Reduced Interferon- Production in Infants with Bronchiolitis and Asthma

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1 Reduced Interferon- Production in Infants with Bronchiolitis and Asthma PAOLO M. RENZI, JEAN P. TURGEON, JACQUES E. MARCOTTE, SUSAN P. DRBLIK, DENIS BÉRUBÉ, MARIE F. GAGNON, and SHELDON SPIER Ste-Justine and C.H.U.M. Research Centers, Pulmonary Units of Ste-Justine and Notre Dame Hospitals, University of Montreal, Respiratory Health Network of Centers of Excellence of Canada, and the Meakins Christie Labs, McGill University, Montreal, Quebec, Canada Infants are at increased risk of developing asthma after acute bronchiolitis. We assessed the hypothesis that cytokine production is related to the development of asthma after bronchiolitis. The smoking history and the presence of atopy or asthma in parents or siblings were recorded and blood mononuclear cell interferon (IFN)- and interleukin (IL)-4 production in response to IL-2 were assessed in 32 infants hospitalized for bronchiolitis and in a subgroup (n 19) in which pulmonary function tests were performed approximately 4.9 mo later. The presence of asthma was determined by the Delphi consensus method 2 yr after hospitalization. Infants were classified as follows: asthma absent (A, n 14), possible (Po, n 9), or probable (Pr, n 9). Infants with possible and probable asthma had lower IFN- production at the time of bronchiolitis and a trend to lower IFN- production 4.9 mo later when compared with those who had no asthma. At the time of bronchiolitis, IFN- production was: versus versus pg/ml, A versus Po versus Pr (p 0.02, ANOVA) and 4.9 mo after bronchiolitis, IFN- production was: versus versus pg/ml, No versus Po versus Pr (p 0.08 ANOVA). IL-4 production did not differ between groups. Infants who went on to develop asthma had more parent smokers (21.4% versus 55.6% versus 55.6%, A versus. Po versus Pr, p 0.04), lower VmaxFRC ( versus 77 7 versus 67 8% predicted, A versus Po versus Pr, p 0.02), lower PC 40 histamine ( versus mg/ml, A versus Po Pr, p 0.03) but no increase in atopy or asthma in their family. Significant. positive correlations were found between IFN- production at the time of bronchiolitis and VmaxFRC (r 0.606) or PC 40 histamine (r 0.648) 4.9 mo after bronchiolitis. Lower IFN- production at the time of bronchiolitis is an indicator of lower pulmonary function and increased responsiveness to histamine 4.9 mo after bronchiolitis and is related to the development of asthma after bronchiolitis in infants. Renzi PM, Turgeon JP, Marcotte JE, Drblik SP, Bérubé D, Gagnon MF, Spier S. Reduced interferon- production in infants with bronchiolitis and asthma. AM J RESPIR CRIT CARE MED 1999;159: Acute bronchiolitis is a common lower respiratory tract infection of infants, usually younger than 6 mo of age, that is often caused by respiratory syncytial virus (RSV) (1 3). Infants with acute bronchiolitis present with wheezing, cough, dyspnea, either with or without fever, rapid breathing, and moderate to severe prostration (1). Although most infants recover in a week from the acute bronchiolitis, up to 50% of these subjects will develop repeated episodes of wheezing or asthma during (Received in original form May 22, 1998 and in revised form December 11, 1998) Supported in part by grant MT from the MRC and by grants from the Quebec Thoracic Society, the Respiratory Health Network of Centers of Excellence of Canada, the Notre Dame Hospital Foundation, and the Ste-Justine Research Institute. Dr. Renzi is the recipient of a Scholar Award from the Fonds de Recherche en Santé du Québec. Correspondence and requests for reprints should be addressed to Dr. P. M. Renzi, University of Montreal Hospitals Research Center, 2065 Alexandre desève, Montreal, PQ, H2L 2W5 Canada. Am J Respir Crit Care Med Vol 159. pp , 1999 Internet address: the 2 yr that follow their infection (1). Bronchiolitis is the most important risk factor for the development of asthma in infants younger than 2 yr of age (4). Subjects who develop bronchiolitis are thus an interesting group to use in order to study the risk factors that are related to the development of asthma. It is not known what differentiates the patients who recover completely from bronchiolitis from the ones who go on to develop asthma afterwards. Several investigators have studied whether a genetic predisposition to atopy increases the