key: cord-009772-pzxvicee authors: GRÜNBERG, K.; TIMMERS, M. C.; SMITS, H. H.; de KLERK, E. P. A.; DICK, E. C.; SPAAN, W. J. M.; HIEMSTRA, P. S.; STERK, P. J. title: Effect of experimental rhinovirus 16 colds on airway hyperresponsiveness to histamine and interleukin‐8 in nasal lavage in asthmatic subjects in vivo date: 2006-04-27 journal: Clin Exp Allergy DOI: 10.1111/j.1365-2222.1997.tb00670.x sha: doc_id: 9772 cord_uid: pzxvicee Background Asthma exacerbations are closely associated with respiratory virus infections. However, the pathophysiological consequences of such infections in asthma are largely unclear. Objective To examine the effect of rhinovirus 16 (RV16) infection on airway hypersensitivity to histamine. and on interleukin‐8 (IL‐8) in nasal lavage. Objective Twenty‐seven non‐smoking atopic, mildly asthmatic subjects participated in a placebo‐controlled, parallel study. A dose of 0.5–2.9 ± 10(4) TCID50 RV16 or placebo was nasally administered. Cold symptoms were recorded by questionnaire throughout the study. Histamine challenges were performed at entry, and on days 4 and 11 after inoculation. Nasal lavages were obtained at entry, and on days 2 and 9. The response to histamine was measured by PC(20) (changes expressed as doubling doses: DD). IL‐8 levels were obtained by ELISA, and were expressed in ng/ml. Results RV infection was confirmed by culture of nasal lavage and/or by antibody titre rise in each of the RV16‐treated subjects. Among the 19 RV16‐treated subjects, eight developed severe cold symptoms. Baseline FEV(1) did not change significantly during the study in either treatment group (P= 0.99). However, in the RV16‐treated subjects there was a decrease in PC(20) at day 4, which was most pronounced in those with a severe cold (mean change ± SEM: –1.14 ± 0.28 DD, P= 0.01). In addition. IL‐8 levels increased in tbe RV16 group at days 2 and 9 (P < 0.001). The increase in nasal IL‐8 at day 2 correlated significantly with the change in PC(20) at day 4 (r=–0.48, P= 0.04). Conclusion We conclude that the severity of cold, as induced by experimental RV16 infection, is a determinant of the increase in airway hypersensitivity to histamine in patients with asthma. Our results suggest that this may be mediated by an infiammatory mechanism, involving the release of chemokines such as IL‐8. virus infections and/or to airborne allergens, potentially leading lo a flare-up of airway inliammation II.2|. Several clinical and epidemiological studies have described a close lemporal as.^ociation of respiratory virus infections with asthma exacerbations [3|. Respiratory viruses can be identified in 10 to 44% of the asthma exacerbations in adults |4.5|. whilst in children identification rates vary from 26 to 83'/f [6] [7] [8] [9] . The use of sen.sitive techniques to detect rhinovirus and coronavirus in the two most recent studies have resulted in the highest identification rates so far [5. 9|. Among the various respiratory viruses identilied, rhinovirus predominates in most of these studies 15.7-91. Interestingly, rbinovirus shedding in the absence of cold symptoms does not seem to be associated with clinical worsening of asthma [7] . The effects of experimental rhinovirus infection on airway responsiveness to inhaled histamine are somewhat controversial. Lemanske et al. demonstrated an induction of hypersensitivity to histamine after experimental rbinovirus 16 (RVI6) infection in non-asthmatic patients with atopic rhinitis [lO] . whereas others have not observed such an effect when using other rhinovirus scrotypes [11.12] . In asthmatic subjects. Halperin et al. found increased hypersensitivity to histamine in only four out of 22 subjects after experimental rhinovirus (serotype 39 and HH strain) infection 113|. whilst in the most recent study by Fraenke! et al. a rather small, but significant increase in sensitivity to histamine could be detected in six asthmatic subjects after infection with RVI6 [ 14|. Since none of these studies was placebo-controlled, it seems mandatory to examine the effect of experimental rhinovirus infection on airway sensitivity to histamine in asthmatic subjects by using such a design. This has successfully been employed in our previous study, showing