Complement C3a and C4a Increased in Plasma of Patients with Aspirin-induced Asthma
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《美国呼吸和危急护理医学》
Genome Research Center for Allergy and Respiratory Diseases, Soonchunhyang University Hospital, Bucheon
Department of Chemistry, Soonchunhyang University, Asan
Yonsei Proteome Research Center, Seoul, Korea
ABSTRACT
Rationale: Aspirin-induced asthma (AIA) is a distinct clinical syndrome that affects up to 10% of adults with asthma. Although eicosanoid metabolites appear to play an important role in AIA, the exact pathogenic mechanism for the syndrome remains obscure. In addition, the proposed mechanism fails to explain why aspirin does not cause bronchoconstriction in all individuals.
Objectives: We aimed to identify proteins that were differentially expressed in between AIA and aspirin-tolerant asthma (ATA) plasma.
Methods and Main Results: By using a proteomics approach, six proteins were found to be differentially expressed in plasma between patients with AIA and patients with ATA at baseline, and eight proteins were significantly up- or down-regulated after aspirin challenge in patients with AIA. These proteins, which were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, can be classified into four groups: complement components, apolipoproteins, modified albumin, and unknown proteins. Among them, the complement component levels in plasma were validated by using ELISA. Plasma concentrations of C3a and C4a were higher in patients with AIA (n = 30) than in patients with ATA (n = 24). After the aspirin challenge, C3 decreased in both patients with AIA and those with ATA, but the C3a concentration increased in the AIA patient group (p = 0.019). Moreover, C3a and C4a levels and the ratios of C3a/C3 and C4a/C4 were correlated with the changes of FEV1 values after aspirin challenge.
Conclusions: Aspirin intolerance may be related to alterations in the levels of complements, as well as those of lipoprotein and other proteins.
Key Words: aspirin-induced asthma complement C3a and C4a two-dimensional electrophoresis
Aspirin-induced asthma (AIA) refers to the development of bronchoconstriction in individuals with asthma after the ingestion of aspirin (acetylsalicylic acid [ASA]) and other nonsteroidal antiinflammatory drugs (NSAIDs). This syndrome has been characterized by aspirin hypersensitivity, bronchial asthma, and chronic rhinosinusitis with nasal polyposis, commonly referred to as the "aspirin triad" (1). AIA affects 5 to 10% of adults with asthma (2). Although the pathogenesis of AIA is not completely understood, the cyclooxygenase (COX) theory is widely accepted (3–5). Asthma attacks initiated by aspirin and NSAIDs are triggered by the specific inhibition of COX (prostaglandin-endoperoxide synthase) in the respiratory tract, which is followed by a reduction of prostaglandin E2 (PGE2) and overproduction of cysteinyl leukotrienes (cysLTs). CysLTs (LTD4, LTC4, and LTE4) are important inflammatory mediators in asthma that can mediate bronchoconstriction and increase mucus secretion, vascular permeability, and cellular infiltration (6, 7). The biological activity of cysLTs occurs through their receptors, CysLTR1 and CysLTR2, which are located on the surfaces of target cells (8). CysLTR1 is a G-protein–coupled seven-transmembrane receptor and is expressed primarily in airway smooth muscle, eosinophils, macrophages, and spleen. CysLTs and their receptors are increased in the airways and peripheral blood cells in patients with AIA compared with levels in patients with aspirin-tolerant asthma (ATA) and normal control subjects (9–13). These data suggest that the overproduction of cysLTs and overexpression of CysLTR1 are fundamental in the pathogenesis of aspirin hypersensitivity (13). However, blockage of the cysLT pathway and CysLTR1 by specific antagonists such as 5-lipoxygenase (5-LO) inhibitors did not completely protect AIA (14, 15). In addition, the proposed mechanism fails to explain why ASA does not cause bronchoconstriction in all individuals (16). These findings suggest the presence of alternative pathways of aspirin hypersensitivity (17, 18). Therefore, studies of triggering factors or polygenic effects are needed to understand the mechanism of AIA.
In this study, we adopted a proteomics approach to investigate the protein basis of the pathology and to develop a serologic marker for ASA hypersensitivity.
METHODS
Subjects
Patients with asthma and healthy control subjects were enrolled from the outpatient clinics of Soonchunhyang University Hospital, Korea. All patients met the American Thoracic Society definition of asthma (19). They had no history of systemic steroid therapy or cysLT receptor antagonist treatment within 6 wk of the study and mild to moderate, persistent asthma based on their symptom severity and initial FEV1 (20, 21). Skin prick tests were performed for 24 common aeroallergens (Bencard Co., Brentford, UK) (22), and atopy was defined as the presence of an immediate skin reaction (> 3 mm in diameter) to one or more of the allergens. Complete blood counts and differential counts were performed automatically using a Coulter counter. Total IgE was measured using the CAP system (Pharmacia Diagnostics, Uppsala, Sweden). Normal control subjects were recruited from the general population; control subjects had no respiratory symptoms, no past history of ASA hypersensitivity, an FEV1 greater than 80% predicted, a PC20 methacholine greater than 25 mg/ml, and normal findings on a simple chest radiogram. All subjects gave informed consent, and the protocols were approved by the local ethics committees.
Aspirin Challenge and Separation of Plasma
Oral aspirin challenge was performed in patients with baseline FEV1 values greater than 70% predicted (23). In all cases, no COX inhibitors were taken in the week preceding the challenge. Venous blood sampling was done in a tube containing ethylenediaminetetraacetic acid, and plasma was separated immediately before the challenge and at the time of symptom appearance in the case of a positive response or at 2 h after ingestion of the last dose of aspirin in the case of a negative response.
