Published ahead of print on May 29, 2008, doi:10.1164/rccm.200712-1818OC
© 2008 American Thoracic Society doi: 10.1164/rccm.200712-1818OC
Nonatopic Children with Multitrigger Wheezing Have Airway Pathology Comparable to Atopic AsthmaDepartments of 1 Cardiac, Thoracic, and Vascular Sciences, 2 Pediatrics, 3 Diagnostic Medical Sciences and Special Therapies, and 4 Environmental Medicine and Public Health, University of Padova, Padova, Italy; and 5 Department of Oncology, Hematology, and Respiratory Diseases, University of Modena and Reggio Emilia, Modena, Italy Correspondence and requests for reprints should be addressed to Marina Saetta, M.D., Unità Operativa di Pneumologia, Dipartimento di Scienze Cardiologiche, Toraciche e Vascolari Università degli Studi di Padova, via Giustiniani 3, 35128 Padova, Italy. E-mail: marina.saetta{at}unipd.it
Rationale: Epidemiologic studies have shown that, in atopic children, wheezing is more likely to persist into adulthood, eventually becoming asthma, whereas it appears to resolve by adolescence in nonatopic children. Objectives: To investigate whether among children with multitrigger wheeze responsive to bronchodilators the airway pathology would be different in nonatopic wheezers, who are often considered nonasthmatic, compared with atopic wheezers, who are more frequently diagnosed as having asthma. Methods: Bronchial biopsies were obtained from 55 children undergoing bronchoscopy for appropriate clinical indications: 18 nonatopic children with multitrigger wheeze (median age, 5 yr; range, 2–10 yr), 20 atopic children with multitrigger wheeze (medan age, 5 yr; range, 2–15 yr), and 17 control children with no atopy or wheeze (median age, 4; range, 2–14 yr). By histochemistry and immunohistochemistry, we quantified epithelial loss, basement membrane thickness, angiogenesis, inflammatory cells, IL-4+, and IL-5+ cells in subepithelium. Measurements and Main Results: Unexpectedly, all pathologic features examined were similar in atopic and nonatopic wheezing children. Compared with control subjects, both nonatopic and atopic wheezing children had increased epithelial loss (P = 0.03 and P = 0.002, respectively), thickened basement membrane (both P < 0.0001), and increased number of vessels (P = 0.003 and P = 0.03, respectively) and eosinophils (P < 0.0001 and P = 0.002, respectively). Moreover, they had increased cytokine expression, which was highly significant for IL-4 (P = 0.002 and P = 0.0001, respectively) and marginal for IL-5 (P = 0.02 and P = 0.08, respectively). Conclusions: This study shows that the airway pathology typical of asthma is present in nonatopic wheezing children just as in atopic wheezing children. These results suggest that, when multitrigger wheezing responsive to bronchodilators is present, it is associated with pathologic features of asthma even in nonatopic children.
Key Words: pediatric asthma • atopy • inflammation • airway remodeling • Th2 cytokines
The natural history of childhood wheezing remains poorly understood. Epidemiologic studies have shown that wheezing in the first years of life may be the clinical manifestation of different conditions (1, 2). In particular, it is common to discriminate between transient wheezing (children who wheeze during the first years of life, but not after the age of 3), nonatopic wheezing (which is believed to be mainly triggered by viral infections), and atopic wheezing (which is often identified with persistent asthma). In this context, several epidemiologic studies have addressed the role of atopy in predicting the persistence of symptoms, emphasizing the difference between nonatopic and IgE-mediated wheezing (2–4). This concept was based on studies showing that the large majority of nonatopic children with repeated wheeze in the preschool period lose their symptoms at school age. By contrast, atopic wheezing children are prone to a chronic course of asthma, characterized by more severe symptoms, presence of airway hyperresponsiveness, and lung function impairment (5). On the basis of these observations, one could predict that the pathology typical of asthma would be present in atopic wheezing children only, whereas nonatopic wheezing children would have a different pathology. Although the relationship between wheezing and allergic sensitization in the first years of life is still controversial (6), atopy is generally believed to be a crucial determinant in the future development of persistent asthma (7, 8). The concept has gone so far that, in clinical practice, atopic children are more frequently diagnosed by their general practitioners as having asthma than are nonatopic children (4, 9). However, it must be highlighted that defining the etiology of wheezing in children, especially in nonatopic preschool children, is extremely difficult and that clinical assessment of symptoms is the crucial step to direct the diagnosis. This study was therefore designed to investigate whether, in children whose pattern of symptoms favors a diagnosis of asthma, airway pathology is influenced by atopic status. Therefore, we evaluated epithelial loss, basement membrane thickness, inflammation, and angiogenesis in atopic and nonatopic children with multitrigger wheeze responsive to bronchodilators. The results were compared with those of control children with no atopy or wheezing. Moreover, in the three groups of children, the Th2-related cytokines interleukin (IL)-4 and IL-5, and vascular endothelial growth factor (VEGF) were examined. Preliminary results of this study have been previously reported in abstract form (10, 11).
