Cyclooxygenase-2 mRNA Is Downexpressed in Nasal Polyps from Aspirin-sensitive Asthmatics CÉSAR PICADO, JOAN C. FERNANDEZ-MORATA, MANEL JUAN, JORDI ROCA-FERRER, MIREIA FUENTES, ANTONI XAUBET, and JOAQUIM MULLOL Servei de Pneumologia i Al?lèrgia Respiratòria, Hospital Clínic, Departament de Medicina, Universitat de Barcelona; Institut d’Investigacions Biomèdiques August Pi i Sunyer; Unitat d’Immunologia, Hospital Germans Trias i Pujol de Badalona, Barcelona, Spain
Exogenous prostaglandin E2 (PGE2) given by inhalation almost completely abrogates aspirin-induced asthma and the accompanying increase in cysteinyl-leukotrienes production. Cyclooxygenase (COX) may be present in cells in both constitutive (COX-1) and inducible (COX-2) forms. To increase the production of the potentially protective endogenous PGE2, COX-2 should be upregulated. We hypothesize that an abnormal regulation of COX-2 will predispose patients with asthma to develop aspirin-intolerant asthma/rhinitis (AIAR). We therefore examined the expression of COX-2 messenger RNA (mRNA) in healthy nasal mucosa (n 5 11) and in nasal polyps from both patients with AIAR (n 5 8) and those with aspirin-tolerant asthma/rhinitis (ATAR) (n 5 20). After total mRNA extraction, COX-1 and COX-2 mRNA expression were measured using a reverse transcriptase (RT)-semiquantitative PCR technique. Hybrid primers of COX-1 ? glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or COX-2 ? GAPDH were used to create PCR products that were cloned and used as internal standard controls in the competitive PCR reaction. Results are presented as mean 6 standard error of 106 molecules of mRNA/mg of total RNA. No differences in COX-1 mRNA expression were found between nasal mucosa and nasal polyps from both patients with ATAR and those with AIAR. However, COX-2 mRNA expression in nasal polyps from the AIAR group (0.38 6 0.10) was markedly and significantly lower than in polyps from the ATAR group (2.93 6 0.52, sevenfold, p , 0.0001) and nasal mucosa (2.10 6 0.54, sixfold, p , 0.01). These findings suggest that an inadequate COX-2 regulation may be involved in AIAR. Picado C, Fernandez-Morata JC, Juan M, Roca-Ferrer J, Fuentes M, Xaubet A, Mullol J. Cyclooxygenase-2 mRNA is downexpressed in nasal polyps from aspirin-sensitive AM J RESPIR CRIT CARE MED 1999;160:291–296. asthmatics.
Aspirin-intolerant asthma/rhinitis (AIAR) is a syndrome characterized by the association of nasal polyps and asthma attacks precipitated by nonsteroidal antiinflammatory drugs. An increase in the release of cysteinyl-leukotrienes (Cys-LT) in patients with AIAR with respect to those with aspirin-tolerant asthma and rhinitis (ATAR) has been detected after lysine-aspirin challenge in bronchial and nasal secretions (1, 2). Although it has been clearly established that asthma attacks are precipitated by the inhibition of cyclooxygenase (COX), it remains unclear why a similar reaction to aspirin is not seen in patients with ATAR and in healthy subjects (3). The recent discovery that COX may be present in cells in both a constitutive (COX-1) and inducible form (COX-2) (4) has opened new avenues in the interpretation of the phenomenon. We hypothesize that an inadequate regulation of COX-2 may predispose to aspirin-induced asthma and rhinitis exacerbations. This hypothesis is based on the observation that pros(Received in original form August 12, 1998 and in revised form January 15, 1999 ) Supported by grants from FIS 95-0595, Sociedad Española de Neumología y Cirugía Torácica (SEPAR), CIRIT (1996 SGR-71), and SEAeIC. Correspondence and requests for reprints should be addressed to César Picado, Servei de Pneumologia, Hospital Clínic, Villarroel 170, 08036 Barcelona, Spain. E-mail: [email protected]
Am J Respir Crit Care Med Vol 160. pp 291–296, 1999 Internet address: www.atsjournals.org
taglandin E2 (PGE2) reduces Cys-LT synthesis in a number of inflammatory cells (3). Moreover, exogenous PGE2 given by inhalation almost completely abrogates AIAR and the accompanying increase in Cys-LT production (5). Based on the concept that the human body usually increases the production of PGE2 by upregulating COX-2, our hypothesis established that an insufficient upregulation of COX-2 in patients with AIAR will leave these patients without the protective effect of endogenous PGE2 and therefore more susceptible to the effect of aspirin. We thus examined the expression of COX-2 mRNA in healthy nasal mucosa and in nasal polyps from both patients with AIAR and those with ATAR.