Two-Dimensional Electrophoresis and Image Analysis
Immobiline DryStrips (Amersham Biosciences, Seoul, Korea) were used for isoelectric focusing. Isoelectric focusing was performed using an IPGphore system (Amersham Biosciences) with 1 mg of plasma protein. After isoelectric focusing separation, the proteins were separated on sodium dodecyl sulfate–polyacrylamide gels. For image analysis, the gels were visualized with Coomassie Brilliant Blue G-250 according to the manufacturer's instructions. Two-dimensional electrophoresis (2-DE) gels were scanned with an ImageScanner (Amersham Biosciences) in transmission mode. Spot detection and matching were performed using ImageMaster 2D version 4.0 (Amersham, Biosciences), as previously described (24). Digitized images were analyzed using ImageMaster program, which calculated the two-dimensional (2-D) spot intensity by integrating the optical density over the spot area (i.e., the spot "volume") and normalized. The normalized values were exported to SPSS 8.0 (SPSS, Inc., Chicago, IL), and then statistical analysis was performed.
Intra-Gel Digestion and Mass Spectrometric Analysis
Differentially expressed protein spots were excised from the gels, cut into smaller pieces, and digested with trypsin (Promega, Madison, WI), as previously described (25). For matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOF MS) analysis, the tryptic peptides were concentrated on POROS R2 columns (Applied Biosystems, Foster City, CA) (26, 27). The spectra for the protein samples were obtained using a Voyager DE PROMALDI-TOF spectrometer (Applied Biosystems). Peptide matching and protein searches against the Swiss-Prot and National Center for Biotechnology Information databases were performed using the Mascot search program (Matrix Science, Boston, MA) and ProFound program (http://prowl.rockefeller.edu/).
Measurement of Complement C3, C4, C3a, and C4a in Plasma
In the plasma of patients with asthma and normal control subjects, complement C3 and C4 were measured by immunoturbidmetric assays (Roche Diagnostics, Mannheim, Germany). FUT-175 (BD Biosciences, Palo Alto, CA) was added as an additive to stabilize plasma samples for complement measurements (final concentration of 50 μg/ml plasma).
The concentrations of C3a and C4a were determined by enzyme-linked immunosorbent assays (Assay Design, Ann Arbor, MI). Inter- and intraassay variations were less than 15%.
Statistical Analysis
Statistical analysis was performed with SPSS 8.0. In the analysis of spot intensity on 2-D gel and concentration of complements, the Kruskal-Wallis test was used to compare the differences of the densities between the three groups (patients with AIA, patients with ATA, and normal control subjects). If significant, the Mann-Whitney test (two-sample rank sum test) was used to analyze differences between the two groups. The change of complement levels after aspirin challenge was analyzed using the Wilcoxon signed rank sum test. Simple linear regression analyses were performed to analyze correlations between variables, and Pearson's correlation coefficients (R) were determined from these analyses. All data are expressed as median values (interquartile range), and significance was defined as p < 0.05.
RESULTS
Characteristics of Patients Participating in the Study
The 54 patients with asthma were classified into two groups (AIA vs. ATA) by the aspirin challenge test. The characteristics of the patients are summarized in Table 1. There were no significant differences in terms of age, sex, initial FEV1, eosinophil %, IgE concentration, PC20 methacholine, and atopy frequency between patients with AIA and ATA. The percentage of fall of FEV1 by aspirin challenge was significantly lower in the AIA group than in the ATA group (p < 0.001).
2-DE Analysis of Patient and Normal Control Plasma
To examine the differential expression of proteins induced by the aspirin provocation test in ATA and AIA, proteomic analysis was performed using high-resolution 2-DE. No significant difference in the average number of spots was observed among the groups (normal control subjects: median, 585; range, 553–599; patients with ATA: median, 590; range, 575–602; patients with AIA: median, 592; range, 572–611; Figure 1). The 54 patients with asthma were classified into two groups (AIA vs. ATA) by the aspirin challenge test. We then selected the six subjects having greater than 25% fall of FEV1 induced by aspirin challenge (typical AIA) and six subjects with a less than 5% fall of FEV1 induced by aspirin challenge (typical ATA), together with six healthy, normal control subjects, for 2-DE. By comparing the differences of protein expression levels in plasma from patients with AIA and ATA and normal control patients, we found protein spots showing statistically significant differences among the three groups. Before the aspirin challenge (Figure 2A), six spots differed in quantity among the three groups; the relative intensities of the spots 11 and 12 were significantly lower in the AIA patient group than in the ATA or normal control group (p < 0.05). In contrast, the intensities of spots 1, 5, 8, and 10 were higher in the AIA patient group than in the normal control group (p < 0.05). After the aspirin challenge (Figure 2B), four spots differed among the three groups. The relative intensities of spots 1 and 9 were significantly higher in the patients with AIA than in the patients with ATA or normal control patients (p < 0.05), and the relative intensities of spots 3 and 10 were higher in the patients with AIA than in the normal control patients (p < 0.05).
The change of intensity from before to after the ASA challenge was calculated as the intensity of the spot post-challenge minus the preaspirin intensity (Figure 2C). The relative intensities of spots 2, 5, and 7 significantly decreased in the AIA group compared with the intensity changes in the other two groups (p < 0.05). The intensity of spot 1 was decreased in patients with AIA and normal control subjects. In contrast, the intensities of spots 4, 6, and 9 significantly increased in the AIA group compared with those in the ATA and normal control groups (p < 0.05).
Identification of Proteins by MALDI-TOF MS Analysis
The proteins were separated by 2-DE and were identified by MALDI-TOF MS, as summarized in Table 2. Three proteins (complement C3 fragments, apolipoprotein, and modified albumin) were found in multiple spots. The spots 1, 2, 3, and 4 were composed of C3 or its fragment and C4B. Spots 8 and 9 were determined to be human serum albumin in a complex with myristic acid. Spots 5, 6, and 7 were identified as apolipoproteins. Except for three unknown proteins, the proteins identified can be summarized as complement components, modified albumin, and apolipoprotein.