risk of developing asthma after bronchiolitis because of the link between atopy and asthma in children. Most studies have not found a positive association between the presence of atopy in the family and the development of asthma after bronchiolitis (4 7). Results linking total serum IgE to the development of asthma have also been inconsistent (4 8). The most convincing work linking atopy with the development of asthma after bronchiolitis has come from Welliver and colleagues (9, 10). They showed that specific mucosal anti-rsv IgE was increased in the patients with bronchiolitis or asthma when compared with those with mild upper respiratory tract illness or pneumonia. The patients with the highest mucosalspecific IgE had the most wheezing episodes. These results

2 1418 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL suggest that a compartmentalized immune response favoring IgE production in the lungs is associated with the development of recurrent wheezing and thus asthma. The mechanism of the increased IgE production in these infants is not known, but it may be related to an imbalance between lymphocyte Th1 and Th2 cytokine production. Indeed, the helper lymphocyte with a Th2 phenotype is a cell involved in B-cell switching to IgE production through cell contact and interleukin (IL)-4 secretion (11). The Th1 cell, through the production of interferon gamma (IFN- ), can inhibit the switching of B cells into IgE producing cells (12). An increase in Th2 cytokine production has also been found in asthma. Interleukin-4 is increased in the airways and serum of patients with asthma (13 15), whereas there is a normal or low production of the cytokine IFN- in cells obtained from the blood (16). The major objective of our study was to test the hypothesis that an imbalance in cytokine production at the time of bronchiolitis could predict the development of asthma afterwards. Our results show that infants with the lowest IFN- production at the time of bronchiolitis are the ones most likely to develop asthma afterwards. In addition, we also show a significant correlation between low IFN- production at the time of bronchiolitis and abnormal pulmonary function 4.9 mo later. METHODS Subjects Thirty-two subjects with acute bronchiolitis were enrolled in the study. Acute bronchiolitis was diagnosed if the subjects had symptoms for fewer than 7 d that included cough, dyspnea, tachypnea, and wheezing with or without fever without any evidence of bacterial infection or pneumonia (on the chest radiograph) and required hospitalization. The diagnosis and decision to admit the patient was made by a pediatrician or a respiratory pediatrician before determining whether the subjects were eligible for the study. All the charts of admitted patients were reviewed from Monday to Friday as soon as the attending physician had decided to hospitalize the patient. Eligible subjects for the study were identified in the emergency room or upon arrival to the wards of Ste-Justine hospital during two consecutive winter seasons (1993, 1994). Enrollment of subjects into the study was done within 24 h of admission. We obtained written informed consent from the parents and then proceeded to blood withdrawal and determination of the presence or absence of RSV on nasal secretions by three different methods as previously described (17). Subjects who had any underlying chronic disease, were born premature, were immunosuppressed, were older than 1 yr of age, or who had to be admitted to the intensive care unit were excluded from enrollment. Infants who had already had a lower respiratory tract infection in the past or had symptoms compatible with asthma prior to the acute event were also excluded from enrollment. This project was approved by the Ethics Committee of the Ste-Justine Hospital, all families consented to regular follow-up for 2 yr, and a subgroup of 19 agreed to another blood test and pulmonary function measurements approximately 4.9 mo after bronchiolitis. A maximum of 7 ml of blood was drawn into a heparinized tube within 24 h of admission for bronchiolitis after it was established that there was no underlying anemia. Subjects received conventional therapy that did not include either Ribavirin or corticosteroids while they were hospitalized. All the families received instructions on how to avoid environmental allergens, and the infants were allowed to receive any