excessive airway narrowing to methacholine after RVl6 infection in atopic asthmatic subjects | LS]. Rhinovirus infection has been shown to lead to infiltration of inflammator>' cells into nasal secretions and mucosa fl6-18| as well us into the bronchia! mucusa |I4] in normal and/or atopic subjects. In vitro, there is evidence that bronchial epithelial cell lines, fibroblasts and mononuclear cells produce pro-inflammatory cytokines in response to infection with rhinovirus 119-211. In vivo, the levels of chemokines such as interleukin-8. RANTES and MIP-lo; were found to be elevated in nasal secretions of asthmatic children during naturally acquired colds [22.23] . Consequently, one can postulate that chemokines such as IL-8 drive recruitment of inflammatory cells |24!. thus promoting airway inflammation, and thereby airway sensitivity to histamine. In the present study, we hypothesized that experimental RVI6 colds in atopic asthmatic patients increase airway sensitivity to histamine. particularly in those with severe cold symptoms. In addition, we postulated that this effect is associated with a rise of IL-8 in nasal secretion. To tbat end. we measured dose-response curves to inhaled histamine and levels of IL-8 in nusul washings before and after placebocontrolled nasal inhalation of wild type RV16 in atopic. mildly asthmatic patients. Twenty-seven non-smoking, atopic asthmatic subjects participated in this study. The subjects had not used inhaled or oral corticosteroids for at least 3 months, nor had they used theophyllines. antihistamines. sodium cromoglycate, or nedocromyl sodium for at least 6 weeks preceding the study. Symptoms of asthma were stable and controlled by on demand usage of inhaled salbutamol alone, that was withheld for at least 8 h before the measurements. There was no history of relevant exposure to allergens from 2 weeks before until the end of the study. The patients were not selected on basis of a history of virus-induced exacerbations. Among the eight subjects who received placebo-inocuiatit^n. four did not have neutralizing antibodies in their undiluted sent against 20-25 tissue culture infective dose (< 1:1). and four had titres between 1:2 and 1:128 serum dilution. Fourteen out of the 19 .subjects who received virus did not have neutralizing antibodies, and live had titres in the range of 1:2 to 1:16 serum dilution. The study was conducted from July to December 1994. The study was approved by the Hospital's Medical Ethics Committee, and informed con.sent was obtained from all participants. The subjects" characteristics are listed in Table I . The study had a double-blind, placebo-controlled parallel design. Prior to tbe study, each subject was screened for inclusion and exclusion criteria. Three days before the experimental inoculation o\ virus or placebo, a histamine inhalation test was carried out. Subsequently, virus or placebo (diluent) was administered on two successive days. Histamine challenges were repeated at days 4 and 11 after the first inoculation of virus or placebo. Nasal washing was performed and a blood sample was taken immediately before the first inoculation of virus or placebo, and then at days 2 and 9. Four weeks after inoculation all subjects returned to the laborator>' for a final nasal washing and a blood sample to determine the convalescent antibody titre. Tbe RVl6 virus strain and stock was the same as used in previous experiments in humans in vivo by others |IOI and K. Griinherg et ai. by ourselves [I5[. The virus was cultured according to standards of good laboratory practice and the int)culum was tested to be safe for human m vivo usage 125|. Nasal inoculation of ihe rhino\ims was performed foIUiuing a previously described method j l()[, that was slightly modified by adding nasal virus inhalation [ I5|. A total dose of 0.5-2.9xin^ TClDs,, diluted in 3ml Hanks' balanced salt solution (HBSS) with O.y/c (w/v) gelatin was administered to each subject. This dose was divided over 2 days. On each day the same procedure for virus inoculation was followed. First, 0.5 ml of the inoculum was inhaled through the nose by using a nebuli/.er (DeVilbiss 646: median mass aerosol diameter (MMAD) 2.4/xm) connected to a lace mask. .Second. 