Concentrations of Complement C3, C3a, C4, and C4a in Plasma
To validate the changes of complement proteins in AIA, case-control studies were conducted. The concentrations of complement C3, C4, C3a, and C4a in plasma from patients with AIA (n = 30), patients with ATA (n = 24), and normal control patients (n = 21) are shown in Figures 3 and 4. At baseline, there were no significant differences in the plasma concentrations of C3 (Figure 3A) and C4 (Figure 3B) among the AIA (C3: 13.3 mg/ml; range, 8.1–20.9 mg/ml; C4: median, 2.26 mg/ml; range, 0.7–4.4 mg/ml), ATA (C3: 13.7 mg/ml; range, 9.9–19.1 mg/ml; C4: median, 2.3 mg/ml; range, 1.3–5.0 mg/ml), and healthy normal control groups (C3: median, 12.8 mg/ml; range, 7.9–16.0 mg/ml; C4: median, 2.1 mg/ml; range, 1.3–3.3 mg/ml). However, the plasma concentrations of C3a (Figure 3C) and C4a (Figure 3D) were significantly higher in patients with AIA than in those with ATA and normal control patients (C3a: normal control, median, 98.08 ng/ml; range, 46.0–157.0 ng/ml; ATA, median, 99.32 ng/ml; range, 14.3–487.0 ng/ml; AIA, median, 148.03 ng/ml; range, 52.3–488.3 ng/ml. C4a: normal control, median, 342.4 ng/ml; range, 198.2–906.3 ng/ml; ATA, median, 274.5 ng/ml; range, 187.4–912.4 ng/ml; AIA, median, 814.6 ng/ml; range, 394.5–1,975.4 ng/ml; p = 0.031 and 0.001, respectively).
Changes in the Plasma Concentrations of Complement C3, C3a, C4, and C4a after Aspirin Challenge
In response to aspirin challenge, the C3 concentration significantly decreased (p = 0.012 for AIA, p = 0.008 for ATA; paired t test), whereas C4 and C4a concentrations did not change (Figure 4A). Only the amount of C3a was elevated in the AIA group in response to aspirin challenge (p = 0.019; Figure 4C). The C4 and C4a concentrations were not changed significantly by the aspirin challenge (Figures 4B and 4D). The C3a and C4a levels were higher in patients with AIA than in those with ATA before and after the aspirin challenge.
Correlations between the Aspirin-induced Change of FEV1 and Complement Components
There was a significant correlation between C4a concentration (at baseline and after aspirin challenge) and changes in FEV1 after aspirin challenge ( = 0.605, p = 0.00008, and = 0.474, p = 0.001, respectively). Similarly, there was a correlation between C3a concentration (after aspirin challenge) and changes in FEV1 after aspirin challenge ( = 0.326, p = 0.018). In addition, the ratios of C3a/C3 and C4a/C4 showed a correlation with changes in FEV1 (Table 3).
DISCUSSION
Although it is reasonable to attribute the effect of aspirin to the liberation of the 5-LO pathway from suppression by endogenous PGE2, it remains unclear why a similar response to aspirin is not seen in patients with ATA and in normal subjects. We were primarily concerned with the causative factors of the differences between AIA and ATA in response to the aspirin challenge test. To better understand the molecular basis of this pathology, we adopted a proteomics approach as a screening tool. Using a screening tool, we could select several target molecules, which were validated in rather large population. We believe that this is the first report to show, in a single map, the different proteins expressed in plasma from patients with AIA and ATA using proteomics.
As the initial step, we tried to screen the target proteins on the 2-D gel to differentiate AIA from ATA. AIA was diagnosed by a positive result on the oral aspirin challenge test, which was performed with increasing doses of aspirin (10–625 mg) according to the modified method previously described (23). The cut-off levels between the two conditions is 20% fall in FEV1 induced by aspirin challenge. However, it is well known that the percentage of fall in FEV1 induced by aspirin challenge is a continuous value.
Because of these reasons, we selected the study subjects having percentage of fall in FEV1 of less than 5% as ASA-tolerant patients with asthma and those having a percentage of fall in FEV1 of greater than 25% as ASA-intolerant patients with asthma to discriminate completely these two clinical phenotypes.
At baseline, the expression levels of two unidentified proteins (spots 11 and 12) were lower in the patients with AIA than in the patients with ATA and normal control subjects. In contrast, the expression levels of complement C3 (spot 1), proapolipoprotein (spot 5), human serum albumin complexed with myristic acid (spot 8), and PRO2619 (spot 10) were higher in patients with AIA than in patients with ATA and normal subjects at baseline. After the aspirin challenge, four proteins were differently expressed among the three groups (spots 1, 3, 9, and 10). However, these were the same proteins that were differentially expressed at baseline, suggesting that these differences between patients with AIA and ATA after the aspirin challenge were caused by differences at baseline rather than by the aspirin challenge. In contrast, the changes in protein expression in response to aspirin differed according to each condition (AIA and ATA). The expression of complement C4B (spot 4), apolipoprotein A2 (apoA2) precursor (spot 6), and human serum albumin complexed with myristic acid (spot 9) was increased by aspirin challenge only in the subjects with AIA, whereas the protein expression levels of complement C3 (spots 1 and 2), pro-apoA1 (spot 5), and apoA2 (spot 7) were decreased in the subjects with AIA.
As a whole, we found 12 spots that were related to AIA, which were composed of eight kinds of proteins according to MALDI-TOF analysis (Table 2). These proteins can be summarized as complement components, modified albumin, apolipoprotein, and unknown proteins. The biological significance of these proteins is analyzed below.
