was significantly inhibited by pretreatment of cells with 10-7M of MK-571 (p=0.004). In contrast, dexamethasone increased promoter activity in both mutant and wild genotype (p<0.001).
CysLTR1 selective antagonist, Pranlukast, significantly decreased the amount of Th2 cytokines (Tohda et al.1999) in peripheral blood mononuclear cells (PBMCs) obtained from asthmatic patients and administration at usual dose has an anti-inflammatory effect by inhibition of NF-κB activation (Tomari S et al 2003;
Ishinaga H et al. 2004). Furthermore, MK-571 inhibited IL-5 and TNF-a production in positive feedback by endogenously produced CysLTs (Eliabeth et al., 2002) and partially inhibited NF-κB activation in 1.3% DMSO- differentiated U-937 and Jurkat cells, and IL-6 release from PBMC (Ichiyama T et al 2003). In addition, administration of CysLTR1 antagonists results in improvement of pulmonary function
on bronchial responsiveness to methacholine in aspirin-intolerant asthmatics treated with corticosteroids and better control of asthmatic symptoms (Ishioka S et al 2000), and it suggests that CysLTR1 selective antagonists have been known to have antibronchoconstrictive and anti-inflammatory effects in asthma (Sampson and Szczeklik, 1998; Picado, 2002; Szczeklik and Kielbasa, 2004), particularly in AIA. These results suggest that IL-4 regulation on CysLTR1 promoter activity may be related to genotype differences. Although a transcription factor which can bind to 3 SNPs position was not exactly identified, it may be possible that nucleotide substitutions at three SNPs, modulate an unknown transcription factor binding sequence by IL-4 regulation, resulting in an increased CysLTR1 protein expression, and enhanced pro-inflammatory effects of CysLTs, which may be
inhibited by leukotriene antagonist, particularly MK-571. Therefore, the asthma patients with mutant forms of CysLTR1 promoter may have increased CysLTR1 gene transcription and their asthma may be more dependent on leukotriene formation.
The finding that dexamethasone upregulate transcriptional activity at ht1[C-A-A] construct in CysLTR1 gene was somewhat surprising, since dexamethasone is a well known inhibitor of inflammation and widely used in the treatement of many inflammatory disease. Glucocorticoids are believed to inhibit the inflammation associated molecules such as cytokine, chemokine, and adhesion molecules in many type of cells, including T-lymphocytes, eosinophils, and macrophages via inhibition of activator protein 1 (AP-1) and nuclear factor kappa B (NF-κB) (Carios and Harlan, 1994; Barnes and Karin, 1997). Glucocorticoids suppress activation, proliferation, and survival of these cells and block the release of inflammatory mediators. On the other hand, the proinflammatory effects of glucocorticoids have also been reported in following cases. Glucocorticoids do not appear to suppress CysLTs production in vitro in either normal or asthmatic patients, even at significant systemic doses (O’Shaughnessy, 1993; Dworski, 1994). Certain cytokines particularly IL-2, IL-4, and IL-13, which show increased expression in bronchial biopsy samples from patients with steroid resistant asthma, may induce a reduction in affinity of glucorcorticoid receptors in inflammatory cells, such as T lymphocytes, resulting in local resistance to the anti-inflammatory actions of corticosteroids (Spahn JD et al., 1996; Szefler SJ et al., 1997; Barnes PJ, 2000). Irusen et al (2002) demonstrated that the combination of IL-2 and IL-4 induces steroid resistance in vitro through the activation of p38 MAP kinase, which phosphorylates glucocorticoid
receptors and reduces corticosteroid binding affinity and steroid-induced nuclear translocation of glucocorticoid receptors. In vitro studies show that glucocorticoids inhibit apoptosis of human neutrophils (Cox G 1995 and Liles WC et al, 1995), enhanced the LTA4 biosynthesis occurred in neutrophils isolated from rheumatoid arthritis patients upon treatment with methylpredisione (Thomas E et al, 1995).
Moreover, the fact similar paradoxical upregulation of leukotriene enzymes including 5-LO, FLAP and LTC4S observed also in monocytic cells such as THP-1 (Riddick CA, 1997) and mast cells such as HMG-1(Laviolette M, 1999). In particular, Riddck et al. (1997) reported that both 5-LO and FLAP expression is increased after in vitro dexamethasone treatment of blood monocytes or THP-1 cells in culture (Goppelt-Struebe Met al and Riddick CA, 1997). In the brain, it has been observed that activation of 5-LO increased leukotriene production, is associated with the translocation of 5-LO immmunoreactive protein from cytosol to membrane (Ohtsuki et al, 1995). Other reported that glucocorticoids also induce 5-LO translocation from cytosol to membrane (Tolga Uz et al, 1999).