METHODS Subjects Human nasal mucosa (NM) was obtained from 11 healthy subjects (eight men, three women; mean age, 34 6 2.5 yr) undergoing nasal corrective surgery for turbinate hypertrophy or septal dismorphy. Nasal polyps (NP) were obtained from 20 aspirin-tolerant patients (15 men, five women; mean age, 53 6 2.3 yr) and from eight aspirin-intolerant patients (six men, two women; age, 49 6 5.3 yr) undergoing nasal polypectomy. At the time of surgery, NM and NP specimens were snap-frozen in liquid nitrogen and stored at 2808 C. Among the 20 patients with NP who were tolerant to aspirin (ATAR), 13 had rhinitis alone, whereas seven had rhinitis and asthma.
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During the course of the disease, all patients with ATAR with NP had received treatment with topical corticosteroids and/or short courses of oral corticosteroids. At the time when polypectomy was carried out, seven patients were receiving topical corticosteroid therapy, and two were also receiving oral corticosteroids (prednisone, 5 to 40 mg/d). The remaining 11 patients with ATAR had discontinued both corticosteroid therapies at least 3 wk prior to polypectomy (Table 1). The eight patients with aspirin intolerance had asthma and rhinitis (AIAR). During the course of the disease, all of them had been treated with topical corticosteroids and/or short courses of oral corticosteroids. At the time of surgery, three patients were receiving topical corticosteroids (budesonide, 200 to 800 mg/d), and two were receiving both topical (budesonide, 200 to 400 mg/d) and oral (prednisone, 5 to 40 mg/d) corticosteroids. The remaining three patients with AIAR
RT Semiquantitative PCR We developed a reverse transcriptase (RT) competitive polymerase chain reaction (PCR) to measure COX-1 and COX-2 messenger RNA (mRNA) expression. This method relies on the addition of known
Nasal polyps (ATAR) P1 P2 P3 P4 P5
48 41 55 51 68
M M M F M
NP NP NP, As NP NP
Negative Negative Negative Negative Negative
P6 P7 P8
61 38 65
M M F
NP, As NP NP
Negative Negative Negative
P9 P10 P11 P12
64 48 68 38
M M M F
NP NP NP, As, AR NP, As
Negative Negative Dpt, grass, olive Negative
P13 P14 P15 P16 P17 P18
51 49 61 40 63 57
M F M F M M
NP NP NP NP, As NP NP, As, AR
Negative Negative Negative Negative Negative Parietaria
None None None Budesonide (nasal) Prednisone (oral) None None None Budesonide (nasal) Budesonide (nasal) Budesonide (nasal)
25 28 56
M F M
NP, As NP, As, AR NP, AR
Negative HDM, Dpt Dpt, Df, Parietaria
65 52 52
M M M
NP, As NP, As, AR NP, As, AR
Negative Dpt Grass-olive
NP, As, AR NP, As
None Budesonide (nasal) Budesonide (nasal) Prednisone (oral) Budesonide (nasal) None Budesonide (nasal) Prednisone (oral) Budesonide (nasal) None
38 35 26 42 24 36 24 25 34 45 46
M M M F M M M M M F F
TH TH TH TH TH TH TH TH TH TH TH
Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative
None None None None None None None None None None None
P24 P25 P26 P27 P28 Nasal mucosa M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11
had discontinued both topical and oral corticosteroid therapies at least 3 wk before polypectomy (Table 1). The diagnosis of aspirin intolerance was made on the basis of both a history of asthma attacks precipitated by nonsteroidal antiinflammatory drugs and a positive aspirin provocation test. Two patients were challenged by the oral route and six by using a nasal challenge according to methods previously reported (6, 7). All subjects agreed to participate in the study, which was approved by the Ethics Committee of our Institution.