Subjects We recruited three groups of children undergoing fiberoptic bronchoscopy for appropriate clinical indications (12–15): (1) 18 nonatopic children with multitrigger wheeze (aged 2–10 yr), (2) 20 atopic children with multitrigger wheeze (aged 2–15 yr), and (3) 17 control children with no atopy or wheeze (aged 2–14 yr). Some of the children examined in the present study were already included in a previous study (14). Multitrigger wheeze was defined as repeated episodes of wheezing, breathlessness, and cough that were present even apart from colds. Moreover, in all wheezing children, symptoms had to be responsive to bronchodilators. Presence and reversibility of symptoms were assessed by parental reports and confirmed by the child's pediatrician. The presence of atopy was defined by an increase in total (paper radioimmunosorbent test [PRIST]) or specific (radioallergosorbent test [RAST]) IgE (14, 15). In particular, specific IgE for all the following aeroallergens were investigated in all children: house dust mite (Dermatophagoides pteronyssinus, D. farinae), molds (Alternaria alternate), cat dander, and grass pollens (Lolium perenne, Poa pratensis, Phleum pratense, Dactylis glomerata, Cynodon dactylon). All children in the three groups underwent routine blood tests, whereas spirometry was performed only in children who were able to cooperate with the test. Bronchoscopy with endobronchial biopsy and bronchoalveolar lavage was conducted in accordance with criteria laid down in the guidelines for fiberoptic bronchoscopy in children (16, 17). Nonatopic wheezing children underwent bronchoscopy for recurrent pneumonia (n = 6), chronic cough (n = 10), stridor (n = 1), or difficult asthma (n = 1); atopic wheezing children underwent bronchoscopy for recurrent pneumonia (n = 11), chronic cough (n = 7), stridor (n = 1), or difficult asthma (n = 1); and control children underwent bronchoscopy for recurrent pneumonia (n = 7), chronic cough (n = 8), stridor (n = 1), or laryngomalacia (n = 1). It has recently been demonstrated that, when a clinically indicated bronchoscopy is performed, bronchial biopsies can be performed safely and ethically (6, 18). Written consent was obtained from the children's parents after they were informed that, during bronchoscopy, one bronchial biopsy would be taken for research purposes. The study was performed according to the Declaration of Helsinki and was approved by the Ethics Committee of Padova City Hospital (Padova, Italy).
Sample Processing and Analysis Bronchial biopsies were considered suitable for examination when there was at least 1.0 mm of basement membrane length and 0.1 mm2 of subepithelial area (14). Briefly, bronchial biopsies were processed and paraffin sections stained with histochemical and immunohistochemical methods (19). In particular, analysis of epithelial loss and reticular basement membrane thickness was performed on sections stained with hematoxylin–eosin (14, 15). The number of inflammatory cells (eosinophils, neutrophils, mast cells, macrophages, and CD4+ T lymphocytes), IL-4+, IL-5+, and VEGF+ cells as well as the number of vessels were quantified in the subepithelium by immunohistochemistry as previously described (14, 15, 19–21). Differences between groups for morphologic data were analyzed using the nonparametric Kruskal-Wallis test. The Mann-Whitney U test was performed after the Kruskal-Wallis test when appropriate. For clinical data, analysis of variance was performed. Correlation coefficients were calculated using the nonparametric Spearman's rank method. Adjusted P values for multiple comparisons were calculated using the Holm method (22, 23). However, to minimize the possibility of introducing type 2 errors (24) when comparing atopic and nonatopic children, results are presented giving the unadjusted P values. A detailed comparison of unadjusted and adjusted P values is included in the online supplement. Presence of bacterial colonization or viral infections was also investigated. All details are reported in the online supplement.