0.5ml was sprayed by atomizer (DeVilbiss 286. powered by a compressor. MMAD: > IO^m)intothe nostrils. And finally, 0.5 ml was instilled into the nostrils by pipette. We considered a four-fold or greater increase in virusspecific neutralizing antibody in the serum and/or recovery of the virus from nasal washes as confinnatitm ^^\ RV16 infection [ 1(M5|. Before and 28 days after virus or placebo inoculation, levels of neutralizing antibodies were determined by a neutralization assay using homologous virus [ 10.I5J. Nasal Unages were obtained before the first virus or placebo administration, and subsequently on days 2. 9 and 28. Human embryonic lung fibroblast (HEL) cultures were inoculated with these lavages and incubated at 32"C for 14 days. If the culture showed the characteristic rhinovirusinduced cytopathic effects. RVl6 was identified by a neutralization assay, using RVl6 specific guinea pig immune serum (1126AS/GP-VR: Atnerican Type Culture Collection. Rdckville. MD). All na.sal washes were also inoculated into rhesus monkey kidney ^C until further analysis. The IL-8 levels were determined by ELISA (CLB. Amsterdam. The Netherlands), according to the manufacturer's direction,*!. The detection limit of this assay was 40pg/mi. Before, and on days 2 and 9 iifter placebo or virus administration, absolute and differential leucocyte counts were assessed by automated blood count analysis (Technicon HI, Technicon, Tarrytown, NY). The highest individual total cold symptom score (referred to as cold score), and the cumulative asthma score recorded from day 0 to 5 after inoculation minus the cumulative scores from 4 days to I day before inoculation (referred to as asthma score) were used for correlation testing [15] . The response of FEV| to histamine was expressed as percentage fall from baseline value |26|, and was plotted against log nebulized concentration of histamine in mg/ml. The concentration-response curves were characterized by their position, expressed as the provocative concentration causing 20% fall in FEVi from baseline value (PCao), which was calculated by log-linear interpolation between the last two adjacent data-points |26]. The logarithm of PC20 was used in the analysis, and changes in PC20 were expressed in doubling doses (DD). IL-8 levels were expressed in ng/ml. peripheral blood leucocyte numbers were expressed in cells/L. Changes in the variables were analysed by repeatedmeasure analysis of variance (MANOVA). with placebo. RV16 treatment or severe cold and mitd eold a.s betweengroup factors and time as a within-group factor. Significant MANOVA effects were explored with Student's /-tests. Differences in PC2(i. IL-f^ levels and leucocyte numbers within the groups between the study days were examined using two-tailed paired r-tests, and differences between the groups were analysed using unpaired /-tests, The summary statistics were expressed as means ± SEM. For evaluation of associations between the variables. Ihe Pearson's correlation test was used. P values less than 0.05 were considered statistically significant. One of the RVl 6-treated subjects (subject 9) dropped out of the study at 7 days after the first inoculation because of a moderate exacerbation of asthma, requiring treatment with oral prednisone, to which she responded well. One nasal washing sample was excluded from the analysis (subject 7, day 9), because of a recent nose bleed. In the plaeebo group all cultures of nasal washes remained negative for RVl6 during each visit. In the virus-treated group RV 16 could not be detected in the nasal lavage before inoculation, whilst at day 2 RVl6 was detected in the nasal lavage of all but one subject (subject 18). At day 9, RVl6 was identified in 10 out of 19 subjects, whereas at day 28 all nasal washings were negative. No other respiratory viruses were identified in any of the nasal washings (Table I) . In the placebo group none of the subjects showed an increase in RV16 neutralizing antibodies. In the RV16 group all subjects but two (subjects 16 and 24) showed at least a four-fold increase in neutralizing antibodies in the convalescent sera (range: 4-fold to 128-fold increase) ( Table 1) . In the placebo group, there was no significant change in cold score or