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Are Rhinovirus-induced Airway Responses in Asthma Aggravated by Chronic Allergen Exposure?

Departments of Pulmonology, Medical Decision Making, and Virology, Leiden University Medical Center, Leiden, The Netherlands; and Respiratory Virus Research Laboratory, University of Wisconsin, Madison, Wisconsin

Correspondence and requests for reprints should be addressed to Peter J. Sterk, M.D., Ph.D., Lung Function Laboratory, C2-P, Leiden University Medical Center, P.O. Box 9600, NL-2300 RC Leiden, The Netherlands. E-mail: p.j.sterk{at}lumc.nl

	ABSTRACT

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Airway inflammation in asthma may represent a favorable environment for respiratory viral infections, augmenting virus-induced exacerbations in asthma. We postulated that repeated low-dose allergen exposure preceding experimental rhinovirus 16 (RV16) infection increases the severity of RV-induced airway obstruction and inflammation. Thirty-six house dust mite–allergic patients with mild to moderate asthma participated in a three-arm, parallel, placebo-controlled, double-blind study. Patients inhaled a low dose of house dust mite allergen for 10 subsequent working days (Days 1–5 and 8–12) and/or were subsequently infected with RV16 (Days 15 and 16). Allergen exposure resulted in a significant fall in FEV₁ (p < 0.001) and provocative concentration of histamine causing a 20% fall in FEV₁ (p < 0.001) and an increase in exhaled nitric oxide (p < 0.001) and percentage of sputum eosinophils (p < 0.001). RV16 infection led to a fall in FEV₁ (p = 0.02) and increases in the percentage of sputum neutrophils (p = 0.01), sputum interleukin-8 (p = 0.04), and neutrophil elastase (p = 0.04). Successive allergen exposure and RV16 infection had no synergistic or additive effect on any of the clinical or inflammatory outcomes. In conclusion, repeated low-dose allergen exposure and RV16 infection induce distinct inflammatory profiles within the airways in asthma without apparent interaction between these two environmental triggers. This suggests that preceding allergen exposure, at the used dose and duration, is not a determinant of the severity of RV-induced exacerbations in patients with mild to moderate asthma.

Key Words: asthma exacerbation • airway hyperresponsiveness • inflammation • sputum • nasal lavage fluid

Despite current guidelines on asthma management, exacerbations of asthma are still prevalent, often requiring additional therapy such as inhaled and/or oral steroids, sometimes including hospital admission. This occurs particularly in patients with relatively severe disease during the fall season (1). It is known that the presence of a common cold and allergen exposure are both associated with such exacerbations (2–4). Epidemiologic evidence has shown that the combination of these two triggers is particularly harmful to patients with asthma (5). However, there is lack of understanding regarding the pathogenic mechanisms involved.

Respiratory viruses are major triggers of asthma exacerbations (2), in which rhinoviruses (RVs) appear to predominate (3, 6). In epidemiologic studies, it has been shown that common colds are associated with an increase in asthma symptoms, variable airways obstruction, airway responsiveness, and features of lower airway inflammation (3, 6, 7). Whereas experimental rhinovirus 16 (RV16) infections mimic these changes in patients with asthma (8–10), the clinical responses to such experimental infections are relatively mild. It has been suggested that host factors such as atopy or airway hyperresponsiveness influence the host susceptibility to virus-induced changes (11). Indeed, after upper respiratory tract infections, patients with asthma suffer from more frequent, severe, and longer lasting lower respiratory tract symptoms as compared with healthy subjects (12). Hence, the asthmatic "airway milieu" may represent a favorable environment for viral infections, which might be associated with allergic airway inflammation.

The patient's natural exposure to environmental allergens can be mimicked using repeated low-dose allergen inhalations (13). Such exposures have been shown to worsen most of the disease features in patients with pre-existing atopic asthma. This has been demonstrated with respect to increased airway hyperresponsiveness (4, 14, 15) as well as for an increase in eosinophilic inflammation (4, 15, 16) and Th2/Th1 cytokine balance (15–17). It can be postulated that such inflammation could facilitate asthma exacerbations caused by subsequent respiratory virus infection.