Albumin is a globular, unglycosylated serum protein that functions primarily as a carrier protein for steroids, fatty acids, and thyroid hormones, and plays a role in stabilizing the extracellular fluid volume. The relationship between aspirin and albumin is continually being studied. Szczeklik and coworkers demonstrated the importance of the albumin-binding property of NSAIDs in causing asthma, but the mechanism was not explained (28). Aarons and colleagues reported that binding to albumin protected aspirin against spontaneous hydrolysis, and the binding affinities varied according to individual phenotypes (29). Williams and coworkers suggested that the albumin-binding property of analgesic drugs may be more important than COX inhibition for inducing asthma (30). When considering these reports, the differences in aspirin-binding affinity between the albumin of patients with AIA and those with ATA might account for the changes of modified serum albumin.
ApoA1 and apoA2 are the major protein components of high-density lipoprotein in the plasma. Given that AIA can be characterized as a disorder of arachidonic acid metabolism (31), apolipoproteins could be a good candidate protein for AIA. Tokizawa and colleagues reported that asthma cases showed low levels of serum cholesterol, triglyceride, low-density lipoprotein, very low-density lipoprotein, and apoB compared with those of a control group, whereas the high-density lipoprotein cholesterol and apoA2 levels were higher than in the control group. (32). Recently, Horani and coworkers showed that COX inhibition with indomethacin or aspirin down-regulates apoA1 protein and mRNA expression at the transcriptional level (33). In addition, the decreased expression rates in response to the aspirin challenge varied with patient phenotype (34). These reports and our data suggest that aspirin down-regulates apoA1 and apoA2 but that the rates are different between AIA and ATA.
Among these three kinds of protein, complement components are the best-known protein related to asthma. C3a, C4a, and C5a are known as anaphylatoxins, which stimulate the release of histamines as part of an immunologic response toward a foreign antigen (35). The participation of complement components in asthma is frequently reported (36–40). In this respect, our proteomics data are quite interesting.
To evaluate whether complement activation contributes to AIA, we determined the plasma concentrations of the complement components in 54 patients with asthma. At baseline, the concentrations of C3 and C4 did not differ among the patient and control groups. However, the plasma concentrations of C3a and C4a in subjects with AIA were higher than those in patients with ATA. We also measured the C5a levels. The median plasma concentration of C5a was higher (p < 0.005) in the AIA (median, 24.0 ng/ml; range, 7.9–88.5 ng/ml) and ATA groups (median, 26.2 ng/ml; range, 8.2–75.0 ng/ml) than in the normal control group (median, 11.7 ng/ml; range, 3.2–43.5 ng/ml). However, there was no difference between AIA and ATA.
In response to aspirin challenge, the C3 concentration significantly decreased in both AIA and ATA groups, whereas C4 and C4a concentrations did not (Figure 4). Only the amount of C3a was elevated in the AIA group in response to aspirin challenge (p = 0.019; Figure 4A), which indicates that aspirin activated precursor complement C3 to an active fragment form (e.g., C3a) in patients with AIA and those with ATA, but did not directly increase C3a concentrations in patients with ATA. These data suggest the possibility of different metabolic pathways for C3 activation or degradation between AIA and ATA. The most important finding of this study is that plasma C3a and C4a levels in the AIA group were significantly higher than those in the patients with ATA and healthy control subjects regardless of aspirin challenge. Considering that C3a and C4a are potent chemotactic factors for inflammatory cells (35, 41, 42) and are able to enhance degranulation of eosinophils, mast cells, and basophils (43, 44), persistently elevated C3a and C4a may explain why aspirin causes bronchoconstriction only in patients with AIA and not in those with ATA and normal subjects. These suggestions are supported by the correlation between the concentrations of C3a or C4a and the changes of FEV1 values on aspirin challenge. As shown in Table 3, there was a significant correlation between C4a concentration and changes in FEV1 after aspirin challenge. Similarly, there was a moderate correlation between C3a concentration after aspirin challenge and changes in FEV1 after aspirin challenge. Thus, the serum concentration of C4a, which did not change in response to aspirin challenge, is believed to be related to aspirin susceptibility. In contrast, it is possible that C3a-related susceptibility in AIA becomes relevant in the disease process only after the AIA attack process has begun.
Aspirin had been reported as an activator of the complement system in vitro and in vitro (45, 46). The COX inhibitors, such as indomethacin and aspirin, cause activation of C3, C4, and C5 via the classical pathway (47, 48), independently from antigen–antibody reaction (49, 50). However, there also were some reports that had been published and that failed to detect an association between aspirin-sensitive asthma and complement activation (51, 52). Pleskow and coworkers showed the absence of detectable complement activation in patients with aspirin-sensitive asthma during aspirin challenge (51). No difference in total hemolytic complement or complement C3 and C4 was observed in patients with AIA and normal control subjects. Delaney and Kay also could not find any relationship between AIA and complement C3 and C4 (52). However, they did not measure the activated fragment of complements such as C3a and C4a. We also did not find any differences between the inactive complement concentration of AIA and normal control subjects (Figures 3A and 3B). In this study, we investigated the production and degradation of the anaphylatoxins C3a and C4a, which have diverse effects on the host immune response.
In conclusion, our study is the first to show differences in protein expression in the plasma of patients with AIA and ATA at baseline and after aspirin challenge. As a result, we found 12 protein spots composed of eight proteins that were significantly up- or down-regulated after aspirin challenge in patients with AIA. Among them, complement component (C3, C3a, C4, and C4a) levels in plasma were validated using a case-control study. The results of our study show significantly elevated levels of C3a and C4a in the plasma of patients with AIA. The values of C3a and C4a in patients with AIA significantly correlated with FEV1. These results suggest that complement is one of the participants in the pathogenesis of AIA, with a possible contribution of apolipoprotein and albumin complexed with myristic acid.
FOOTNOTES
Supported by grants from the Korean Health 21 R&D project, Ministry of Health and Welfare, Republic of Korea (01-PJ10-PG6-01GN14-0003 and 03-PJ10-PG6-GP01-0002).