asthma score (MANOVA, P > 0.52). In the RVl6 group, there was a significant increase in cold score (MANOVA. P<0.00\). that peaked I day after the first inoculation, gradually returning to baseline within 5 days. The highest cold scores were significantly different between the groups (P < 0.001). Eight of the RVI6-infected subjects had a severe cold as shown by a symptom score > 11 ( Table 1 ). In the RVl 6-treated subjects there was a significant increase in asthma symptoms {MANOVA, P < 0.001) that peaked on the second and third day after the first inoculation, and returned to baseline within 5 days. The asthma score in the subjects with a severe cold was significantly higher than the asthma score in those with a mild cold and the placebo-treated subjects (/»< 0.001) ( Table 1) . Cold score and asthma score were significantly correlated in the RVI6 group (r^O.92. /'< 0.001). The use of salbutamol did not change significantly within the groups at any time point (MANOVA, P = 1.00). Before RV 16 or placebo inoculation, FEV i % predicted was slightly higher in the RVl6 group as compared to the placebo group (P = 0.04) ( Table 1 ). During the course of the study, there were no significant effects on baseline FEV| within either the placebo group or in the RVl 6-treated subjects with a mild or severe cold (MANOVA, P -0.99) ( Figure 1 ). The maximal change in FEV, after infection did not correlate significantly with the asthma score (P = 0.98). Before inoculation of RVl6 or placebo, the mean PC20 was not different between the two treatment groups (P ^ 0.93). In the placebo group, there was no significant change in PC20 during the study (MANOVA, P = 0.67) (Figure 2 ). In the RVl6 group, there was a significant decrease in PC20 at day 4 (mean difference ± SEM: -0.65 ± 0.25 DD, P = 0.02), which was no longer significant at day 11 (mean difference ± SEM: -0.40 ± 0.30 DD, P = 0.19). These changes were not significantly different from placebo (P = 0.10 and P = 0.27, respectively). However, in the subjects with a severe cold, this decrease was more pronounced: mean difference ± SEM: -1.14 ± 0.28 DD, P -0.005 at day 4. with a trend towards a decrease at day II (mean difference ± SEM: -0.75 ± 0.34DD. P = 0.07) {Figure 2). This change was significantly different from placebo at day four, but not at day \i (P = O.OI and P = 0.09. respectively). In the tnild cold group there was neither a change in PC2(, at day 4 (mean difference ± SEM: -0.30 ± 0.35 DD. P = 0.42). nor at day II (mean difference ± SEM: -0.18 ± 0.44 DD, P = 0.68) (Figure 2) . The changes in PC20 in the five subjects with pre-existing neutralizing antibodies against RV16 were not statistically different from the changes in those without such antibodies {MANOVA. P ^ 0.33) {day 4: mean change ± SEM: -0.53 ± 0.46 DD. P = 0.31. and -0.69 ± 0.31 DD. F = 0.04. respectively. Day 11: -1.10 ±0.28DD. P = 0.02. and -0.13 ± 0.38 DD. P = 0.73, respectively). IL-8 in the nasal washings did not change significantly in the placebo group (MANOVA. P = 0.06) {Figure 3). In the RVl6 group, IL-8 increased both at days 2 and 9 {P 0.17). The number of neutrophils at day 2 correlated significantly with cold score (r = 0.59. P = 0.008). asthma score {r^O.65, P = 0.002), the change in PC2,) at day 4 (r --0.49, P = 0.03), whilst there was a trend towards a significant correlation with the change in IL-8 levels at day 2 (r-0.39. P-O.IO) (Figure 4c) . Furthermore, the number of lymphocytes at day 2 was also significantly related to the cold scores histamine. particularly in those patients who develop a severe cold. In addition, we have demonstrated that the levels of the pro-infiammatory chemokine IL-8 in nasal secretions rise after infection. This rise is ass(x:iated with cold score, change in airway hyperresponsiveness. and numbers of neutrophils and lymphwytes in peripheral bltHxi after infection. These findings suggest that the severity of the cold is a major determinant of rhinovirus-indiiced airway hypenesponsiveness in asthma. Our results fit in with the hypeithesis that this is mediated through an inflammatory mechanism inviil\ing locally produced chemokines. This is the first placebo-controlled study showing the development of airway hypersensitivity to