therapy that their physician judged necessary within the hospital and after they had been discharged from the hospital. Cell Purification, Culture, and Cytokine Assays Mononuclear cells were obtained by centrifugation of the buffy coat over ficoll-hypaque and plastic adherence as previously described (18). The percentage of monocytes remaining as assessed by nonspecific esterase staining was %. The remaining cells were resuspended in enriched RPMI medium at a concentration of /ml and cultured for 3 d at 37 C with 5% CO 2 in medium alone or medium with 125 U/ml of IL-2. Interleukin-2 was chosen over the stimulation of mononuclear cells with virus in order to avoid the effects of cell death on cytokine production. The concentration of IL-2 employed has been previously shown in subjects with asthma to stimulate lymphocytes to release Th2 cytokines that increase eosinophil proliferation and survival (18). In addition, IL-2 induces IFN- production in resting lymphocytes through a CD40 ligand/il-12-mediated mechanism (19). The measurement of IL-4 and IFN- levels was performed by commercial enzyme-linked immunoassays (ELISA). The ELISAs were purchased from Genzyme Co. (Cambridge, MA). The sensitivities of the various assays were: 25 pg/ml for IL-4 and 20 pg/ml for IFN-, and every measurement was performed in duplicate. Pulmonary Function Measurements Pulmonary function was evaluated with methods recommended by the American Thoracic Society (20 23). Maximal expiratory flow at functional residual capacity ( maxfrc) was assessed by the rapid thoracoabdominal compression technique (RTC) using the following procedure. Patients previously sedated with chloral hydrate 100 mg/kg body weight (maximal dose, 1,000 mg) were placed supine with the neck slightly extended in an inflatable jacket covering the abdomen and thorax and connected to a pressure reservoir. Starting from a pressure of 30 cm H 2 O and using increments of 5 cm H 2 O, measurements of expiratory flow at FRC were obtained until maxfrc was achieved. Flows were measured with a soft cushion mask connected to a Fleisch no. 1 pneumotachograph and integrated. Three additional technically correct maneuvers were performed at this pressure from which the highest value was chosen to represent baseline maxfrc. All subsequent maxfrc maneuvers were carried out using the same procedure. Bronchial reactivity to histamine was assessed by using a Hudson updraft no. 2 nebulizer driven at 8 L/min to administer doubling concentrations of histamine starting at mg/ml to a maximum of 8.0 mg/ml for 1 min at 5-min intervals. maxfrc was determined after each nebulization. The challenge test ended when a decrease in maxfrc of at least 40% from baseline value had been reached, or the maximum concentration of histamine had been given. Heart rate and oxygen saturation were continuously monitored throughout the study with an Ohmeda BIOX 3740 pulse oximeter (Ohmeda, Boulder, CO). Data Retrieval and Statistical Analysis We questioned the parents at the time of bronchiolitis in order to determine if atopy or asthma were present in parents or siblings, whether the parents smoked, and whether the mother had smoked during pregnancy. A modified respiratory questionnaire was administered to the parents at the end of 2 yr of follow-up as previously performed (24). Inpatient and outpatient charts and a list of all medication that was received were collated and this information was presented to three respiratory pediatricians who were blinded to cytokine production and pulmonary function so that they could determine whether the infants had no, possible, or probable asthma. The definition of asthma was obtained from criteria previously employed in this age group (25). The physicians then met and assigned each infant to one of the three groups by the Delphi consensus method. Results were then compared between no, possible, and probable asthma by analysis of variance or by the Kruskall-Wallis analysis of variance followed by Student s t test or the Mann-Whitney U test to assess differences between groups. Comparison of certain demographic data was performed by chi-square analysis. Results for PC 40 histamine were presented as geometric means, and statistical analysis were performed with logarithmic values. Regression analyses were performed using Spearman s rank correlation coefficient. Results are presented as mean standard error of the mean (SEM). The level of significance was set at a p RESULTS Demographics, Atopy, Second-hand Smoking, and Asthma Thirty-two families were enrolled and their infants were followed for 2 yr after bronchiolitis. The average age of the in-