These authors contributed equally to this article.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.200505-740OC on November 17, 2005
Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
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Delaney JC, Kay AB. Complement components and IgE in patients with asthma and aspirin idiosyncrasy. Thorax 1976;31:425–427.(Seung-Ha Lee, TaiYoun Rhim, Yun-Sung Cho)
Department of Chemistry, Soonchunhyang University, Asan
Yonsei Proteome Research Center, Seoul, Korea
ABSTRACT
Rationale: Aspirin-induced asthma (AIA) is a distinct clinical syndrome that affects up to 10% of adults with asthma. Although eicosanoid metabolites appear to play an important role in AIA, the exact pathogenic mechanism for the syndrome remains obscure. In addition, the proposed mechanism fails to explain why aspirin does not cause bronchoconstriction in all individuals.
Objectives: We aimed to identify proteins that were differentially expressed in between AIA and aspirin-tolerant asthma (ATA) plasma.
Methods and Main Results: By using a proteomics approach, six proteins were found to be differentially expressed in plasma between patients with AIA and patients with ATA at baseline, and eight proteins were significantly up- or down-regulated after aspirin challenge in patients with AIA. These proteins, which were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, can be classified into four groups: complement components, apolipoproteins, modified albumin, and unknown proteins. Among them, the complement component levels in plasma were validated by using ELISA. Plasma concentrations of C3a and C4a were higher in patients with AIA (n = 30) than in patients with ATA (n = 24). After the aspirin challenge, C3 decreased in both patients with AIA and those with ATA, but the C3a concentration increased in the AIA patient group (p = 0.019). Moreover, C3a and C4a levels and the ratios of C3a/C3 and C4a/C4 were correlated with the changes of FEV1 values after aspirin challenge.
Conclusions: Aspirin intolerance may be related to alterations in the levels of complements, as well as those of lipoprotein and other proteins.
Key Words: aspirin-induced asthma complement C3a and C4a two-dimensional electrophoresis
Aspirin-induced asthma (AIA) refers to the development of bronchoconstriction in individuals with asthma after the ingestion of aspirin (acetylsalicylic acid [ASA]) and other nonsteroidal antiinflammatory drugs (NSAIDs). This syndrome has been characterized by aspirin hypersensitivity, bronchial asthma, and chronic rhinosinusitis with nasal polyposis, commonly referred to as the "aspirin triad" (1). AIA affects 5 to 10% of adults with asthma (2). Although the pathogenesis of AIA is not completely understood, the cyclooxygenase (COX) theory is widely accepted (3–5). Asthma attacks initiated by aspirin and NSAIDs are triggered by the specific inhibition of COX (prostaglandin-endoperoxide synthase) in the respiratory tract, which is followed by a reduction of prostaglandin E2 (PGE2) and overproduction of cysteinyl leukotrienes (cysLTs). CysLTs (LTD4, LTC4, and LTE4) are important inflammatory mediators in asthma that can mediate bronchoconstriction and increase mucus secretion, vascular permeability, and cellular infiltration (6, 7). The biological activity of cysLTs occurs through their receptors, CysLTR1 and CysLTR2, which are located on the surfaces of target cells (8). CysLTR1 is a G-protein–coupled seven-transmembrane receptor and is expressed primarily in airway smooth muscle, eosinophils, macrophages, and spleen. CysLTs and their receptors are increased in the airways and peripheral blood cells in patients with AIA compared with levels in patients with aspirin-tolerant asthma (ATA) and normal control subjects (9–13). These data suggest that the overproduction of cysLTs and overexpression of CysLTR1 are fundamental in the pathogenesis of aspirin hypersensitivity (13). However, blockage of the cysLT pathway and CysLTR1 by specific antagonists such as 5-lipoxygenase (5-LO) inhibitors did not completely protect AIA (14, 15). In addition, the proposed mechanism fails to explain why ASA does not cause bronchoconstriction in all individuals (16). These findings suggest the presence of alternative pathways of aspirin hypersensitivity (17, 18). Therefore, studies of triggering factors or polygenic effects are needed to understand the mechanism of AIA.
In this study, we adopted a proteomics approach to investigate the protein basis of the pathology and to develop a serologic marker for ASA hypersensitivity.
METHODS
Subjects
Patients with asthma and healthy control subjects were enrolled from the outpatient clinics of Soonchunhyang University Hospital, Korea. All patients met the American Thoracic Society definition of asthma (19). They had no history of systemic steroid therapy or cysLT receptor antagonist treatment within 6 wk of the study and mild to moderate, persistent asthma based on their symptom severity and initial FEV1 (20, 21). Skin prick tests were performed for 24 common aeroallergens (Bencard Co., Brentford, UK) (22), and atopy was defined as the presence of an immediate skin reaction (> 3 mm in diameter) to one or more of the allergens. Complete blood counts and differential counts were performed automatically using a Coulter counter. Total IgE was measured using the CAP system (Pharmacia Diagnostics, Uppsala, Sweden). Normal control subjects were recruited from the general population; control subjects had no respiratory symptoms, no past history of ASA hypersensitivity, an FEV1 greater than 80% predicted, a PC20 methacholine greater than 25 mg/ml, and normal findings on a simple chest radiogram. All subjects gave informed consent, and the protocols were approved by the local ethics committees.
Aspirin Challenge and Separation of Plasma
Oral aspirin challenge was performed in patients with baseline FEV1 values greater than 70% predicted (23). In all cases, no COX inhibitors were taken in the week preceding the challenge. Venous blood sampling was done in a tube containing ethylenediaminetetraacetic acid, and plasma was separated immediately before the challenge and at the time of symptom appearance in the case of a positive response or at 2 h after ingestion of the last dose of aspirin in the case of a negative response.