Clinical Findings The characteristics of the children studied are shown in Table 1. The three groups of children were similar with regard to age. Pulmonary function testing was successfully performed in 11 of 18 nonatopic wheezing children, 10 of 20 atopic wheezing children, and 7 of 17 control children. FEV1 and the ratio between FEV1 and FVC (FEV1/FVC) were not significantly different in nonatopic and atopic wheezing children. However, FEV1 was reduced in nonatopic wheezing children as compared with control subjects. The duration of symptoms was not significantly different in the two groups of wheezing children examined. As for age at onset of symptoms, there was a trend for an earlier onset in nonatopic as compared with atopic wheezing children, but the difference did not reach the levels of statistical significance (P = 0.06). Finally, as expected, total IgE values were increased in atopic wheezing children as compared with both nonatopic wheezing children and control subjects.
All wheezing children were receiving bronchodilators as needed and those with recurrent pneumonia (n = 24) were treated with antibiotics. Twenty-five wheezing children (11 nonatopic and 14 atopic) had mild symptoms and were not currently being treated with corticosteroids on a regular basis, but some of those children had received intermittent therapy with steroids in the past. Eleven wheezing children (6 nonatopic and 5 atopic) had a moderate pattern of symptoms requiring a regular combined treatment with equivalent daily doses of beclomethasone ranging from 200 and 400 µg, and the two patients with difficult asthma had also been treated with oral betamethasone. Only a minority of children were also treated with antileukotrienes (n = 3). When we examined clinical history, we found early evidence of bronchiolitis (at 5 and 6 mo) in only 2 out of 18 nonatopic children. Information on atopic status (total and specific IgE) is included in the online supplement. The bronchoscopy procedure was well tolerated and no complications were encountered.
Biopsy Findings
All pathologic features examined were similar in nonatopic and atopic children with multitrigger wheeze (Figures 1–3
For the main parameters examined (epithelial loss, basement membrane thickness, number of eosinophils and vessels), the differences in median values between wheezing children and control subjects were comparable to those observed in our previous studies (14, 17). By contrast, the differences in median values between atopic and nonatopic wheezing children were as follows: 15% for epithelial loss, 0.2 µm for basement membrane thickness, 5 eosinophils/mm2, and 48 vessels/mm2. These differences were well below the median differences that were found to be significant in previous studies (i.e., 40% of epithelial loss, 1.8 µm of basement membrane thickness, 30 eosinophils/mm2, and 126 vessels/mm2). When the analysis was performed using a more stringent definition of atopy (both high total IgE and positive RAST), the results remain unchanged: that is, atopic children with high total IgE and positive RAST (n = 12) had increased basement membrane thickness, epithelial damage, eosinophils, and IL-4 as compared with control subjects and, again, were not different from nonatopic wheezing children. Details are included in the online supplement. When we compared nonatopic and atopic children within the group with mild symptoms and within that with moderate symptoms, we found no difference in any of the parameters examined (Table 3). When we compared all children with mild symptoms with those with moderate symptoms, they had similar values of epithelial loss (median [interquartile range]: 57% [39–74%] vs. 53% [37–74%]), basement membrane thickness (4.9 [4.2–6.0] vs. 5.5 [4.6–6.2] µm), number of eosinophils (28 [21–87] vs. 47 [14–222] cells/mm2), number of vessels (252 [112–276] vs. 206 [124–313] vessels/mm2), IL-4+ cells (131 [99–176] vs. 144 [62–183] cells/mm2), and IL-5+ cells (315 [307–474] vs. 366 [211–413] cells/mm2). The differences in median values between children with mild and moderate symptoms were as follows: 4% for epithelial loss, 0.6 µm for basement membrane thickness, 19 eosinophils/mm2, and 46 vessels/mm2. These differences were well below the median differences that were found to be significant in previous studies (14, 17). The only significant difference observed was the number of mast cells that was lower in children with moderate symptoms compared with those with mild ones (median [interquartile range]: 0 [0–71] vs. 131 [59–263] cells/mm2; P = 0.001), probably because of steroid therapy.