In this study, dexamethasone upregulated transcriptional activity at ht1[C-A-A]
construct in CysLTR1 gene, is likely to enhance the biosynthesis of equally potent proinflammatory mediator, particularly leukotrienes, in A549 cells.
Our results in the present study that corticosteroids fail to reduce leukotriene synthesis in CysLTR1 gene with major ht1[C-A-A] haplotype, although physiological meaning of these finding are presently difficult to understand, these action of glucocorticoids, probably, because of paradoxical up-regulation of leukotriene synthetic enzyme including 5-LO, LTC4S and FLAP and (or) reduce
corticosteroid binding affinity.
Further studies will be needed to investigate the unknown factors that are involved in the regulation of IL-4 inducible transcription of the CysLTR1 promoter by methods such as antibody supershift assay, and whether a significant therapeutic benefit can be expected by combining two agents, corticosteroids and leukotriene modifiers.
V. CONCLUSION
In conclusion, we have identified novel 3 SNPs in CysLTR1 gene, and these novel genetic variants of the CysLTR1 promoter are associated with AIA risk in male patients. Also, we have shown that the minor haplotype, ht2 [T-C-G], affects the transcriptional activity of CysLTR1 gene which may contribute to an increase of CysLTs expression. Furthermore, this expression was amplified by IL-4, which was significantly inhibited by leukotriene modifier, MK-571.
Our study results suggest that CysLTR1 is a potentially important gene in AIA susceptibility in the Korean population and leukotriene modifier may be more beneficial in patients carrying variant genotype of CysLTR1 promoter polymorphism.
REFERENCES
1. Antczak A, Montuschi P, Kharitonov S, Gorski P, Barnes PJ: Increased exhaled cysteinyl-leukotrienes and 8-isoprostane in aspirin-induced asthma.
Am J Respir Crit Care Med 166: 301-306, 2002
2. Barnes PJ: Steroid-resistant asthma. Eur Resp Rev 10:74-78, 2000
3. Barnes PJ, Karin M: Nuclear factor (NF-kB) a pivotal transcriptional factor in chronic inflammatory diseases. N Engl J Med 336: 1066–1071, 1997
4. Barrett JC, Fry B, Maller J, Daly MJ: Haploview analysis and visualization of LD and haplotype maps. Bioinformatics 21:263–5, 2005
5. Becler K, Hakansson L, Rak S: Treatment of asthmatic patients with a Cysteinyl leukotriene receptor-1 antagonist montelukast (Singulair), decreases the eosinophil survival-enhancing activity produced by peripheral blood mononuclear leukocytes in vitro. Allergy 57:1021-28, 2002
6. Biagaard H: Role in lekotrienes in asthma pathophysiology. Pediatr palmonol 30: 166-76, 2000
7. Busse ww, Lemanske RF Jr: Asthma. N Engl J Med 344:350-62, 2001
8. Carios TM, Harlan JM: Leukocyte-endothelial adhesion molecules. Blood 84: 2068– 2101, 1994
9. Celik G, Bavbek S, Misirligil Z, Melli M: Release of cysteinyl leukotrienes with aspirin stimulation and the effect of prostaglandin E2 on this release from peripheral blood leucocytes in aspirin-induced asthmatic patients. Clin Exp Allergy 31: 1615-1622, 2001
10. Chibana K, Ishii Y, Asakura T, Fukuda T: Up-regulation of cysteinyl leukotriene 1 receptor by IL-13 enables human lung fibroblasts to respond to leukotriene C4 and produce eotaxin. J Immunol 170: 4290-4295, 2003
11. Choi JH, Park HS, Oh HB, Lee JH, Suh YJ, Park CS, Shin HD: Leukotriene related gene polymorphisms in ASA-intolerant asthma: an association with a haplotype of 5-lipoxygenase. Hum Genet. 114:337-344, 2004
12. Coleman RA, Eglen RM, Jones RL, Narumiya S, Shimizu T, Smith WL, Dahlen S, Drazen JM, Gardiner PJ, Jackson WT, Jones TR, Krell RD, Nicosia S: Prostanoid and leukotriene receptors: A progress report from the IUPHAR working parties on classification and nomenclature. Adv Prostaglandin Thromboxane Leukot Res 23:283–285, 1995
13. Cowburn AS, Sladek K, Soja J, Adamek L, Nizankowska E, Szczeklik A,
Lam BK, Penrose JF., Austen FK, Holgate ST, Sampson AP: Overexpression of leukotriene C4 synthase in bronchial biopsies from patients with aspirin-intolerant asthma. J Clin Invest 101:834-846, 1998
