Genetic Mechanisms in Aspirin-induced Asthma

ANDRZEJ SZCZEKLIK and MAREK SANAK

Department of Medicine, Jagiellonian University School of Medicine, Kraków, Poland

	INTRODUCTION

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Although cases of violent, acute bronchospasm after aspirin ingestion were reported shortly after introduction of aspirin into therapy, only in more recent years has this type of asthma attracted the growing interest of clinicians, pharmacologists, biochemists, and molecular biologists. It has distinct features that differentiate it clearly from other types of asthma. Its course is characteristic, suggesting a common principle as the driving force. And in the background there is an alteration of arachidonic acid metabolism that contributes to the clinical presentation. Indeed, aspirin-induced asthma appears to be a cysteinyl-leukotriene (Cys-LT)-driven syndrome. Its association with leukotriene-C₄ synthase polymorphism points to a genetic influence. Would patients with this type of asthma benefit most from treatment with antileukotriene drugs, rapidly reaching the market? This question and others related to the genetic features of aspirin-induced asthma are discussed here.

	NATURAL HISTORY AND CLINICAL PRESENTATION

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The distinct clinical syndrome, called aspirin-induced asthma (AIA) (1), affects about 10% of adults with asthma, more often women than men. In most patients, the first symptoms occur during the third decade of life as intense rhinitis. Over a period of months, chronic nasal congestion with rhinorrhea develops; physical examination often reveals nasal polyps and anosmia. Bronchial asthma and intolerance to aspirin develop subsequently, on average within 4 yr. The intolerance presents as a unique picture: within 1 h of ingestion of aspirin, an acute asthma attack occurs, often accompanied by rhinorrhea, conjunctival irritation, and scarlet flushing of head and neck. Aspirin is a common precipitating factor of life-threatening attacks of asthma; in a large survey, 25% of patients with asthma who required emergency mechanical ventilation were found to be aspirin intolerant (5).

Asthma runs a protracted course, despite the avoidance of aspirin and cross-reactive drugs. The blood eosinophil count is raised, and eosinophils are present in airways. Atopy traits are not rare, and may be even more common than in the general population (6). While a patient's clinical history might raise suspicion of aspirin-induced asthma, the diagnosis can be established with certainty only by aspirin challenge. For the majority of patients, aspirin intolerance, once developed, remains for the rest of their lives.

	FAMILIAL OCCURRENCE

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Only a few families have been described as presenting with "aspirin triad" (asthma, aspirin intolerance, and nasal polyposis). The Mennonite family reported by Lockey and colleagues (7), and the pair of sisters reported by Miller (8), have much in common in that they conformed in large part to the classic description of aspirin-intolerant asthma. In contrast, the family presented by von Maur and coworkers (9) included four of six affected individuals under 17 yr of age at onset; none had polyposis. A considerable discordance in clinical manifestation of the disease was noted in identical twin sisters, pointing to the influence of environmental factors (7, 10). In a non-Mennonite family, studied by Lockey and coworkers (7), two siblings suffered from aspirin triad, while the third had intrinsic asthma that improved after ingestion of aspirin. The rare syndrome of asthma relieved by aspirin bears much clinical resemblance to aspirin-intolerant asthma, and in a sporadic patient one form may evolve into the other (11).

AIA is not a single Mendelian trait. Familial occurrence of aspirin hypersensitivity was reported by 5.1% of 365 AIA patients studied in the European Network on Aspirin-Induced Asthma (12, 13). The affected members were usually siblings, had a high incidence of rhinitis, and the course of their asthma, as judged by the number of hospital admissions, appeared to be more severe than in the group without familial occurrence. Studies of genetic polymorphism are advisable in such families. We have seen two sisters (27 and 20 yr old) with aspirin intolerance. In the older, who was asthmatic, aspirin challenge precipitated an asthma attack. In the younger, suffering from perennial rhinitis with blood eosinophilia but without asthma, oral aspirin challenge triggered marked nasal and occular symptoms, was accompanied by a profound fall in nasal flow, but bronchial obstruction was absent. Both sisters had a leukotriene-C₄ synthase (LTC4S) synthase allelic variant predisposing to aspirin intolerance, and in both the disease started as rhinitis at the age of 12 yr. Although their mother was asthmatic, the father was atopic, and some other members of the family had moderate eosinophilia and/or reversible bronchial obstruction, the aspirin challenge was negative in all (Figure 1).