We then decided to investigate pathologic changes in atopic and nonatopic children considering separately those at preschool and those at school age. We found that all pathologic changes were similar in nonatopic and atopic wheezing children younger than 6 years (Table 4) as well as in those older than 6 years (Table 5). For the majority of these parameters, there was an increase as compared with control subjects; however, some failed to reach a statistical significance, probably due to the low number of subjects examined in each subgroup (Tables 4 and 5).
When Holm correction for multiple comparison was performed, most of the parameters that, in the overall analysis, were increased in atopic and nonatopic children without adjusting remained increased even after adjustments. The differences that were not confirmed after adjustment were as follows: IL-5 and VEGF expression in nonatopic wheezers as compared with control subjects (P = 0.072 and P = 0.08, respectively). Most of the differences that were borderline in the subanalyses (<6 yr,  6 yr, mild, and moderate) were not confirmed after adjustment. Detailed information is included in the online supplement. Finally, when all wheezing children were grouped together, we observed a number of weak correlations between physiologic and morphometric parameters, which are reported in the online supplement.
The purpose of this study was to investigate whether the airway pathology of children with multitrigger wheeze responsive to bronchodilators would be different in nonatopic wheezers, who are often considered nonasthmatic, compared with atopic wheezers, who are more frequently diagnosed as having asthma. Unexpectedly, we found that all inflammatory and structural changes characteristic of asthma (i.e., epithelial loss, basement membrane thickening, angiogenesis with increased VEGF, eosinophilia, and even up-regulation of IL-4 and IL-5) were present to a similar degree in nonatopic and atopic children with multitrigger wheeze. Asthma is a heterogeneous condition with different phenotypes and clinical expressions. The presence of different conditions that may be associated with lower airway obstruction during childhood complicates our understanding of the pathogenesis of asthma (3). Wheezing is the major clinical expression of the disease, although it is widely recognized to be a nonspecific sign associated with airway narrowing. Nonetheless, wheezing is the most frequent symptom in children with asthma and often the one we have to rely on in the youngest children. Therefore, to clarify the nature of wheezing in children is a crucial issue in understanding the natural history of asthma. Several different wheezing patterns have been described in children (1–8) and have recently been investigated using a multidimensional approach, resulting in three major phenotypes: "transient viral wheeze," "atopic persistent wheeze," and "nonatopic persistent wheeze" (25). All wheezing children examined in the present report had a persistent pattern of symptoms that were present even apart from colds, indicating a multiple trigger phenotype. The study demonstrated that, when these specific symptoms are present, the airway pathology is that typical of asthma in both atopic and nonatopic individuals. Although a group of children with transient viral wheeze was not examined, there is evidence from the literature that the airway inflammation may be different in these children (26). This does not rule out the possibility that viral infections may play a role in the pathogenesis of asthma. Indeed, early exposure to infective agents combined with a defective innate immune response (27, 28) may be an important factor that adds to other alterations, such as polymorphisms in the gene coding for IL-4 and IL-13 (29, 30). Therefore, environmental exposure, maturation of the immune system, and development of airway structure in the first years of life are strongly implicated in the subsequent persistence of wheezing. Of interest, it has been shown that neither eosinophilic inflammation nor basement membrane thickening is present in symptomatic infants (<2 yr of age), even in the presence of atopy and reversible airflow obstruction (31). By contrast, both eosinophilia and basement membrane thickening are definitely present in preschool-age in children with difficult asthma (32). This observation is in agreement with our study because, when we performed an age-stratified analysis, we observed that these pathologic changes could be detected even in preschool children with milder forms of the disease. Moreover, we observed that the pathologic changes present in children with mild symptoms were similar to those observed in children with a moderate pattern of symptoms, who are more promptly identified as asthmatics. Altogether, the observations discussed above indicate that the airway pathology characteristic of asthma, although not present at birth, develops early in life and occurs at all stages of disease severity. Even if all the pathologic parameters examined were on average higher in the groups of wheezing children, we should acknowledge that there was a wide within-group dispersion, with some wheezing children having values within the normal range. Nevertheless, a high degree of variability is a common finding when analyzing pathologic