Two-Dimensional Electrophoresis and Image Analysis
Immobiline DryStrips (Amersham Biosciences, Seoul, Korea) were used for isoelectric focusing. Isoelectric focusing was performed using an IPGphore system (Amersham Biosciences) with 1 mg of plasma protein. After isoelectric focusing separation, the proteins were separated on sodium dodecyl sulfate–polyacrylamide gels. For image analysis, the gels were visualized with Coomassie Brilliant Blue G-250 according to the manufacturer's instructions. Two-dimensional electrophoresis (2-DE) gels were scanned with an ImageScanner (Amersham Biosciences) in transmission mode. Spot detection and matching were performed using ImageMaster 2D version 4.0 (Amersham, Biosciences), as previously described (24). Digitized images were analyzed using ImageMaster program, which calculated the two-dimensional (2-D) spot intensity by integrating the optical density over the spot area (i.e., the spot "volume") and normalized. The normalized values were exported to SPSS 8.0 (SPSS, Inc., Chicago, IL), and then statistical analysis was performed.
Intra-Gel Digestion and Mass Spectrometric Analysis
Differentially expressed protein spots were excised from the gels, cut into smaller pieces, and digested with trypsin (Promega, Madison, WI), as previously described (25). For matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOF MS) analysis, the tryptic peptides were concentrated on POROS R2 columns (Applied Biosystems, Foster City, CA) (26, 27). The spectra for the protein samples were obtained using a Voyager DE PROMALDI-TOF spectrometer (Applied Biosystems). Peptide matching and protein searches against the Swiss-Prot and National Center for Biotechnology Information databases were performed using the Mascot search program (Matrix Science, Boston, MA) and ProFound program (http://prowl.rockefeller.edu/).
Measurement of Complement C3, C4, C3a, and C4a in Plasma
In the plasma of patients with asthma and normal control subjects, complement C3 and C4 were measured by immunoturbidmetric assays (Roche Diagnostics, Mannheim, Germany). FUT-175 (BD Biosciences, Palo Alto, CA) was added as an additive to stabilize plasma samples for complement measurements (final concentration of 50 μg/ml plasma).
The concentrations of C3a and C4a were determined by enzyme-linked immunosorbent assays (Assay Design, Ann Arbor, MI). Inter- and intraassay variations were less than 15%.
Statistical Analysis
Statistical analysis was performed with SPSS 8.0. In the analysis of spot intensity on 2-D gel and concentration of complements, the Kruskal-Wallis test was used to compare the differences of the densities between the three groups (patients with AIA, patients with ATA, and normal control subjects). If significant, the Mann-Whitney test (two-sample rank sum test) was used to analyze differences between the two groups. The change of complement levels after aspirin challenge was analyzed using the Wilcoxon signed rank sum test. Simple linear regression analyses were performed to analyze correlations between variables, and Pearson's correlation coefficients (R) were determined from these analyses. All data are expressed as median values (interquartile range), and significance was defined as p < 0.05.
RESULTS
Characteristics of Patients Participating in the Study
The 54 patients with asthma were classified into two groups (AIA vs. ATA) by the aspirin challenge test. The characteristics of the patients are summarized in Table 1. There were no significant differences in terms of age, sex, initial FEV1, eosinophil %, IgE concentration, PC20 methacholine, and atopy frequency between patients with AIA and ATA. The percentage of fall of FEV1 by aspirin challenge was significantly lower in the AIA group than in the ATA group (p < 0.001).
2-DE Analysis of Patient and Normal Control Plasma
To examine the differential expression of proteins induced by the aspirin provocation test in ATA and AIA, proteomic analysis was performed using high-resolution 2-DE. No significant difference in the average number of spots was observed among the groups (normal control subjects: median, 585; range, 553–599; patients with ATA: median, 590; range, 575–602; patients with AIA: median, 592; range, 572–611; Figure 1). The 54 patients with asthma were classified into two groups (AIA vs. ATA) by the aspirin challenge test. We then selected the six subjects having greater than 25% fall of FEV1 induced by aspirin challenge (typical AIA) and six subjects with a less than 5% fall of FEV1 induced by aspirin challenge (typical ATA), together with six healthy, normal control subjects, for 2-DE. By comparing the differences of protein expression levels in plasma from patients with AIA and ATA and normal control patients, we found protein spots showing statistically significant differences among the three groups. Before the aspirin challenge (Figure 2A), six spots differed in quantity among the three groups; the relative intensities of the spots 11 and 12 were significantly lower in the AIA patient group than in the ATA or normal control group (p < 0.05). In contrast, the intensities of spots 1, 5, 8, and 10 were higher in the AIA patient group than in the normal control group (p < 0.05). After the aspirin challenge (Figure 2B), four spots differed among the three groups. The relative intensities of spots 1 and 9 were significantly higher in the patients with AIA than in the patients with ATA or normal control patients (p < 0.05), and the relative intensities of spots 3 and 10 were higher in the patients with AIA than in the normal control patients (p < 0.05).
The change of intensity from before to after the ASA challenge was calculated as the intensity of the spot post-challenge minus the preaspirin intensity (Figure 2C). The relative intensities of spots 2, 5, and 7 significantly decreased in the AIA group compared with the intensity changes in the other two groups (p < 0.05). The intensity of spot 1 was decreased in patients with AIA and normal control subjects. In contrast, the intensities of spots 4, 6, and 9 significantly increased in the AIA group compared with those in the ATA and normal control groups (p < 0.05).
Identification of Proteins by MALDI-TOF MS Analysis
The proteins were separated by 2-DE and were identified by MALDI-TOF MS, as summarized in Table 2. Three proteins (complement C3 fragments, apolipoprotein, and modified albumin) were found in multiple spots. The spots 1, 2, 3, and 4 were composed of C3 or its fragment and C4B. Spots 8 and 9 were determined to be human serum albumin in a complex with myristic acid. Spots 5, 6, and 7 were identified as apolipoproteins. Except for three unknown proteins, the proteins identified can be summarized as complement components, modified albumin, and apolipoprotein.