CHARACTERISTICS OF PATIENTS UNDERGOING TURBINECTOMY (M) OR POLYPECTOMY (P)
P20 Nasal polyps (AIAR) P21 P22 P23
None Budesonide (nasal) Budesonide (nasal) None Budesonide (nasal) Prednisone (oral) Budesonide (nasal) None Budesonide (nasal)
Aspirin (oral) None None None None None None Budesonide (inh) Formoterol None None Ranitidin None None None Captopril None None Budesonide (inh) Salbutamol (inh) Budesonide (inh) Ipratrop. Br. (inh) Salmeterol Insulin None BDP (inh) None None None Budesonide (inh) Budesonide (inh) Budesonide (inh) None None None None None None None None None None None
Definition of abbreviations: AIAR 5 aspirin-intolerant asthma/rhinitis; AR 5 allergic rhinitis; As 5 asthma; ATAR 5 aspirin-tolerant asthma/ rhinitis; Df 5 Dermatophagoides farinae; Dpt 5 Dermatophagoides pteronyssinus; HDM 5 house dust mite; NP 5 nasal polyp; TH 5 turbinate hypertrophy.
Picado, Fernandez-Morata, Juan, et al.: Cyclooxygenase-2 in Nasal Polyps and Mucosa amounts of a complementary DNA (cDNA) competitor molecule (internal standard control, SC) in the amplification reactions (8). Hybrid primers of COX-1 ? glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or COX-2 ? GAPDH were used to create PCR products, which were cloned and used as internal SC in the PCR. To set the RT semiquantitative PCR, we carried out the following procedures (Figure 1). First, internal SC, which basically was a GAPDH fragment with the COX-2 (or COX-1) sequence at both ends, was amplified by single PCR using hybrid primers. Secondly, total RNA was extracted from samples and reverse transcribed to cDNA. Thirdly, after the previous addition of different concentrations of SC to a constant amount of sample cDNA, a competitive PCR was performed using specific primers for COX-1 and COX-2, which coamplified the mRNA of both COX-1 and COX-2 and the internal SC. Finally, after amplification, PCR products were resolved by gel electrophoresis, and the yields of amplified SC and sample products were quantified.
Construction of Competitors and Design of the Internal Standard Controls Internal SC obtainment by PCR. To obtain SC, we designed hybrid primers with two regions: the 59 region anneals to COX-2 or COX-1 and the 39 region anneals to GAPDH (Table 2). Amplification conditions for 35 cycles of composite primers were: denaturing at 958 C for 1 min, annealing for 2 min (at 558 C for COX-1 ? GAPDH and at 588 C for COX-2 ? GAPDH), and extension at 728 C for 1 min. These primers amplified a band of 641 base pairs, which was basically a GAPDH (the glyceraldehyde-3-P-dehydrogenase constitutive gene) fragment with the COX-2 or COX-1 sequences at both ends. We used a previous amplified GAPDH fragment as a PCR template. These COX-1 ? GAPDH (SC1) and COX-2 ? GAPDH (SC2) fragments were electrophoresed and electroeluted with the Biotrap Starterkit (Schleichker & Schuell, Dassel, Germany) for cloning. Internal SC cloning. To clone the PCR products of SC1 and SC2, we used the Original TA cloning kit (Invitrogen, Santa Ana, CA) following the manufacturer’s instructions. Briefly, electroeluted fragments were ligated into pCR2.1 plasmid (Invitrogen) and cloned into INVaF’ Escherichia coli strain. Selected colonies were analyzed by automatic sequencing, with the complete PCR product being amplified to obtain large quantities of the corresponding SC. After plasmid
TABLE 2 AMPLIFIED DNA SIZE (BASE PAIRS) AND SEQUENCES OF SPECIFIC PRIMERS USED FOR SEMIQUANTITATIVE RT–PCR Gene
Sequence of “Primers” 5’→3’
5’ TGC CCA GCT CCT GGC CCG CCG CTT 3’ GTG CAT CAA CAC AGG CGC CTC TTC
5’ TTC AAA TGA GAT TGT GGG AAA ATT GCT 3’ AGA TCA TCT CTG CCT GAG TAT CTT
5’ CCA CCC ATG GCA AAT TCC ATG GCA 3’ TCT AGA CGG CAG GTC AGG TCC ACC
EcoRI digestion, SC fragments were resolved in a 1% agarose gel and electroeluted. Internal SCs were electrophoresed and loaded in parallel with serial dilutions of Lamda phage (5, 10, 20 mg) digested with HindIII. Bands were compared by densitometric analysis, and the concentration of each SC dilution could be determined.