The interaction between repeated low-dose allergen exposure and subsequent RV infection is not only plausible from an epidemiologic standpoint (1, 5), but is also likely to occur at the cellular level within the airways. For instance, the presence of specific cytokines of ongoing allergic inflammation could lead to an increased expression of intercellular adhesion molecule-1, the receptor for the major group of RVs (18), and increased susceptibility to subsequent RV infection (19). Other possible mechanisms of interaction could be (1) switching of virus-specific CD8+ T cells from IFN- to interleukin (IL)-5 production in the presence of IL-4 (20, 21); (2) worsening of an asthma exacerbation by combined eosinophilic and neutrophilic inflammation (9, 22–24), possibly because of the degranulation of eosinophils by neutrophil proteases (25); and/or (3) eosinophils participating in an RV-induced immune response through antigen presentation and T-cell activation (26).

Therefore, in this study, we postulated that repeated low-dose allergen exposure preceding experimental RV16 infection increases the severity of an RV-induced asthma exacerbation and thus leads to a flare-up of symptoms, medication use, variability of airways obstruction, airway hyperresponsiveness, and airway inflammation. To that end, we exposed patients with mild to moderate atopic asthma to low doses of house dust mite allergen for 2 weeks, RV16 infection, or the combination of the two.

	METHODS

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The extended version of the methods is available in the online supplement. Thirty-six steroid-naive atopic patients with mild to moderate asthma participated (Table 1) (see Table E1 in the online supplement for details of the patients' characteristics). The Medical Ethics Committee of the Leiden University Medical Center approved the study, and the subjects gave written informed consent before entering. The study had a placebo-controlled, double-blind, parallel, three-armed design (Figure 1) . The thirty-six patients were randomly assigned to either low-dose allergen exposure with placebo infection (allergen + placebo; n = 12), placebo exposure with RV16 infection (placebo + RV16; n = 12), or low-dose allergen exposure with subsequent RV16 infection (allergen + RV16; n = 12).

View larger version (21K): [in this window] [in a new window]   Figure 1. Study design and schedule. Each week shows the five separate working days and the performed measurements. A/P = low-dose allergen or placebo inhalation; NL = nasal lavage; PC20 = airway responsiveness to histamine (provocative concentration causing a 20% fall in FEV1); RV/P = rhinovirus 16 inoculation or placebo; Sput = sputum induction.

RV16 inoculation was done by nasal administration of a total dose of 0.4–8.6 x 10⁴ 50% tissue culture infective dose (TCID₅₀) diluted in 3 ml of Hanks' balanced salt solution to each subject according to a previously described procedure (8, 29). Confirmation of RV16 infection was established by a fourfold or greater increase in virus-specific neutralizing antibody titer in serum and/or by recovery of the virus from the nasal lavages (8, 29).

FEV₁, provocative concentration of histamine causing 20% fall in FEV₁ (PC₂₀FEV₁) to histamine (27), exhaled nitric oxide (NO) measurements (30), sputum inductions, and nasal lavages were performed according to the study schedule represented in Figure 1. Asthma symptom scores, cold scores, salbutamol usage, and PEF measurements were recorded three times daily in diary cards during the 7 weeks of the study (16, 22, 31, 32).

Sputum was induced and processed according a protocol that has been validated in our laboratory (16, 33). In the sputum supernatant, concentrations of eosinophil cationic protein and levels of neutrophil elastase and IL-8 were determined by enzyme immunoassay (16, 22, 33–35).

Nasal lavages were obtained using a modified "nasal pool device" (9, 22). The lavages recovered from the first (Hanks' balanced salt solution) and second nostril (phosphate-buffered saline) were used for confirmation of RV16 infection and to determine the cell numbers and mediator level, respectively. Levels of IL-8 were determined by ELISA.