14. Cox G: Glucocorticoid treatment inhibits apoptosis in human neutrophils.
Separation of survival and activation outcomes. J Immunol 154: 4719–4725, 1995
15. Crooke ST, Sarau H, Saussy D, Winkler J, Foley J: Signal transduction processes for the LTD4 receptor. Adv Prostaglandin Thromboxane Leukot Res 20:127–137, 1990
16. Dahlen SE, Hedqvist P, Hammarstrom S, Samuelsson B: Leukotrienes are potent constrictors of human bronchi. Nature 288: 484-486, 1980
17. Del Prete: IL-4 is essential co-factor for the IgE synthesis induced in vitro by human T cells clones and their supernatants. J Immunol 140: 4193-4198, 1999
18. Drazen JM ,Austen KF: Leukotrienes and airway responses. Am Rev Respir Dis 136:985–998, 1987.
19. Elizabeth AM, Frank Austen K, Joshu AB: CysLTs and Uridine diphosphate
induce cytokine generation by human mast cells through an IL-4-regulated pathway that is inhibited by leukotriene receptor antagonist. J Exp Med :583-592, 2002
20. Evans JM, Piper PJ, Costello JF.
The pharmacological profile of SK&F 104353-Z2, a potent, selective inhaled antagonist of cysteinyl leukotrienes, in normal man. Adv Prostaglandin Thromboxane Leukot Res 21A:469-72,1991
21. . Figueroa DJ, Breyer RM, Defoe SK, Kargman S, Daugherty BL, Waldburger K, Liu Q, Clements M, Zeng Z, O'Neill GP, Jones TR, Lynch KR, Austin CP, Evans JF: Expression of the cysteinyl leukotriene 1 receptor in normal human lung and peripheral blood leukocytes. Am J Respir Crit Care Med 163:226-233, 2001
22. Fregonese L, Silvestri M, Sabatini F, Rossi GA.: Cysteinyl leukotrienes induce human eosinophil locomotion and adhesion molecule expression via a CysLT1 receptor-mediated mechanism. Clin Exp Allergy 32(5):745-750, 2002
23. Frieri M, Therattil J, Wang SF, Huang CY, Wang YC: Montelukast inhibits interleukin-5 mRNA expression and cysteinyl leukotriene production in ragweed and mite-stimulated peripheral blood mononuclear cells from patients with asthma. J Allergy Clin Immunol 103:203s, 1999
24. Goppelt-Struebe M, Schaefer D, Habenicht AJ: Differential regulation of cyclo oxygenase-2 and 5-lipoxygenase-activating protein (FLAP) expression by glucocorticoids in monocytic cells. Br J Pharmacol 122: 619–624, 1997
25. Hay DW, Torphy TJ, Undem BJ: Cysteinyl leukotrienes in asthma: Old mediators up to new tricks. Trends Pharmacol Sci 16:304–309, 1995
26. Henderson WR: The role of leukotriene inflammation. Ann intern Med 121:
684-697, 1994
27. Hedrick PW: Gametic disequilibrium measures: proceed with caution.
Genetics 117: 331–341, 1987
28. Heise CE, O'Dowd BF, Figueroa DJ, Sawyer N, Nguyen T, Im DS, Stocco R, Bellefeuille JN, Abramovitz M, Cheng R, Williams DL Jr, Zeng Z, Liu Q, Ma L, Clements MK, Coulombe N, Liu Y, Austin CP, George SR, O'Neill GP, Metters KM, Lynch KR, Evans JF: Characterization of the human
cysteinyl leukotriene 2 receptor. J Biol Chem 275:30531-30536, 2000
29. Hirata F, Schiffmann E, Venkatasubramanian K, Salomon D, Axelrod J: A Phospholipase A2 inhibitory protein in rabbit neutrophils induced by glucocorticoids. Proc Natl Acad Sci USA 77: 2533–2536, 1980
30. Horwitz RJ, McGill KA, Busse WW: The role of leukotriene modifiers in the treatment of asthma. Am J Respir Crit Care Med 157:1363–1371, 1998
31. Ichiyama T, Hasegawa S, Umeda M, Terai K, Matsubara T, Furukawa S:
Pranlukast inhibits NF-kappa B activation in human monocytes/macrophages and T cells: Clin Exp Allergy 33(6):802-807,2003
32. Ishioka S, Hozawa S, Haruta Y, Maeda A, Tamagawa K, Watanabe T, Hiyama K, Yamakido M: Effects of pranlukast, a cysteinyl leukotriene antagonist, on bronchial responsiveness to methacholine in aspirin-intolerant asthmatics treated with corticosteroids. : Hiroshima J Med Sci 49(2):105-108,2000
33. Irusen E, Matthews JG, Takahashi A, Barnes PJ, Chung KF, Adcock IM: p38 Mitogen-activated protein kinase-induced glucocorticoid receptor phosphorylation reduces its activity: role in steroid-insensitive asthma. J Allergy Clin Immunol 109:649-57, 2002
34. Ishinaga H, Takeuchi K, Kishioka C, Suzuki S, Basbaum C, Majima Y:
Pranlukast inhibits NF-kappaB activation and MUC2 gene expression in cultured human epithelial cells. Pharmacology 73(2):89-96, 2005
35. Jones TR, Labelle M, Belley M, Champion E, Charette L, Evans J,
Ford-Hutchinson AW, Gautheir J-Y, Lord A, Masson P, McAuliffe M, McFarlane CS, Metters KM, Pickett E, Piechiuta H, Rochette C, Rodger IW, Sawyer N, Young RN, Zamboni R, Abraham WM: Pharmacology of montelukast sodium (Singulair), a potent and selective leukotriene D4 receptor antagonist.