Concentrations of Complement C3, C3a, C4, and C4a in Plasma
To validate the changes of complement proteins in AIA, case-control studies were conducted. The concentrations of complement C3, C4, C3a, and C4a in plasma from patients with AIA (n = 30), patients with ATA (n = 24), and normal control patients (n = 21) are shown in Figures 3 and 4. At baseline, there were no significant differences in the plasma concentrations of C3 (Figure 3A) and C4 (Figure 3B) among the AIA (C3: 13.3 mg/ml; range, 8.1–20.9 mg/ml; C4: median, 2.26 mg/ml; range, 0.7–4.4 mg/ml), ATA (C3: 13.7 mg/ml; range, 9.9–19.1 mg/ml; C4: median, 2.3 mg/ml; range, 1.3–5.0 mg/ml), and healthy normal control groups (C3: median, 12.8 mg/ml; range, 7.9–16.0 mg/ml; C4: median, 2.1 mg/ml; range, 1.3–3.3 mg/ml). However, the plasma concentrations of C3a (Figure 3C) and C4a (Figure 3D) were significantly higher in patients with AIA than in those with ATA and normal control patients (C3a: normal control, median, 98.08 ng/ml; range, 46.0–157.0 ng/ml; ATA, median, 99.32 ng/ml; range, 14.3–487.0 ng/ml; AIA, median, 148.03 ng/ml; range, 52.3–488.3 ng/ml. C4a: normal control, median, 342.4 ng/ml; range, 198.2–906.3 ng/ml; ATA, median, 274.5 ng/ml; range, 187.4–912.4 ng/ml; AIA, median, 814.6 ng/ml; range, 394.5–1,975.4 ng/ml; p = 0.031 and 0.001, respectively).
Changes in the Plasma Concentrations of Complement C3, C3a, C4, and C4a after Aspirin Challenge
In response to aspirin challenge, the C3 concentration significantly decreased (p = 0.012 for AIA, p = 0.008 for ATA; paired t test), whereas C4 and C4a concentrations did not change (Figure 4A). Only the amount of C3a was elevated in the AIA group in response to aspirin challenge (p = 0.019; Figure 4C). The C4 and C4a concentrations were not changed significantly by the aspirin challenge (Figures 4B and 4D). The C3a and C4a levels were higher in patients with AIA than in those with ATA before and after the aspirin challenge.
Correlations between the Aspirin-induced Change of FEV1 and Complement Components
There was a significant correlation between C4a concentration (at baseline and after aspirin challenge) and changes in FEV1 after aspirin challenge ( = 0.605, p = 0.00008, and = 0.474, p = 0.001, respectively). Similarly, there was a correlation between C3a concentration (after aspirin challenge) and changes in FEV1 after aspirin challenge ( = 0.326, p = 0.018). In addition, the ratios of C3a/C3 and C4a/C4 showed a correlation with changes in FEV1 (Table 3).
DISCUSSION
Although it is reasonable to attribute the effect of aspirin to the liberation of the 5-LO pathway from suppression by endogenous PGE2, it remains unclear why a similar response to aspirin is not seen in patients with ATA and in normal subjects. We were primarily concerned with the causative factors of the differences between AIA and ATA in response to the aspirin challenge test. To better understand the molecular basis of this pathology, we adopted a proteomics approach as a screening tool. Using a screening tool, we could select several target molecules, which were validated in rather large population. We believe that this is the first report to show, in a single map, the different proteins expressed in plasma from patients with AIA and ATA using proteomics.
As the initial step, we tried to screen the target proteins on the 2-D gel to differentiate AIA from ATA. AIA was diagnosed by a positive result on the oral aspirin challenge test, which was performed with increasing doses of aspirin (10–625 mg) according to the modified method previously described (23). The cut-off levels between the two conditions is 20% fall in FEV1 induced by aspirin challenge. However, it is well known that the percentage of fall in FEV1 induced by aspirin challenge is a continuous value.
Because of these reasons, we selected the study subjects having percentage of fall in FEV1 of less than 5% as ASA-tolerant patients with asthma and those having a percentage of fall in FEV1 of greater than 25% as ASA-intolerant patients with asthma to discriminate completely these two clinical phenotypes.
At baseline, the expression levels of two unidentified proteins (spots 11 and 12) were lower in the patients with AIA than in the patients with ATA and normal control subjects. In contrast, the expression levels of complement C3 (spot 1), proapolipoprotein (spot 5), human serum albumin complexed with myristic acid (spot 8), and PRO2619 (spot 10) were higher in patients with AIA than in patients with ATA and normal subjects at baseline. After the aspirin challenge, four proteins were differently expressed among the three groups (spots 1, 3, 9, and 10). However, these were the same proteins that were differentially expressed at baseline, suggesting that these differences between patients with AIA and ATA after the aspirin challenge were caused by differences at baseline rather than by the aspirin challenge. In contrast, the changes in protein expression in response to aspirin differed according to each condition (AIA and ATA). The expression of complement C4B (spot 4), apolipoprotein A2 (apoA2) precursor (spot 6), and human serum albumin complexed with myristic acid (spot 9) was increased by aspirin challenge only in the subjects with AIA, whereas the protein expression levels of complement C3 (spots 1 and 2), pro-apoA1 (spot 5), and apoA2 (spot 7) were decreased in the subjects with AIA.
As a whole, we found 12 spots that were related to AIA, which were composed of eight kinds of proteins according to MALDI-TOF analysis (Table 2). These proteins can be summarized as complement components, modified albumin, apolipoprotein, and unknown proteins. The biological significance of these proteins is analyzed below.
