INTRODUCTION

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The inflammatory bowel diseases, namely Crohn's disease (CD) and ulcerative colitis are immunologically mediated illnesses of unknown etiology. Although studies in children are limited, epidemiologic data of CD indicate that individuals younger than 20 yr of age are increasingly diagnosed (1).

Extraintestinal manifestations including symptomatic lung disease are described in CD (1). Latent pulmonary abnormalities evident either at pulmonary function tests (PFTs) or at bronchoalveolar lavage have been also reported in adults with CD in the absence of clinical evidence of airway disease (5). However, the pathogenesis of lung involvement in CD remains obscure.

Recently, subclinical bronchial hyperresponsiveness (BHR) has been described in atopic or nonatopic adults with either CD or ulcerative colitis (8). No studies on bronchial responsiveness in the pediatric population with CD are currently available.

The present study was designed to evaluate the bronchial responsiveness to methacholine (MCh) in children with CD. In order to investigate its long-term evolution and its relationship with possible changes in the underlying disease activity or the therapeutic program, bronchial responsiveness was reevaluated in a subgroup of patients at least 2 yr after the first assessment.

	METHODS

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We studied 14 patients with CD diagnosed on the basis of standard clinical, radiological, endoscopic, and histological findings (median age 12 yr; range, 9.3 to 16.9 yr; 7 males, 7 females). In eight patients the disease was localized both in the small bowel and in the colon. No patient presented with the involvement of the small bowel alone; the colon alone was involved in six subjects. None of the examined subjects had extraintestinal manifestations.

Clinical details of the patients are shown in Table 1. The length of CD at the onset of the study ranged from 1 to 80 mo (median 28.5 mo). The relative severity of CD was based on the Pediatric Crohn's Disease Activity Index (PCDAI) in which a final value  10 indicated quiescent disease, 11  PCDAI < 30 mild active CD disease, and > 30 moderate to severe CD disease (9). In our patients median PCDAI was 10 (range, 2.5 to 22.5); two patients had mild active CD disease (Cases 10 and 13; PCDAI 22.5 and 20, respectively). Therapy consisted of pharmacological treatment, i.e., oral mesalazine (50 mg/ kg/d) in two patients, azathioprine (1.5 mg/kg/d) in one subject, enteral nutrition with semi-elemental diet in five cases, semi-elemental diet plus oral methylprednisolone (1 mg/kg/d) in one subject; five patients with quiescent disease at the onset of the study had not been receiving any treatment in the last 6 mo. Forced expiratory volume in one second (FEV₁) was obtained from all subjects at the entry into the study (Model 2200; Sensor Medics Inc., Yorba Linda, CA) and expressed as percentage of the predicted value (10). Infection or parasites were excluded by stool cultures, microscopic stool examination, and serology. Other metabolic diseases, namely diabetes mellitus and cystic fibrosis were excluded. Physical examination and chest X-ray were normal in all patients.

Ten healthy nonatopic subjects (median age, 10.5 yr; range, 8.7 to 15; 6 males, 4 females), and 10 patients with diagnosed mild bronchial asthma (median age, 9.9 yr; range, 8.7 to 13.7 yr; 4 males, 6 females) were also entered into the study. Mean FEV₁ in healthy control subjects and asthmatics was 95 ± 3% and 90 ± 2% (SEM), respectively. Five asthmatic subjects had positive skin-prick tests to the most common aeroallergens. At the time of testing all patients and control subjects were clinically stable and none of them was receiving treatment for asthma. Apart from one boy with CD treated also with methylprednisolone (Case 7), no patient was receiving any other drug known to affect bronchial responsiveness. None of the subjects had ever smoked.