Isolation and Quantification of Target RNA Total RNA extraction. NM and NP specimens were homogenized with a polytron (Kinematica AG). Total RNAs from tissues were obtained with TRI-reagent total RNA extraction kit (MRC, Cincinnati, OH). Tissue samples were incubated with 2 U of RNAse-free DNAse (Promega, Madison, WI) for 30 min at 378 C in a buffer containing 40 mM TRIS-Cl (pH, 7.8), 10 mM NaCl, and 6 mM MgCl2. RNA was finally resuspended in 0.05% diethylpyrocarbonate-treated (RNAsefree) water. To check RNA integrity, samples were resolved in a 1% agarose denaturing gel, and prequantified by comparison with 28s and 18s bands. Finally, total RNA samples were quantified by densitometric analysis with respect to four concentrations of control RNA loaded in parallel. Reverse transcription (RT). Four micrograms of total RNA was reverse transcribed to cDNA in a 30-ml final volume reaction using 400 U of Moloney murine leukemia virus reverse transcriptase (MmLVRT). The reaction was carried out at 378 C for 1 h, with a 13 RT buffer (50 mM TRIS-Cl, 40 mM KCl, 4 mM MgCl2, and 10 mM dithiotreitol at pH 8.3), 3 mM dNTPs, 20 U RNAsin (Promega), and 1.5 mg random primers (hexanucleotides). The reaction was heated (958 C for 10 min) and the samples kept on ice.
Competitive PCR Using Internal SC
Figure 1. Schematic diagram of RT semiquantitative competitive PCR using a competitor DNA (internal standard control). Serial dilutions (three for COX-1 ? GAPDH; four for COX-2 ? GAPDH) of the competitor (standard control, SC) were added to a constant amount of target cDNA. After amplification, PCR products were resolved by gel (1% agarose) electrophoresis and stained with ethidium bromide in TRIS borate/EDTA buffer. The relative amounts of target and SC products of varying sizes in each sample from nasal mucosa or polyps were analyzed and compared. After correction for size differences, the initial amounts of target and competitor (SC) products were assumed to be equal in the reactions when their molar ratio was considered to be equal (log ratio 5 0). In the present diagram, an electrophoresed gel of an ATAR polyp is displayed. The molecular weight standard (Column 1) and four decreasing dilutions (Columns 2 to 5) of internal SC2 (COX-2 ? GAPDH, 641 bp), which compete with a constant amount of the target cDNA (COX-2 mRNA, 305 bp), are shown. The relative quantities of COX-2 or COX-1 to competitors (SCs) were quantified and compared by densitometric analysis (see METHODS for more details).
A previous prequantification of this PCR led us to use the synthesized cDNA diluted 1:8 times. Specific primers for COX-2, COX-1 were selected by their optimal properties by oligo 4.DR (Table 3). To establish a semiquantitative PCR, we introduced serial dilutions of the internal SC into the reaction. Known quantities of the SC (SC1 or SC2) were loaded into each PCR tube with a constant amount of the diluted target cDNA. Three SCs were selected for COX-1 (103, 104, 105 copies) and four for COX-2 (103, 104, 105, 106 copies). The SC had complementary sequences to COX-1 and COX-2 primers, so there was a competitive reaction to coamplify the SC and
TABLE 3 SEQUENCES OF HYBRID PRIMERS (hCOX-1 ? GAPDH AND hCOX-2 ? GAPDH) USED TO GENERATE INTERNAL STANDARD CONTROLS (SC) FOR SEMIQUANTITATIVE PCR Gene hCOX-1 ? GAPDH
Sequence of SC “Hybrid Primers” 5’→3’ 5’ TGC CCA GTC CCT GGC CCG CCG CTT ? ? CCA CCC ATG GCA AAT TCC ATG GCA 3’ GTG CAT CAA CAC AGG CGC CTC TTC ? ? TCT AGA CGG CAG GTC AGG TCC ACC
hCOX-2 ? GAPDH
5’ TTC AAA TGA GAT TGT GGG AAA ATT GCT ? ? CCA CCC ATG GCA AAT TCC ATG GCA 3’ AGA TCA TCT CTG CCT GAG TAT CTT ? ? TCT AGA CGG CAG GTC AGG TCC ACC
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the target cDNA. The competitive PCR was carried out with a buffer containing: 10 mM TRIS-HCl (pH, 8.3), 50 mM KCl, 2 mM MgCl2, 0.2 mM dNTPs, 0.5 mM primers, and 1 U Taq Pol, in a 50-ml final volume. Amplification was performed for 30 cycles using the following cycle conditions: denaturing at 958 C for 1 min, annealing for 2 min with different temperatures for each specific amplification (608 C for COX-1 and GAPDH, and 588 C for COX-2), and extension at 728 C for 1 min. Solutions were covered with mineral oil and reactions were carried out with a mini thermal cycler (MJ Research, Watertown, MA). Thus, SC was coamplified with each target cDNA by specific COX-1 or COX-2 primers (Figure 1). PCR number cycles were defined to determine the proportional amplification zone, and the quantity of DNA stained with ethidium bromide was evaluated by using a linear range value in the densitometer.