histamine after experimental infection with wild type rhinovirus. The change in airway hypersensitivity in the asthmatic subjects with severe colds was about 1 doubling dose, which is similar to what is usually observed after allergen challenge |28|. Rhinovinis-induced hypersensitivity of such magnitude has also been demonstrated in patients with atopic rhinitis by Lemanske et ai |l()|. However, in the latter subjects the histamine hypersensitivity lasted up lo 4 weeks after infection, whereas in our study the effect was no longer significant at day 11. One could speculate that the prolonged effect in atopic rhinitis may have been due to the additional allergen challenges during that study. Two previous studies on experimental rhinovirus infection in asthma [!3.14| showed small and variable changes in airway hypersensitivity. Our findings suggest that this might be explained by the severity of the colds that were induced. After taking this into account, it appears that experimental rhinovirus infection in asthma does lead to substantial worsening of PCio to histamine. Interestingly, this also occurred in the small number of subjects who had preexisting RVl6 neutralizing, but possibly cross-reactive circulating antibodies. The latter may not be surprising, since atopic .subjects with low titres of neutralizing antibodies, as opposed to normal subjects with such titres, have been shown to develop severe cold symptoms after experimental RV16 inoculation [29] . The design of the present study allowed us to differentiate the responses to rhinovirus inoculation from normal fluctuations in symptoms and airway physiology that are characteristic to asthma. The study was performed in the months July to December, but no attempt was made to exclude coinciding allergen exposure (pollen, house dust mite), since this would have been hard to accomplish. However, the present circumstances can be considered as those encountered during naturally occurring infections. Despite the fact that the subjects were clinically stable as assessed by history, and by symptom control with p.r.n. /ST adrenergic medication alone, a moderate exacerbation of asthma developed in the subject who had the lowest PC^o at entry into the study. This underlines the potential of exacerbations after rhinovirus infection in patients with asthma (3.9,301, despite the usually small accompanying changes in lung function [13] [14] [15] . In this study, we applied validated procedures for inoculation and measuring the responses to rhinovirus infection. First, by using a combination of three methods of virus admini.stration, including nasal inhalation, the natural ways of transmission were mimicked [31] . In this way, the virus may even have reached the intrapulmonary airways [32] . Second, commonly used and well-standardized methods for lung function testing and histamine challenges were used [26] . Third, we applied a validated method for nasal lavage [27] , which allowed lL-8 to diffuse into the lavage fluid during a 5-min exposure period of the nasal epithelium, resulting in IL-8 levels well above the detection limit of the IL-8 ELISA. How can the present results be interpreted? The increase in airway hypersensitivity, in the absence of a significant decrease in lung function, during the acute phase of infection might be explained by physiological phenomena such as airway wall swelling, potentiating the airway narrowing effect of smooth muscle shortening [33] . Such an explanation would be in keeping with the observations by Cheung et al. [15] , who showed that experimental RV16 infection leads to excessive airway narrowing in response to inhaled methacholine in subjects with asthma. Airway wall swelling in asthma is generally considered to be a consequence of inflammation [2] . Indeed, Fraenkel et al. [14] recently described the infiltration of inflammatory cells, particularly lymphocytes and eosinophils into the bronchial mucosa in patients with asthma after experimental RVI6 infection. The presently observed correlation between the numbers of © 1997 Blackwell Science Lid, Clinical and Experimental Allergy. 