Bronchial responsiveness was measured in the morning by MCh challenge according to a dosimetric method currently used in our laboratory (11). Briefly, at baseline the subject performed three consistent forced expiratory maneuvers to ensure reproducibility of the observed pulmonary function. Patients with FEV₁ greater than 80% of the predicted value underwent the MCh challenge. Methacholine chloride (Lofarma, Milan, Italy) at 0.125% and 1% concentration in phosphate buffer saline were inhaled through a mouthpiece connected to a dosimeter (Mefar, Brescia, Italy; air driving pressure: 1.5 kg/cm²; particle size of the aerosol: between 0.5 and 5 µ). Nebulization was activated by the subject's inspiratory maneuver beginning at functional residual capacity. The nebulization time (0.4 ± 0.2 s) was regulated to obtain an output of 5 µl. Spirometry was obtained after the inhalation of the diluent control solution. If the FEV₁ did not change more than 5% from the baseline value, increasing doses of MCh from 6.25 µg to 2,600 µg as cumulative doses were delivered. FEV₁ was measured again 1 min after each inhalation. The test was continued until either a fall of at least 20% in FEV₁ was obtained or the last dose was reached. Results were expressed in terms of provocation dose (PD₂₀), i.e., micrograms of MCh causing a 20% fall in FEV₁ from the postdiluent value. This was calculated on the cumulative log dose-response curve by linear interpolation between the last two points. In our laboratory a PD₂₀ greater than 2,600 µg is regarded as normal.

To evaluate possible changes of bronchial responsiveness to MCh in subjects with CD, five unselected CD patients underwent a second MCh challenge after a median period of 25 mo (range, 24 to 28 mo) from the first test. During this interval in three cases the therapeutic regimen consisting of mesalazine (Cases 1 and 5) and azathioprine (Case 2) remained unmodified; in the remaining subjects therapy with mesalazine was started in a previously untreated girl (Case 3), and treatment with semi-elemental diet was tapered off in an additional boy (Case 4) 4 and 2 mo before the second MCh, respectively. In order to avoid any effect of circadian or seasonal variation on airway responsiveness or bronchial tone, the MCh challenge was repeated in the same conditions (time of the day; month of the year) of the first challenge.

On completing the MCh, 200 µg of albuterol was given to all patients and healthy subjects by inhalation by a spacer device to restore airway caliber.

A standardized pediatric questionnaire devised by the American Thoracic Society (12) and adapted to an Italian population was administered to subjects with CD to obtain a personal and family history of respiratory disease and allergic symptoms. In all patients with CD the existence of an atopic state was evaluated by skin-prick test using common allergen extracts. Total blood eosinophil count and the serum IgE levels, the latter measured by an enzyme-linked immunosorbent assay (ELISA) and expressed as kU · L¹ were also determined in CD subjects.

Results were given as geometric means with 95% confidence interval (CI), unless otherwise stated. Because the raw data were not normally distributed, we compared PD₂₀ among groups after logarithm transformation. This allowed us to use parametric tests. Correlations were calculated by Pearson's product moment coefficient. All negative tests (nonresponders to the cumulative dose of 2,600 µg) were assigned a value of 2,601. A p value < 0.05 was considered significant. A standard statistical package was used for data analysis (SPSS-PC, release 6; SPSS Inc., Chicago, IL).

Informed consent was obtained from the patients and/or their families. The study protocol was approved by the hospital ethics committee.

	RESULTS

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In patients with CD mean total blood eosinophil count and serum IgE levels were 0.20 × 10⁹ · L¹ (95% CI  1.68 to 2.08) and 138.4 kU · L¹ (95% CI 18.84 to 257.96), respectively. In three of 14 patients with CD skin-prick tests were positive to common aeroallergens (Cases 8 and 10, positive to Dermatophagoides pteronyssinus, Dermatophagoides farinae, and Parietaria officinalis; Case 9 positive to D. pteronyssinus and D. farinae).

Results of the MCh challenge in the control group were considered negative because no subject responded to the cumulative last dose of 2,600 µg of MCh. The distribution of bronchial responsiveness to MCh in patients with CD and asthmatics is shown in Figure 1. In 10 of the 14 CD patients PD₂₀ was less than 2,600 µg (cases 1 to 10) and seven of these had a PD₂₀  1,000 µg; the remaining four patients did not respond to the cumulative last dose of 2,600 µg of MCh (Cases 11 to 14). These findings indicate a prevalence of BHR in our CD population equal to 71%. In patients with CD PD₂₀ was not related to the baseline FEV₁ (r = 0.32, p = not significant [NS]).