Can J Physiol Pharmacol 73:191–201, 1995
36. Kawano T, Matsuse H, Kondo Y, Machida I, Saeki S, Tomari S, Mitsuta K, Obase Y, Fukushima C, Shimoda T, Kohno S. Cysteinyl leukotrienes induce nuclear factor kappa b activation and RANTES production in a murine model of asthma. J Allergy Clin Immunol 112: 369-74, 2003
37. Krell RD, Aharony D, Buckner CK, Keith RA, Kusner EJ, Snyder DW, Bernstein PR, Matassa VG, Yee YK, Brown FJ, Hesp B, Giles RE : The preclinical pharmacology of ICI 204,219. A peptide leukotriene antagonist.
Am Rev Respir Dis 141:978–987, 1990
38. Lam BK, Frank Austen K: Leukotriene C4 synthase. A pivotal enzyme in the biosynthesis of the cysteinyl leukotrienes. Am J Respir Crit Care Med 161:
S16-9, 2000
39. Lewis RA, Austen KF, Soberman RJ: Leukotrienes and other products of the 5-lipoxygenase pathway: biochemistry and relation to pathobiology in human diseases. N Engl J Med 323: 645-655, 1990
40. Liles WC, Dale DC, Klebanoff SJ: Glucocorticoids inhibit apoptosis of human neutrophils. Blood 86:3181–3188, 1995
41. Lynch KR, O’Neill GP, Liu Q, Im DS, Sawyer N, Metters KM, Coulombe N, Abramovitz M, Figueros DJ, Zeng Z, Connolly BM, Bai C, Austin CP, Chateauneuf A, Stocco R, Greig GM, Kargman S, Hooks SB, Hosfield E, Williams DL Jr, Ford- Hutchinson AW, Caskey CT, Evans JF : Characterization of the human cysteinyl leukotriene CysLT1 receptor. Nature (Lond) 399:789–793, 1999
42. Metters KM: Leukotriene receptors. J Lipid Mediat Cell Signal 1:413–427, 1990
43. Obata T, Okada Y, Motoishi M, Nakagawa N, Terawaki T, Aishita H: In vitro antagonism of ONO-1078, a newly developed anti-asthma agent,
against peptide leukotrienes in isolated guinea pig tissues. Jpn J Pharmacol 60:227–237,1992
44. Ohd JF, Nielsen CK, Campbell J, Landberg G, Lofberg H, Sjolander A:
Expression of the leukotriene D4 receptor CysLT1, COX-2, and other cell survival factors in colorectal adenocarcinomas. Gastroenterology 124: 57-70, 2003
45. Ohtsuki T, Matsumoto M, Hayashi Y, Yamamoto K, Kitagawa K, Ogawa S, Yamamoto S, Kamada T: Reperfusion induces 5-lipoxygenase translocation and leukotriene C4 production in the ischemic brain. Am J Physiol 268:
H1249–H1257, 1995
46. Peters-Golden M, Sampson AP: Cysteinyl leukotriene interactions with other mediators and with glucocorticosteroids during airway inflammation. J Allergy Clin Immunol 111: S37-42, 2003
47. Picado C: Aspirin-intolerant asthma: role of cyclooxygenase enzymes.
Allergy S72: 58-60, 2002
48. Piper PJ: Formation and actions of leukotrienes. Physiol Rev 64, 744-761, 1984
49. Piper PJ, Conroy DM, Costello JF, Evans JM, Green CP, Price JF, Sampson AP, Spencer DA: Leukotrienes and inflammatory lung disease. Ann NY Acad Sci 629:112-119, 1991
50. Pierzchalska M, Mastalerz L, Sanak M, Zazula M, Szczeklik A: A moderate and unspecific release of cysteinyl leukotrienes by aspirin from peripheral blood leucocytes precludes its value for aspirin sensitivity testing in asthma.
Clin Exp Allergy 30: 1785-1791, 2000
51. Pierzchalska M, Szabo Z, Sanak M, Soja J, Szczeklik A.: Deficient prostaglandin E2 production by bronchial fibroblasts of asthmatic patients,
51. Pierzchalska M, Szabo Z, Sanak M, Soja J, Szczeklik A.: Deficient prostaglandin E2 production by bronchial fibroblasts of asthmatic patients,