3 Renzi, Turgeon, Marcotte, et al.: Bronchiolitis, IFN-, and Asthma 1419 fants upon enrollment was mo, 53% were male and this profile was not significantly different among the groups (Table 1). Asthma was judged as absent in 44% of the infants 2 yr after bronchiolitis and the remainder of the infants were split between having possible and probable asthma. RSV was present in the nasal secretions of 53% of the infants upon enrollment, not significantly different among the three groups. We assessed the possible effects of atopy on the development of asthma after bronchiolitis by having the family members complete a questionnaire and by measuring serum IgE levels of infants during bronchiolitis. Serum IgE levels were normal in more than 90% of the infants upon enrollment and we found no difference in the percentage of parents or siblings with atopy or asthma among the three groups (Table 1). We determined whether the mother had smoked during pregnancy and whether one or both of the parents were current smokers at the time of bronchiolitis. There was as much as a 2.5-fold increase in the percentage of infants with possible and probable asthma who had parents who smoked at the time of bronchiolitis when compared with the infants who did not develop asthma (p 0.05). This difference was significant whether we considered smoking in the mother during pregnancy, smoking in the mother at the time of bronchiolitis, or smoking in both parents, although the difference seemed more significant when smoking in both parents was considered (Table 1). We repeated blood tests and performed pulmonary function studies on average mo after bronchiolitis in a subgroup of 19 infants whose parents had consented at the time of bronchiolitis. There was no difference in the average age ( mo), sex (58% male), or in RSV positivity (58%) between this subgroup and the 32 infants who were analyzed at the time of bronchiolitis. There was also no differ- Cytokine Production and Asthma Interferon- production in response to IL-2 by blood mononuclear cells obtained at the time of bronchiolitis was significantly lower in both the possible and the probable asthma groups when compared with infants with no asthma (p 0.022, Kruskall-Wallis) (Figure 1a). When IFN- production at the time of bronchiolitis was assessed only in the infants that were RSV positive (seven with no asthma, four with possible asthma, and five with probable asthma) there was no significant difference by analysis of variance (p 0.30 Kruskall- Wallis) but IFN- production was higher in the infants with no asthma when compared with those with possible and probable asthma ( pg/ml versus pg/ml no versus possible and probable asthma, respectively, p 0.04 Mann- Whitney U test). The level of production of IL-4 at the time of bronchiolitis was not significantly different among the three groups (65 16 versus versus pg/ml for no, possible, and probable asthma, respectively). TABLE 1 BASELINE CHARACTERISTICS IN INFANTS Asthma Absent (n 14) Possible (n 9) Probable (n 9) Age, mo* M/F 7/7 6/3 4/5 RSV, % IgE (normal), %* Atopy in one of the parents, % Asthma in one of the parents, % Asthma in one of the siblings, % Smoking in mother during pregnancy, % Smoking in mother, %* Smoking in one of the parents, %* * At time of bronchiolitis. By three different methods on nasal secretions (36). If one test was positive, the patient was considered RSV. Normal IgE levels are less than 25 KU/L. p 0.05, no asthma versus possible and probable. Figure 1. Interferon gamma production in response to IL-2 by blood mononuclear cells. Mononuclear cells obtained at the time of bronchiolitis (a, n 32) or 4.9 mo later (b, n 19) were partially depleted of monocytes and cultured with IL-2 for 3 d. The supernatant was retrieved and IFN- production was measured by ELISA. Results are presented for patients evaluated 2 yr after bronchiolitis as having no asthma (no), possible asthma (possible), and probable asthma (probable). *p 0.05, Kruskall-Wallis test and Mann-Whitney U test, possible and probable versus no asthma; **p 0.08, Kruskall-Wallis; p 0.05 Mann-Whitney U test, possible and probable asthma versus no asthma.