View larger version (13K): [in this window] [in a new window]   Figure 1.   Pedigree of the family in which two sisters have aspirin-intolerant rhinitis/asthma (marked with arrows). C and A represent LTC4S alleles.

	CHRONIC EOSINOPHILIC INFLAMMATION OF AIRWAY

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The airways of AIA patients show signs of persistent inflammation with marked eosinophilia, epithelial disruption, cytokine production, and upregulation of adhesion molecules (14). In bronchial biopsies eosinophils are fourfold more numerous than in aspirin-tolerant subjects with asthma, and 15-fold more numerous than in normal biopsies (15). Both increase (14) and suppression (15) of mast cell numbers as compared with normal individuals were reported, the discrepancies probably reflecting mast cell activation.

Eosinophil infiltration of airway tissue appears to be a central feature of AIA. The airway expression of interleukin 5 (IL-5), known to be involved in recruitment, activation, maturation and perpetuation of survival of eosinophils, is markedly increased in patients with AIA (4, 15). Immunohistochemical analysis of bronchial biopsies indicates that eosinophils are the predominant site for leukotriene-C₄ synthase and that this enzyme is distinctly overrepresented in patients with AIA (16).

The persistent airway inflammation could result from a non-IgE-mediated reaction to an endogenous or exogenous antigen, possibly an autoantigen or a chronic viral infection (17). These possibilities are supported by the finding of elevated markers of autoimmunity, enhanced IgG₄ synthesis, and HLA association with AIA (18).

	CENTRAL ROLE OF EICOSANOIDS IN PATHOGENESIS

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Although clinical reactions precipitated by aspirin in patients with asthma are reminiscent of immediate-type hypersensitivity reactions, numerous attempts to demonstrate specific antibodies against aspirin or its derivatives have been unsuccessful. Furthermore, in patients with AIA, asthmatic attacks can be precipitated not only by aspirin, but by several other analgesics with different chemical structures, a fact that makes immunological cross-reactivity most unlikely (19). Over the years, the importance of arachidonic acid metabolism in the pathogenesis of AIA became apparent, and led to the development of the cyclooxygenase (COX) theory (20). The theory proposes that precipitation of asthma attacks by aspirin is not based on antigen-antibody reactions, but stems from the pharmacological action of the drug, namely specific inhibition in the respiratory tract of the enzyme COX. The original observations that the drug intolerance can be predicted on the basis of its in vitro inhibition of COX have been consistently reaffirmed over the ensuing years. After aspirin desensitization, cross-desensitization to other nonsteroidal antiinflammatory drugs (NSAIDs) that inhibit COX also occurs.

The cyclooxygenase response to aspirin is altered in AIA (21). Such alteration may result from an acquired change in the COX gene(s), perhaps as a consequence of a latent viral infection (17). An altered COX molecule in the reaction with aspirin could produce unknown metabolites that stimulate 5-lipoxygenase (5-LO). It might also be imagined that in AIA, the signal for 5-LO activation stems from removal by aspirin of prostaglandin E₂ (PGE2), a prostaglandin that inhibits leukotriene biosynthesis and appears to play a special role in this type of asthma. All these explanations are conjectural and await experimental testing.

Cys-LTs have emerged as major mediators in AIA (4, 22, 23). Most patients with AIA excrete 2- to 10-fold higher amounts of LTE₄ in urine than do other patients with asthma who tolerate aspirin well (4, 23). Aspirin challenge results in a temporary, although significant, increase in urinary LTE₄ excretion (24). Cys-LTs are also released into the nasal cavity after nasal challenge with aspirin (25, 26) and into the bronchi after challenge with lysine-aspirin (21).