Analysis of PCR Products PCR products (amplified cDNA) were resolved by gel (1% agarose) electrophoresis and stained with ethidium bromide in TRIS borate/ ethylenediaminetetraacetic acid (EDTA) buffer. Images from electrophoresed gels were captured by a camera in a computer-assisted imaging system (photometer-integrator), and the relative quantities of COX-2 or COX-1 to competitors (SCs) were quantified and compared by densitometric analysis using the software Bio-Profil (Vilber Lourmat, Marne La Vallée, France). To correct the differences in nucleotide number, the density ratio of the SC band to the target band was multiplied by a correction factor that was 0.44 for COX-2 (305/ 691) and COX-1 (303/691) (8). The logarithm of the corrected ratio was then plotted versus the logarithm of the initial amount of SC added in the competitive PCR (8). At the competition equivalence point (log ratio 5 0), the initial concentration of the target (COX-2 or COX-1) corresponds to the initial concentration of the added SC (Figure 1) (8). The relative amounts of target and SC products, differing in molecular weight size (COX-2, 305 bp; COX-1, 303 bp; SCs, 691 bp), were compared in each tissue sample from nasal mucosas and polyps.
Figure 2. Expression of COX-1 mRNA in nasal mucosas and nasal polyps. Expression of COX-1 mRNA levels in nasal mucosa (n 5 11), and nasal polyps from aspirin-tolerant asthma/rhinitis patients (ATAR, n 5 20) and aspirin-intolerant asthma/rhinitis patients (AIAR, n 5 8). Columns for polyps from patients who were (1) and who were not (2) receiving corticosteroid treatment are displayed. Results are presented as 10 6 molecules of COX-1 mRNA/ mg of total RNA. No significant differences in mean COX-2 mRNA expression were found (Kruskal-Wallis test). No significant differences were found between patients who were receiving corticosteroid treatment and those who were not.
Data Analysis Results are expressed as arithmetic mean 6 SEM of 106 molecules of COX-2 or COX-1 mRNA/mg of total RNA. Kruskal-Wallis test was used to analyze the means of COX-1 and COX-2 mRNA of the three groups (nasal polyps from AIAR and ATAR, and nasal mucosas). Mann-Whitney U test was used for between-group comparisons. A p value , 0.05 was regarded as statistically significant.
RESULTS The characteristics of the subject population are presented in Table 1. Nasal mucosa was obtained from subjects significantly younger than patients with nasal polyposis. However, no correlation was found between age and either COX-1 mRNA or COX-2 mRNA levels. As shown in Figure 2, no differences in COX-1 mRNA expression were found between nasal mucosa (0.36 6 0.13) and nasal polyps from either patients with ATAR (0.71 6 0.14) or those with AIAR (0.48 6 0.21). In contrast, the mean levels of COX-2 mRNA expression in nasal polyps from the AIAR group (0.38 6 0.10) were significantly lower than in polyps from the ATAR group (2.93 6 0.52, sevenfold, p , 0.0001) and in nasal mucosas (2.10 6 0.54, sixfold, p , 0.01) (Figure 3). There were no differences in COX-2 mRNA mean levels between ATAR nasal polyps and nasal mucosas. The COX-2 mRNA levels in polyps from patients who were receiving corticosteroid therapy at the time when polypectomy was done were higher (ATAR, 3.17 6 0.73 and AIAR, 0.52 6 0.11) than COX-2 mRNA levels (ATAR, 2.73 6 0.75 and AIAR, 0.15 6 0.10) in polyps from patients
Figure 3. Expression of COX-2 mRNA in nasal mucosa and nasal polyps. Expression of COX-2 mRNA in nasal mucosa (n 5 11), and nasal polyps from aspirin-tolerant asthma/rhinitis patients (ATAR, n 5 20) and aspirin-intolerant asthma/rhinitis patients (AIAR, n 5 8). Columns for polyps from patients who were (1) and who were not (2) receiving corticosteroid treatment are displayed. Results are presented as 106 molecules of COX-2 mRNA/mg of total RNA. Significant differences were found in COX-2 mRNA expression between polyps from AIAR and polyps from ATAR and nasal mucosa (**p , 0.01, ***p , 0.001, Kruskal-Wallis test and Mann-Whitney U test for between-group comparisons). No significant differences were found between patients who were or who were not receiving corticosteroid treatment.