4 1420 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL Asthma TABLE 2 RELATIONSHIP BETWEEN MEASUREMENTS PERFORMED 4.9 MONTHS AFTER BRONCHIOLITIS AND ASTHMA No (n 7) Possible (n 5) Probable (n 7) IL-4, pg/ml maxfrc, % * PC 40 histamine, mg/ml * * p 0.05, no asthma versus possible and probable. p 0.05, ANOVA. DISCUSSION Asthma is an inflammatory disease of the airways that affects approximately 5% of the population (26). There appears to be a link between an index viral infection, IgE production, altered cellular immunity, and the subsequent development or aggravation of asthma (27, 28). Interestingly, recent studies have shown that lymphocyte mediators, key agents in cellular and humoral immunity, may have a role in asthma (13 16). Subjects with asthma have signs of T-lymphocyte activation in their blood (18, 19) and these cells favor IgE production and eosinophilia through increased release of the Th2 cytokines ence in the distribution of infants among all three groups (seven no asthma, five possible asthma, and seven probable asthma) and in cytokine production at the time of bronchiolitis and 4.9 mo later (IFN- : versus pg/ml and IL-4: versus pg/ml, t 0 versus t 4.9 mo, p 0.05). The difference in IFN- production 4.9 mo after bronchiolitis did not reach statistical significance between no, possible, and probable asthma groups (Kruskall-Wallis p 0.08) (Figure 1b). However, when possible and probable asthma were compared with no asthma, there was a significant difference (p 0.05, Mann-Whitney U test). IL-4 production in response to IL-2 was not different between no, possible, and probable asthma (Table 2). Pulmonary Function and Asthma Pulmonary function measurements obtained 4.9 mo after bronchiolitis showed a significantly lower maxfrc and PC 40 histamine in the infants who were later classified as having possible and probable asthma when compared with infants without asthma (Table 2). Comparisons Between Second-hand Smoking, Pulmonary Function, and Cytokine Production Comparisons were performed among smoking in parents, pulmonary function, and cytokine production (Table 3). Infants who were exposed to second-hand smoke had lower max- FRC and PC 40 histamine 4.9 mo after bronchiolitis than did those who were not. However, exposure to second-hand smoke did not seem to affect IFN- or IL-4 production in vitro at both time points. Correlations were performed between pulmonary function and IFN- production. Significant correlations were found between IFN- production at the time of bronchiolitis and pulmonary function 4.9 mo after bronchiolitis (p 0.008) (Figure 2), but no significant correlations were found between IFN- production 4.9 mo after bronchiolitis and pulmonary function (r for maxfrc, r for PC 40 ; p 0.05). TABLE 3 COMPARISONS BETWEEN SMOKING IN PARENTS, PULMONARY FUNCTION, AND CYTOKINE PRODUCTION* Smoking in Parents Yes (n 8) No (n 11) p Value maxfrc, % PC 40 histamine, mg/ml IFN- (t 0), pg/ml NS IFN- (t 4, 9 mo), pg/ml NS IL-4 (t 0), pg/ml NS IL-4 (t 4, 9 mo), pg/ml NS * Values shown for Smoking Parents are means SEM. Figure 2. Correlation between interferon gamma production and maxfrc (a) or PC 40 histamine (b). Cytokine production was measured at the time of bronchiolitis, and pulmonary function was measured 4.9 mo later.

5 Renzi, Turgeon, Marcotte, et al.: Bronchiolitis, IFN-, and Asthma 1421 IL-4 and IL-5 and a normal or decreased production of IFN- (13 16). Whether these abnormalities are present prior to or at the first attack of asthma has not been assessed. We studied infants during and after bronchiolitis because it is an important risk factor for the development of asthma (4). We assessed whether abnormalities in cytokine production are related to the development of asthma by measuring IFN- and IL-4 production in response to IL-2 at the time of bronchiolitis and comparing the results obtained between the infants that went on to develop asthma and those who did not. We found a significantly lower IFN- production at the time of bronchiolitis in infants who went on to develop asthma. The lower IFN- production was not a temporary event because, in a subgroup of infants whose parents accepted to have repeated tests 4.9 mo after bronchiolitis, the ones who went on to develop asthma still had a lower IFN- production. The lower IFN- production was thus either present prior to bronchiolitis or induced by bronchiolitis and persisted for at least 4.9 mo. The finding that IFN- production is decreased in mononuclear cells obtained from children with asthma when compared with normal children (16) would suggest that this defect is permanent. It has been shown that a reduced production of IFN- in neonates or at 9 mo of age is a risk factor for the development of subsequent atopy (29, 30). In addition, we have previously reported in a different group of infants with similar inclusion/ exclusion criteria that lower IFN- production is associated with early wheezing after bronchiolitis (31). Although it is possible that IFN- is only a marker for the development of asthma, the current literature suggests that a defect in IFN- production may be directly involved in the development of asthma through different