The expression of LTC₄ synthase, the terminal enzyme for Cys-LT production, was 5-fold higher in bronchial biopsies from patients with AIA than in patients with asthma who tolerated aspirin well, and 19-fold higher than in normal biopsies (15, 16). In contrast, expression of other leukotriene pathway enzymes (5-LO and 5-LO-activating protein [FLAP]) was similar in all subject groups. Increased LTC₄ synthase expression in AIA biopsies was the only enzyme or cell marker that correlated significantly with bronchial hyperresponsivness to inhaled lysine-aspirin, a definitive clinical measure of aspirin sensitivity. Overexpression of LTC₄ synthase protein in eosinophils infiltrating the bronchial mucosa of patients with AIA could result from genetic polymorphism of the enzyme and/or intense cytokine signaling within the local environment. We investigated both these possibilities.

	GENETIC POLYMORPHISM OF THE ENZYMES CONTROLLING BIOSYNTHESIS OF LEUKOTRIENES

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Oxygenation of arachidonic acid (AA) at position C-5 of the molecule is performed by two proteins5-LO and FLAP acting in tandem. Genes encoding both proteins have numerous regulatory elements in their promoter regions, which can control the cell-specific expression and regulate the transcription rate. Protein studies and enzymatic activity assays are complicated by translocations of proteins on activation from the cytosol to the cell membrane, while reaction products have short lifetimes. As an alternative approach, a semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) technique has been used to measure messenger RNA and estimate the change in the transcription rate of the genes. In patients with asthma, increased expression of FLAP and 5-LO mRNAs has been reported in peripheral blood mononuclear cells (PBMCs) by this method (27). Analysis of the promoter region of the 5-LO enzyme revealed a polymorphism in a variable number of tandem repeats (VNTRs) of potential influence on the transcription rate of the gene (28). However, no difference in 5-LO gene variability was found between subjects with and without asthma. This genetic variation does not seem to contribute to susceptibility to AIA, the observation was confirmed by normal expression of 5-LO in AIA bronchial biopsies (16).

The product of 5-lipoxygenation of AA, leukotriene A₄ (LTA₄), is released by granulocytes. Avid intra- and transcellular metabolism of LTA₄ and its nonenzymatic conversion to LTB₄, a potent chemoattractant, seem to be intact in patients with AIA.

The enzyme leukotriene-C₄ synthase (LTC4S), present in eosinophils, basophils, macrophages, and platelets, alternatively converts LTA₄ to Cys-LTs. The LTC4S gene has been cloned and its molecular organization, including the promoter region has been published (29, 30). A single-nucleotide transversion of adenine to cytosine 444 bases upstream of the translation start creates a common diallelic variability of the LTC4S gene (31). On the basis of population studies (Figure 2) we demonstrated an allelic association between the promoter C variant and aspirin-induced asthma. In more recently studied (more than 60 individuals each) groups of asthmatic and control subjects we found allele C of the LTC4S gene to be more than 50% more common in AIA (q = 0.39). The allelic frequency in aspirin-tolerant subjects with asthma (ATAs) (q = 0.26) and healthy control subjects (q = 0.25) did not differ. Patients with AIA also had upregulated LTC4S mRNA expression in peripheral blood eosinophils, and an increased number of gene transcripts was most pronounced in carriers of the 444 C-allele (32).

View larger version (18K): [in this window] [in a new window]   Figure 2.   MspI restriction sites (M, upper panel ) within the promoter of LTC4S and fluorogram of RFLP fragments. [See Sanak, M., H.-U. Simon, and A. Szczeklik. Lancet 1997;350:1599-1560 (31). Reproduced with permission from the publisher.]