Picado, Fernandez-Morata, Juan, et al.: Cyclooxygenase-2 in Nasal Polyps and Mucosa
who had discontinued corticosteroid therapy before polypectomy. However, the differences were not statistically significant. Most asthmatic patients were receiving bronchodilators and inhaled corticosteroids (Table 1). There were no significant differences in COX-2 mRNA levels between polyps from ATAR patients with asthma (2.53 6 0.94) and those without asthma (3.14 6 0.64). One patient from the ATAR group was receiving regular low-dose aspirin (150 mg/d). The COX-2 mRNA (3.23) value of this patient was close to the mean of the group.
DISCUSSION The four main findings of our study are: (1) COX-2 is expressed in the nasal mucosa; (2) the inflammatory process present in nasal polyps is not accompanied by an upregulation of COX-2 mRNA expression; (3) corticosteroid treatment does not have an inhibitory effect on polyp COX-2 mRNA expression; and (4) COX-2 mRNA is markedly downregulated in polyps from patients with AIAR. It is generally believed that the COX-1 gene is constitutively expressed, whereas COX-2 is an inducible gene (4). However, COX-2 expression is detected at basal state in gastric mucosa (9) and in the central nervous system (10). We report that COX-2 mRNA is also expressed in nasal mucosa. These findings suggest that COX-2 induction has diverse pathophysiologic roles depending on the tissues where its expression is induced. Why COX-2 is induced in some tissues is unclear, but its constant presence suggests that this enzyme may play a physiologic and complementary role to COX-1. COX-2 is induced in most cells in response to proinflammatory stimuli, suggesting that the induction of the enzyme plays a role in generating inflammatory prostanoids (4). Nasal polyps represent an inflamed tissue that contains a high number of activated eosinophils and degranulated mast cells (11). It is interesting to note that we did not find any differences in COX-2 mRNA expression between nasal mucosa and nasal polyps from patients with ATAR. These results are in keeping with previous studies that reported that normal mucosa and nasal polyp synthesize similar amounts of PGE2 (12). COX-2 mRNA expression was dramatically downregulated in nasal polyps from patients with AIAR. In keeping with our finding, Kowalski and colleagues (13) have recently reported that cultured epithelial cells obtained from patients with AIAR produce much less PGE2 than do those cultured from patients with ATAR. Taken together, these findings suggest that patients with AIAR could not adequately respond to inflammatory insults by releasing PGE2. PGE2 reduces LT synthesis in a number of inflammatory cells, including human eosinophils (14) and basophils (15). We hypothesize that an inadequate regulation of COX-2 will cause a chronic failure in the production of PGE2, which is the brake that prevents an exaggerated production of LTs in AIAR exacerbations. Our findings reporting a low COX-2 expression in polyps from patients with AIAR support this hypothesis. The results of our study differ with respect to previous studies that show that immunostaining for COX-2 is not different in bronchial mucosa from patients with AIAR with respect to that from those with ATAR (16, 17). Differences in the methodology used to evaluate COX-2 (RT–PCR versus immunostaining) and/or in the tissues (nasal mucosa and polyps versus bronchial biopsies) may account for discrepancies in the results. It is interesting to note that although Cowburn and colleagues (16) did not find any statistically significant differences in COX-2 expression, the lowest value of COX-2 immunostaining corresponded to AIAR. The limited number of patients studied could account for this lack of significance.