11, 36-45 neutrophils and lymphocytes in peripheral blood after infeetion and the change in airway hypersensitivity indirectly supports an active role of these cells in the virus-induced airway inllammation. We found a marked rise in lL-8 in nasal secretions after RVl6 infection. In general, this confirms the ability of rhinovirus to increase the release of a number of proinflammatory mediators and/or cytokines within the airways, such as kinins [16, 34] and interleukin-1 [35] in nasal secretions, and histamine in broncho-alveolar lavage fluid [36] . The present results obtained by experimental rhinovirus infection are in keeping with the preliminary data of Teran et al. [22] , who showed that levels of IL-8 in nasal secretions were elevated in nasal secretions during a naturally acquired cold in children with asthma. Our results extend these previous observations by showing an association between the increase in IL-8 in nasal washings and cold or asthma symptoms, as well as the degree of worsening of airway hyperrespon.sivene.ss. IL-8 is a CXC-chemokine, produced by tissue cells {epithelial ceils, fibroblast and endothelial cells), leucocytes, macrophages and mast cells [24, 37] and displays various activities, such as chemotactic activity for neutrophils, lymphocytes and basophils [38] . In addition, IL-8 may be involved in the recruitment of primed eosinophils, implicating its involvement in allergic inflammation |39]. Since rhinovirus in vitro induces the production of IL-8 in epithelium, fibroblasts and peripheral blood mononuclear cells 120,21,40], our findings support the hypothesis that the release of mediators, such as the chemokine IL-8, can drive the airway inflammation, and thereby the hypersensitivity to histamine after rhinovirus infection in allergic asthma. What are the clinical implications of this study? First, a common cold aggravates airway hypersensitivity in patients with asthma, fitting in with the close epidemiological association between rhinovirus infections and exacerbations of asthma [5, 9] . And .second, it appears that atopie asthmatic patients with low titres of neutralizing antibodies may not be fully protected against experimental RVI6 infection, and its detrimental effects on their asthma. This ohservation in a small number of subjects in the present study first needs confirmation in larger series of experimental or naturally occurring rhinovirus infections in patients with allergic asthma. In conclusion, experimental RV16 infection can be employed as a useful laboratory model for the development of airway hypersensitivity during an asthma exacerbation. One of the potential mechanisms for this might be the rhinovirus-induced release of pro-inflammatory chemokines. This hypothesis needs further testing in models of rhinovirus infection in vitro and in vivo, focusing on the pathological mechanisms in the intrapulmonary airways in patients with asthma. International consensus report on diagnosis and treatment of asthma Wilson JW ci at. Mucosal inflammation in asthma Viruses as precipitanis ot asthma synipioni.s. I. Epidemiology Viral respiratory tract infection and exacerbations of asthma in adult patients Respiratory viruses and exacerbations of asthma in adults The association of viral and bacterial respiratory inlections with exacerbations of wheezing in young asthmatic children Viruses as precipitants of asthmatic attacks in children Respiratory viral infection and whee/y bronchitis in childhood A community study of the role of virus infections in exacerhaiions of asthma in 9-11 ye:ir old children Rhinovirus upper respirator;' infection increases ainvay hyperreactivity and late asthmatic reactions Ht)lgate ST. 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A common cold vini.s, rhinovirus 16. potentiates airway inflammation after ,segmental antigen bronchoprovocation in allergic subjects lL-8 is expressed by human peripheral blood eosinophils. Evidence for increased secretion in asthma CC chemokines in allergic inflammation Upregulation of formyl-peptide and interleukin-8-induced eosinophil chemotaxis in patients with allergic asthma Rhinovirus stimulation of inter!eukin-6 in vivo and in vino. Evidence for nuclear factor kB-dependent transcriptional activation This study was supported by a grant of The Nelherlands Asthma Foundation (project no. 93.17).