Albumin is a globular, unglycosylated serum protein that functions primarily as a carrier protein for steroids, fatty acids, and thyroid hormones, and plays a role in stabilizing the extracellular fluid volume. The relationship between aspirin and albumin is continually being studied. Szczeklik and coworkers demonstrated the importance of the albumin-binding property of NSAIDs in causing asthma, but the mechanism was not explained (28). Aarons and colleagues reported that binding to albumin protected aspirin against spontaneous hydrolysis, and the binding affinities varied according to individual phenotypes (29). Williams and coworkers suggested that the albumin-binding property of analgesic drugs may be more important than COX inhibition for inducing asthma (30). When considering these reports, the differences in aspirin-binding affinity between the albumin of patients with AIA and those with ATA might account for the changes of modified serum albumin.
ApoA1 and apoA2 are the major protein components of high-density lipoprotein in the plasma. Given that AIA can be characterized as a disorder of arachidonic acid metabolism (31), apolipoproteins could be a good candidate protein for AIA. Tokizawa and colleagues reported that asthma cases showed low levels of serum cholesterol, triglyceride, low-density lipoprotein, very low-density lipoprotein, and apoB compared with those of a control group, whereas the high-density lipoprotein cholesterol and apoA2 levels were higher than in the control group. (32). Recently, Horani and coworkers showed that COX inhibition with indomethacin or aspirin down-regulates apoA1 protein and mRNA expression at the transcriptional level (33). In addition, the decreased expression rates in response to the aspirin challenge varied with patient phenotype (34). These reports and our data suggest that aspirin down-regulates apoA1 and apoA2 but that the rates are different between AIA and ATA.
Among these three kinds of protein, complement components are the best-known protein related to asthma. C3a, C4a, and C5a are known as anaphylatoxins, which stimulate the release of histamines as part of an immunologic response toward a foreign antigen (35). The participation of complement components in asthma is frequently reported (36–40). In this respect, our proteomics data are quite interesting.
To evaluate whether complement activation contributes to AIA, we determined the plasma concentrations of the complement components in 54 patients with asthma. At baseline, the concentrations of C3 and C4 did not differ among the patient and control groups. However, the plasma concentrations of C3a and C4a in subjects with AIA were higher than those in patients with ATA. We also measured the C5a levels. The median plasma concentration of C5a was higher (p < 0.005) in the AIA (median, 24.0 ng/ml; range, 7.9–88.5 ng/ml) and ATA groups (median, 26.2 ng/ml; range, 8.2–75.0 ng/ml) than in the normal control group (median, 11.7 ng/ml; range, 3.2–43.5 ng/ml). However, there was no difference between AIA and ATA.
In response to aspirin challenge, the C3 concentration significantly decreased in both AIA and ATA groups, whereas C4 and C4a concentrations did not (Figure 4). Only the amount of C3a was elevated in the AIA group in response to aspirin challenge (p = 0.019; Figure 4A), which indicates that aspirin activated precursor complement C3 to an active fragment form (e.g., C3a) in patients with AIA and those with ATA, but did not directly increase C3a concentrations in patients with ATA. These data suggest the possibility of different metabolic pathways for C3 activation or degradation between AIA and ATA. The most important finding of this study is that plasma C3a and C4a levels in the AIA group were significantly higher than those in the patients with ATA and healthy control subjects regardless of aspirin challenge. Considering that C3a and C4a are potent chemotactic factors for inflammatory cells (35, 41, 42) and are able to enhance degranulation of eosinophils, mast cells, and basophils (43, 44), persistently elevated C3a and C4a may explain why aspirin causes bronchoconstriction only in patients with AIA and not in those with ATA and normal subjects. These suggestions are supported by the correlation between the concentrations of C3a or C4a and the changes of FEV1 values on aspirin challenge. As shown in Table 3, there was a significant correlation between C4a concentration and changes in FEV1 after aspirin challenge. Similarly, there was a moderate correlation between C3a concentration after aspirin challenge and changes in FEV1 after aspirin challenge. Thus, the serum concentration of C4a, which did not change in response to aspirin challenge, is believed to be related to aspirin susceptibility. In contrast, it is possible that C3a-related susceptibility in AIA becomes relevant in the disease process only after the AIA attack process has begun.
Aspirin had been reported as an activator of the complement system in vitro and in vitro (45, 46). The COX inhibitors, such as indomethacin and aspirin, cause activation of C3, C4, and C5 via the classical pathway (47, 48), independently from antigen–antibody reaction (49, 50). However, there also were some reports that had been published and that failed to detect an association between aspirin-sensitive asthma and complement activation (51, 52). Pleskow and coworkers showed the absence of detectable complement activation in patients with aspirin-sensitive asthma during aspirin challenge (51). No difference in total hemolytic complement or complement C3 and C4 was observed in patients with AIA and normal control subjects. Delaney and Kay also could not find any relationship between AIA and complement C3 and C4 (52). However, they did not measure the activated fragment of complements such as C3a and C4a. We also did not find any differences between the inactive complement concentration of AIA and normal control subjects (Figures 3A and 3B). In this study, we investigated the production and degradation of the anaphylatoxins C3a and C4a, which have diverse effects on the host immune response.
In conclusion, our study is the first to show differences in protein expression in the plasma of patients with AIA and ATA at baseline and after aspirin challenge. As a result, we found 12 protein spots composed of eight proteins that were significantly up- or down-regulated after aspirin challenge in patients with AIA. Among them, complement component (C3, C3a, C4, and C4a) levels in plasma were validated using a case-control study. The results of our study show significantly elevated levels of C3a and C4a in the plasma of patients with AIA. The values of C3a and C4a in patients with AIA significantly correlated with FEV1. These results suggest that complement is one of the participants in the pathogenesis of AIA, with a possible contribution of apolipoprotein and albumin complexed with myristic acid.
FOOTNOTES
Supported by grants from the Korean Health 21 R&D project, Ministry of Health and Welfare, Republic of Korea (01-PJ10-PG6-01GN14-0003 and 03-PJ10-PG6-GP01-0002).
These authors contributed equally to this article.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.200505-740OC on November 17, 2005
Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
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