We prepared a set of molecular constructs in which either variant of the LTC4S allele promoter was ligated to a reporter gene, -galactosidase (33). These expression plasmids were transfected into eukaryotic cells (COS-7) (Figure 3). In several experiments we observed a consistent increase in reporter enzyme activity in the cells to which plasmid with allele C of LTC4S was introduced. The overexpression, in unstimulated cells, was moderate (~ 25%) and lower than in the control, intact cytomegalovirus promoter. Analysis of the DNA sequence affected by nucleotide transversion indicates that some recognition sites of transcription factors may be affected by the polymorphism. Preliminary studies of eosinophil nuclear extracts, which were incubated with radioactively labeled oligonucleotides corresponding to the two common alleles, demonstrate a difference in the binding of the AP2 transcription factor. It is highly probable that altered by nucleotide transversion, the AP2 consensus motif responds inadequately, or even engages in cross-talk with other transcription factors. The effect of such an illegitimate transcription of LTC4S may be noticeable only in these individuals, who either develop eosinophilic infiltrates in their bronchial mucosa or severe hypereosinophilia. If eosinophils circulating in the peripheral blood of individuals with asthma have overexpression of LTC4S, as suggested by our mRNA quantitation (and upregulation of the enzyme would be dependent on the promoter C allele trait), the difference in Cys-LT pathway should be recognized in a clinical study. Indeed, an inhaled provocation test with lysine aspirin led to a significant increase in urinary output of LTE₄ (Figure 4), but only in carriers of the 444 C allele (34). It is interesting, that the genotype of patients with AIA correlated with the cumulative dose of lysine-aspirin required to precipitate bronchoconstriction. Patients with AIA who were AA homozygotes reacted to relatively lower doses of aspirin (6.65 ± 7.45 mg [SD]) while those with the allele C trait required higher and more unpredictable doses (30.0 ± 47.0 mg). This negative correlation of PD₂₀ aspirin dose with genotype (Kendall coefficient of concordance, 0.55) was significant when tested with the Friedman ANOVA test (p < 0.001).

View larger version (35K): [in this window] [in a new window]   Figure 3.   (Left panel ) LTC4S promoter fragments of 300 bp, corresponding to either of the allelic variants, were cloned into the eukaryotic expression vector pcDNA 2.1, in which the original cytomegalovirus promoter has been truncated. (Right panel ) Enhancer properties of LTC4S alleles were tested as -galactosidase activity (reporter gene) in COS-7 cells transfected with the constructs. Expression of allele C was on average 25% greater than allele A and almost half that of the intact CMV promoter. [Adapted from Sanak, M., and A. Szczeklik. Eur. Respir. J. 1998;12(Suppl. 28):373s (33). Reproduced with permission from the publisher.]

View larger version (48K): [in this window] [in a new window]   Figure 4.   Urinary LTE4 samples collected, as 2 h fractions, before and after inhalatory challenge of aspirin-sensitive subjects with asthma with a PD20 dose of lysyl-aspirin. Patients homozygous for allele A (n = 17) had significantly lower (p < 0.03) excretion of LTE4 than did those who inherited allele C (n = 25). [See Szczeklik, A., M. Sanak, E. Nizankowska, L. Mastalerz, G. Bochenek, G. Pulka, and A. Bestynska. Allergy 1998;53(Suppl. 43):61 (34). Reproduced with permission from the publisher.]

	CONCLUSIONS

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In bronchi of patients with AIA, whose asthma is characterized by increased production of Cys-LTs, there is an overexpression of LTC₄ synthase. This phenomenon is explained, at least in part, by a genetic polymorphism of the LTC₄ synthase gene. A common promoter variant of the gene creates a predisposition to aspirin-sensitive asthma by reinforcing the effector mechanism of bronchoconstriction. The exact mechanism by which aspirin triggers bronchoconstriction by acting on cyclooxygenase remains elusive. Aspirin challenge studies, coupled with estimation of LTC4S polymorphism and LTC₄ urinary excretion point to some heterogeneity between patients with AIA. It remains to be explained why almost 30% of patients with AIA have no predisposing variant of the LTC4S gene, while 25% of controls do have it, without consequences to their health. Such a finding is common in studies of genetic predisposition to multifactorial diseases, and is predictable on the basis of non-Mendelian, low inheritance of AIA. Studies of triggering factors, such as persistent viral infection, or of polygenic effects, including Cys-LT receptors and lipoxins, might uncover the mysteries of aspirin-induced asthma.

	Footnotes

Correspondence and requests for reprints should be addressed to Andrew Szczeklik, M.D., Department of Medicine, School of Medicine, Jagiellonian University, 8 Skawinska Street, 31-066 Kraków, Poland.

	References

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