features of asthma, not only in children but in adults as well. Our findings that airway pathology characteristic of asthma is unrelated to atopy apparently contrast with our previous observation showing that a similar airway pathology was present even in atopic children without symptoms of asthma. It should be highlighted, however, that some, but not all, pathologic traits of asthma were present in atopic nonasthmatic children. Indeed, these children had eosinophilia and an increased number of vessels (13, 14), but not the epithelial damage and the degree of basement membrane thickening observed in children with asthma (13, 14). In fact, thickening of the basement membrane, which is definitely present in children with asthma, is only marginal in atopic children without symptoms (13, 14). Nevertheless, the observation that atopy per se may be associated with some pathologic features of asthma does not rule out the possibility that this pathology is present in children with asthma and without atopy. It is possible that the same immune response may be activated by diverse mechanisms (not necessarily IgE mediated) because the type of response may be determined by the tissue itself rather than by the kind of stimulus (33). In our study, we found that children with either atopic or nonatopic wheezing show infiltration of the bronchial mucosa not only by eosinophils but also by cells expressing the Th2 cytokines IL-4 and IL-5, which are traditionally believed to be strictly related to atopic status (34). On the one hand, it is possible that atopy may not be immediately apparent and nonatopic children may become atopic later on. However, when we performed a separate analysis of children older than 6 years, we found that the pathologic alterations present in nonatopic children at preschool age were present even at school age. In this context, it is interesting to note that studies in adults have demonstrated that even nonatopic individuals with asthma show increased IL-4 and IL-5 expression to a similar degree as atopic individuals with asthma (35). In adults, the evidence that airway pathology is the same in atopic and nonatopic individuals with asthma is so convincing that the two traditional phenotypes of "intrinsic" and "extrinsic" asthma are considered a single disease (36, 37). In our study, all children were recruited on the same clinical bases (i.e., the pattern of symptoms); nevertheless, nonatopic children had lower values of FEV1 and a trend for an earlier onset of symptoms. This observation is in line with studies in adults with asthma whose clinical presentation is typically more severe in nonatopic individuals than in atopic ones. Moreover, we reasoned that the earlier onset of symptoms may be related to the presence of early bronchiolitis, which could have been more frequent in nonatopic children. However, this possibility seems unlikely because only 2 out of 18 nonatopic wheezing children had a clinical history of bronchiolitis. Other causes of wheezing, such as helminth infections that may be frequent in nondeveloped countries, are also improbable in the context of our study (38). It is interesting to note that the evolution of symptoms between childhood, adolescence, and midadult life is still a matter of debate. Indeed, symptoms can persist, remit conclusively, or eventually relapse (8, 39), and there is recent evidence that complete clinical remission of childhood wheezing is less frequent than previously believed (40). Furthermore, even when clinical remission apparently occurs, airway inflammation and remodeling are still present and may promote a relapse of symptoms later on (41). We should acknowledge, however, that ours is a cross-sectional study examining airway biopsies at a single time point in childhood, whereas only longitudinal studies examining airway pathology both in childhood and puberty might provide definite conclusions on this issue. However, these studies cannot be proposed for ethical reasons, and we tried to gain some new insight by combining our pathologic approach to the epidemiologic debate. In conclusion, this study shows that the airway pathology characteristic of asthma—that is, epithelial loss, basement membrane thickening, angiogenesis, eosinophilia, and up-regulation of IL-4 and IL-5—is present in nonatopic wheezing children. Unexpectedly, all the pathologic features were similar to those present in the airways of atopic wheezing children, whose symptoms are more likely to persist into adult life. Although we do not know what the evolution of symptoms will be in these nonatopic children, our study shows that, when suggestive symptoms are present in nonatopic children, the airway pathology is that characteristic of asthma.
The authors thank Christina A. Drace for assistance in editing the manuscript.
Supported by the University of Padova, Italian Ministry of University and Research, and the Italian Society of Childhood Respiratory Diseases (SIMRI). This article has an online supplement, which is accessible from the issue's table of contents at www.atsjournals.org Originally Published in Press as DOI: 10.1164/rccm.200712-1818OC on May 29, 2008 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form December 13, 2007; accepted in final form May 29, 2008
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