A recent study has reported an increase in leukotriene C4 (LTC4) synthase expression in AIAR bronchial biopsies (16). It has been suggested that this finding can account for the increased capacity of aspirin to generate Cys-LT both basally and after aspirin challenge in AIAR. Because aspirin can reduce PGE2 production to the same extent in AIAR and in ATAR, it has been suggested that this finding may also resolve the paradox that aspirin does not trigger a similar rise in Cys-LT in ATAR. According to this hypothesis, aspirin should reduce PGE2 synthesis with the same efficacy in all asthmatics, but more LTC4 is released by cells after the suppression of the braking effect of PGE2 in AIAR, leading to an overproduction of Cys-LT in these patients only. We have shown a marked downregulation of COX-2 mRNA in polyps from patients with AIAR. Whether this abnormality is somehow linked to an enhanced LTC4 synthase expression deserves further investigation. Corticosteroids have been shown to significantly attenuate COX-2 upregulation in vitro (18). However, we found no effect of inhaled and oral corticosteroid therapy on COX-2 expression in nasal polyps. This observation is in keeping with a recent study reporting that prednisolone treatment results in a significant increase of COX-2 mRNA and protein in blood monocytes and alveolar macrophages obtained from atopic subjects (19). However, dexamethasone effectively blocked the stimulatory effect of endotoxin on COX-2 expression in in vitro monocytes obtained from the same patients, demonstrating the existence of a dichotomy in the in vivo response as opposed to the in vitro one (19). In summary, we report a marked downregulation of COX-2 mRNA expression in nasal polyps from aspirin-sensitive asthmatics. The origin of the decreased expression of COX-2 mRNA in nasal polyps from patients with AIAR is unclear. COX-2 mRNA expression is regulated by cytokines, which activate the nuclear factor kappa B (NF-kB) transcription factor (20). Further studies are needed to explain whether an alteration in the NF-kB and/or other regulatory mechanism is responsible for the abnormal expression of COX-2 mRNA in patients with AIAR. References 1. Sladek, D., R. Dworski, J. Soja, J. R. Sheller, E. Nizankowska, J. A. Oates, and A. Szczeklik. 1994. Eicosanoids in bronchoalveolar lavage fluid of aspirin-intolerant patients with asthma after aspirin challenge. Am. J. Respir. Crit. Care Med. 149:940–946. 2. Picado, C., I. Ramis, J. Roselló, J. Prat, O. Bulbena, V. Plaza, J. M. Montserrat, and E. Gelpí. 1992. Release of peptide leukotriene into nasal secretions after local instillation of aspirin in aspirin-sensitive asthmatic patients. Am. Rev. Respir. Dis. 145:65–69. 3. Szczeklik, A. 1997. Mechanisms of aspirin-induced asthma. Allergy 52: 613–619. 4. Smith, W. L., and D. L. De Witt. 1996. Prostaglandin endoperoxide H synthases-1 and -2. Adv. Immunol. 62:167–215. 5. Sestini, P., L. Armetti, G. Gambaro, M. G. Pieroni, R. M. Refini, A. Sala, G. C. Folco, S. Bianco, and M. Robuschi. 1996. Inhaled PGE 2 prevents aspirin-induced broncho-constriction and urinary LTE 4 excretion. Am. J. Respir. Crit. Care Med. 153:572–575. 6. Castillo, J. A., and C. Picado. 1986. Prevalence of aspirin-intolerance in a hospital population. Respiration 50:153–157. 7. Casadevall, J., J. Mullol, and C. Picado. 1997. Acoustic rhinometry in the diagnosis of aspirin-induced asthma by lysine-aspirin challenge (abstract). Eur. Respir. J. 10(Suppl. 25):476. 8. Auboeuf, D., and H. Vidal. 1997. The use of the reverse transcriptioncompetitive polymerase chain reaction to investigate the in vivo regulation of gene expression in small tissue samples. Anal. Biochem. 245: 141–148. 9. Iseki, S. 1995. Immunochemical localization of cyclo-oxygenase-1 and cyclo-oxygenase-2 in the rat stomach. Histochem. J. 27:323–328. 10. Yamagata, K., K. I. Andreasson, W. E. Kaufman, C. A. Barnes, and P. F. Worley. 1993. Expression of a mitogen inducible cyclooxygenase in
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