mechanisms. A decreased production of IFN- may lead to an increase in IgE production by either permitting the switching of B-cells into IgE producing plasma cells or by permitting more CD-4 T-cell progenitors to differentiate into Th2 cells (12). A decreased IFN- production may also be involved in the development of asthma by permitting the development of pulmonary allergic responses (32). In addition, since interferon was first described as antiviral, it is also possible that lower IFN- permitted a longer period of infection, with detrimental effects on pulmonary function. Interleukin-4 is a major cytokine implicated in IgE production and is increased in asthma (11, 13 15). We had previously measured the production of IL-4 in response to IL-2 in infants with bronchiolitis and found a trend for increased IL-4 production at the time of bronchiolitis in infants with early wheezing (31). In this study we found no relationship between IL-4 production at the time of bronchiolitis and the development of asthma 2 yr after bronchiolitis. The number of patients in the current study were not sufficient to conclude with certainty that IL-4 is not related to the development of asthma after bronchiolitis. However, it may be that early wheezing after bronchiolitis does not equate with the development of asthma per se. The balance between Th1 (IFN- ) and Th2 (IL-4) cytokine production is also important in atopy. Although atopy has been linked to asthma, especially in children (33), we found no relationship between the presence of atopy or asthma in the parents or siblings or between serum IgE levels at the time of bronchiolitis and the development of asthma after bronchiolitis. These findings are in agreement with most published studies (4 8). Although pulmonary function seems to return to levels comparable to that in normal subjects within 2 wk after bronchiolitis (34), we assessed whether it is the infants with the lowest pulmonary function and the highest airway responsiveness who would be classified as having asthma 2 yr after bronchiolitis. For this, we measured pulmonary function in 19 infants 4.9 mo after bronchiolitis. As would be expected, it is the infants with the lowest maxfrc and the lowest PC 40 histamine 4.9 mo after bronchiolitis who were those considered as having possible and probable asthma almost 2 yr later. Among the environmental factors that have been linked to the development of asthma, exposure to second-hand tobacco smoke has been shown to be a significant factor, especially in infants. Exposure to second-hand tobacco smoke increases the incidence of airway hyperresponsiveness (35), the number of exacerbations of asthma (36), and the chances of being hospitalized for bronchiolitis (37). We found that more infants who developed asthma after bronchiolitis had parents who were smokers at the time of bronchiolitis, showing that exposure to tobacco smoke is also a risk factor for the development of asthma after bronchiolitis. There is no available data comparing exposure to second-hand smoke and immune function in infants. Because smoking increases IgE levels in adults and children (38, 39), we assessed whether second-hand exposure to tobacco smoke affected cytokine production. We found no effect of exposure to second-hand tobacco smoke on IFN- or IL-4 production at the time of bronchiolitis or 4.9 mo later. Martinez and colleagues (40) have shown that there seems to be two groups of infants who have recurrent wheezing, one that will grow out of their disease by 6 yr of age, and one that will have a persistent disease that is related to atopy and IgE production. These results would suggest that there are independent risk factors for the development of asthma in infants. In subjects who have suffered from bronchiolitis, it may be that exposure to tobacco smoke and lower IFN- production are independent risk factors for the development of asthma. Our findings would suggest this since we found no relationship between smoking and cytokine production. However, the relatively small number of infants enrolled in our study has not permitted us to conclude with certainty whether this is the case. We did find a positive correlation between IFN- production at the time of bronchiolitis and maxfrc or PC 40 histamine 4.9 mo later. Because we found no correlation between pulmonary function and IFN- production 4.9 mo after bronchiolitis, it seems that the immune response at the time of bronchiolitis may affect pulmonary function up to 4.9 mo afterwards. An association has been shown between immune function and airway responsiveness in mice (41); however, no information is available to confirm or infirm this hypothesis in humans. In summary, our results show that lower IFN- production at the time of bronchiolitis is linked to abnormal pulmonary function afterwards and to the development of asthma. These results suggest that there is a link between the immune response and pulmonary function in infants. References 1. Wohl, M. E., and V. Chernick State of the art: bronchiolitis. Am. Rev. Respir. Dis. 118: McIntosh, K Bronchiolitis and asthma: possible common pathogenetic pathways. J. 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