View larger version (9K): [in this window] [in a new window]   Figure 1.   Distribution of PD20 in atopic (closed diamonds) and nonatopic (open diamonds) patients with Crohn's disease (geometric mean 699 µg [95% CI 238 to 1,115]), and atopic (closed circles) and nonatopic (open circles) subjects with asthma (geometric mean 104 µg [95% CI 37.35 to 293]) (p < 0.05).

All asthmatic subjects showed a PD₂₀ less than 2,600 µg; Kolgomorov Smirnov nonparametric test showed a significant difference between mean PD₂₀ of the whole study population of CD patients and asthmatic subjects (699 µg [95% CI 238 to 1,115] versus 104 µg [95% CI 37.35 to 293]; p < 0.05). In patients with CD PD₂₀ was not related either to serum IgE levels or to total blood eosinophil count (r = 0.5, p = NS; r = 0.15, p = NS, respectively).

BHR was evident both in atopic patients with CD and in seven out of 10 nonatopic CD subjects (Cases 1 to 7), as shown by PD₂₀ of 97.04 µg (95% CI 58.88 to 1,592) and 537 µg (95% CI 326.8 to 882), respectively. Mean IgE concentrations were 507 kU · L¹ (95% CI 195 to 819) and 59.7 kU · L¹ (95% CI 21.1 to 98.3) in atopic and nonatopic CD patients with BHR, respectively.

Answers to the questionnaire showed that only two of the 14 subjects with CD (14% of the total) had suffered from asthma, but not in the year preceding the study; both patients were also atopic (Cases 8 and 10). None of the patients had ever had rhinitis. Nine of the 14 patients with CD (64% of the total), including six nonatopic subjects and three atopic patients had a family history of atopy (asthma, rhinitis, eczema), but only one nonatopic boy with CD (7% of the total: Case 7) reported a family history of allergic asthma.

Bronchial responsiveness to MCh was not affected by the disease activity at the time of the study because median PCDAI was 9 (range, 2.5 to 22.5) and 8 (range, 5 to 20) in the subgroups of CD patients with BHR and normal bronchial reactivity, respectively. Finally, the duration of the disease at the time of the MCh did not seem to influence the prevalence of BHR because CD patients with either BHR or normal bronchial responsiveness had a median duration of 33 (range, 1 to 80) and 28 (range, 8 to 68) mo, respectively.

When the bronchial responsiveness was reevaluated, mean PD₂₀ at the second MCh challenge appeared significantly greater than basal PD₂₀ (1,941 µg versus 575 µg, p < 0.05; Wilcoxon's rank sum test). All patients who were retested were nonatopic. In the same subgroup median PCDAI at the first and at the second MCh challenge were 6.5 (range, 2.5 to 10) and 5 (range, 2.5 to 7.5), respectively.

	DISCUSSION

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The results of this study show that BHR occurs in children and adolescents with CD in a very high proportion (71%) even in the absence of clinical, radiological, and functional evidence of airway disease. These data are in agreement with those obtained by Louis and colleagues who demonstrated BHR unrelated to disease activity in a group of adults with either CD or ulcerative colitis; the prevalence of BHR in patients with CD observed by these investigators (48%) appears lower than the prevalence seen in our study population (8). This finding is likely to depend on the different mean age of the patients enrolled in the two studies and is in keeping with the observation that BHR may progressively decrease with age (13).

Clinical signs and symptoms of respiratory disease have been reported in patients with CD (1). Most of the anecdotal accounts of moderate to severe lung damage characterized by granulomatous infiltrates of the bronchioles or lung interstitium involved adults (2). However, children or adolescents with variable lung involvement have also been described (2, 14, 15). Striking abnormalities at PFTs, mainly consisting of decreased diffusing capacity and restrictive defects have also been documented at all ages (16). Sensitive techniques like the measurement of the volume of equal flow revealed significant abnormalities of the small airways in asymptomatic subjects without resting airflow limitation (5). Finally, pharmacological treatment with mesalazine might also contribute to the development of pulmonary symptoms (19), but in some cases lung disease progressed even after the discontinuation of the treatment (3).

A high incidence of subclinical lymphocytic alveolitis has also been reported in adults with CD either with or without abnormalities of pulmonary permeability (6, 7). Pathogenesis of alveolitis in CD is controversial. Few studies have revealed an increased CD4/CD8 ratio resembling the abnormalities found in sarcoidosis, suggesting an underlying disorder of the mucosal immune system common to both entities (20). It has been hypothesized that the alveolar macrophage may participate directly in the injury to the alveolar wall by releasing increased amounts of superoxide anions (21). Finally, because stimulation of the alveolar macrophage by immune complexes may contribute to maintaining alveolitis, a role for circulating immune complexes in the pathogenesis of CD alveolitis has also been suggested (18).

We cannot provide an exhausting explanation for the pathogenesis of BHR in CD. In our study bronchial responsiveness was not influenced by either the activity of the disease at the time of the study, or its duration. None of the investigated subjects had extraintestinal manifestations. Current therapy for CD was unlikely to affect BHR because even individuals who were not receiving any treatment were hyperresponsive. Although the relatively small sample size does not allow one to reach definite conclusions, atopy does not appear a satisfactory explanation because BHR was demonstrated in both atopic and nonatopic patients with CD; however, there was a trend to lower values of PD₂₀ in CD patients with positive skin-prick test and high serum IgE levels. The only patient treated with oral steroids was hyperresponsive (Case 7; PD₂₀ 416 µg); this boy who had negative skin-prick test and normal serum IgE, never suffered from asthma but had a family history of asthma. Data from the questionnaire showed that in patients with CD the prevalence of either family or personal history of asthma is not different from the prevalence recently seen in a schoolchildren population from central Italy (7% versus 8.3%, and 14% versus 15%, respectively) (22). CD atopic subjects with BHR and previous episodes of asthma were clinically stable at the time of the study and had no symptoms of acute respiratory tract disease. These data, according with the findings from Louis and coworkers indicate that the mechanism of BHR in CD is still obscure (8).

Despite extensive efforts, both the cause and pathogenesis of CD have yet to be established. There is strong evidence that deranged intestinal permeability plays a central role in the pathogenesis of the disease by enhancing the uptake of toxic luminal bacterial products. Cytokine regulation of epithelial permeability may play a role in the pathogenesis of mucosal damage in CD as abnormal and uncontrolled immune responses to microbial products and luminal antigens have been implicated in tissue damage (1). Interestingly, a recent study has shown that patients with CD have increased pulmonary permeability which appears unrelated to the disease activity (7). Increased pulmonary permeability might be the expression of a generalized, genetically determined abnormality of epithelial permeability in CD. This would provide a satisfactory explanation even for BHR: lymphocytes and/or macrophages sensitized to antigens in the intestinal mucosa would more easily enter the bronchial mucosa and start the inflammatory reaction in the airway. The absence of any signs or symptoms of respiratory disease even in hyperreactive patients with CD supports the supposition of subclinical airway involvement which may be alternately responsible for the development of small airways dysfunction or lymphocytic alveolitis or increased pulmonary permeability, as previously reported (5) or even BHR.

Bronchial hyperreactivity significantly decreased over a 2-yr period in a subgroup of patients with CD unrelated to disease status or treatment. This finding confirms that no relationship between bronchial responsiveness and the activity of the inflammatory bowel process exists. Nevertheless, criticism has been made on the limitation of PCDAI in reflecting adequately the disease activity (9). For this reason, the determination of intestinal permeability or the measurement of inflammatory mediators such as cytokines originating in the inflamed tissue itself have been proposed for monitoring the disease even in the absence of clinical activity (9, 23). Should additional laboratory tests be included in the score, more detailed information about continued bowel inflammation would be obtained, thus providing the opportunity of relating adequately bronchial responsiveness to the activity of the disease. Finally, because the prevalence of BHR may decrease with age (13), we cannot exclude an effect of age on reducing BHR.

In summary, the large proportion of patients with CD who may be found hyperreactive at any age even in the absence of symptoms or signs of respiratory disease indicates that BHR either related or unrelated to atopy must be included among the manifestations of lung involvement in CD. BHR is likely to be the expression of subclinical airway inflammation, a multifaceted phenomenon which can be responsible for the development of various pulmonary manifestations in CD.