For sample size estimation and statistical power, see the online supplement. The cold score was analyzed using the Mann-Whitney U test to test between-group differences (SPSS 10.0; SPSS Inc., Chicago, IL). The longitudinal data were analyzed using a random-effects model to explore allergen-induced, RV16-induced, and allergen + RV16–induced changes in asthma symptoms, ß₂-agonist usage, PEF values, FEV₁, (log-transformed) PC₂₀FEV₁, exhaled NO, and sputum and nasal lavage data (36). The STATA 6.0 xtreg procedure (StataCorp, College Station, TX) was used to fit the applied random-effects model. A p value of 0.05 or less was considered statistically significant.

	RESULTS

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In the majority of patients (21 out of 24) who were inoculated with RV16, RV infection was confirmed, whereas in three (patients 6, 13, and 20), it was not. Patients 28 and 29 in the allergen group had an intercurrent spontaneous "common cold" with RV. Two patients dropped out of the study because of an asthma exacerbation. Patient 24 had a spontaneous exacerbation during the beginning of the study, during the placebo-allergen period. Patient 3 developed an RV-induced exacerbation on the 2nd day of RV16 inoculation (Day 16), after 2 weeks of allergen exposure. The sums of the cold scores and asthma scores that day were 42 and 30, respectively. PEF decreased to 28% of personal best. The patient was withdrawn from the study and admitted to our hospital for 1 night. The patient was treated with oxygen, inhaled ipratropium/salbutamol, and prednisone orally and recovered quickly during the night and following days. The results from these seven patients were excluded from the analysis (see Table E1 for details). There were no differences in baseline values for any of the measurements between the groups (p > 0.06).

The acute response to a low dose of allergen exposure as assessed by the mean maximal fall in FEV₁ ± SD within 30 min after low-dose allergen exposure was 5.97 ± 1.68% in the combined allergen + placebo and allergen + RV16 groups as compared with 1.40 ± 0.63% in the placebo + RV16 group (p < 0.001).

Between-intervention differences with corresponding 95% confidence intervals are outlined in Table 2 . There were significant changes during the 2 weeks of low-dose allergen exposure in most of the parameters as compared with placebo inhalations (discussed later here). However, the differences between the interventions were not maintained beyond the 2 weeks of allergen exposure for any of the parameters.

View larger version (19K): [in this window] [in a new window]   Figure 2. Asthma symptom score (mean score ± SEM) (A), lowest morning/highest PEF (mean in % ± SEM) (B), and ß2-agonist usage (mean in puffs ± SEM; one puff is 100 µg) (C) per week. Data are presented for the allergen (All) + placebo (Plac) group by squares, placebo + rhinovirus 16 (RV16) group by open circles, and allergen + RV16 group by closed circles. During the allergen exposure, the asthma symptom score (p = 0.001) and ß2-agonist usage (p < 0.001) increased significantly, whereas the lowest morning/highest PEF decreased significantly (p = 0.009) (random-effects model).

View larger version (20K): [in this window] [in a new window]   Figure 3. Cold score per time point (mean ± SEM) from Days 15 to 21. Data are presented for the allergen + placebo group by squares, placebo + RV16 group by open circles, and allergen + RV16 group by closed circles. Using the Mann-Whitney U test, the cold score increased significantly after RV16 infection, in both the allergen + RV16 group (p = 0.048), as well as in the placebo + RV16 group (p = 0.006) as compared with the allergen + placebo group (depicted by an asterisk). There were no significant differences in scores between the allergen + RV16 group and the placebo + RV16 group.

View larger version (19K): [in this window] [in a new window]   Figure 4. FEV1 (mean FEV1 in percentage predicted ± SEM). Data are presented for the allergen + placebo group by squares, placebo + RV16 group by open circles, and allergen + RV16 group by closed circles. Both allergen exposure (p < 0.001) and RV16 infection (p = 0.02) induced a significant decrease in FEV1 (random-effects model).

View larger version (20K): [in this window] [in a new window]   Figure 5. Provocative concentration of histamine causing a 20% decrease in FEV1 (provocative concentration of histamine causing a 20% fall in FEV1 [PC20]) (geometric mean in mg/ml ± geometric SEM). Data are presented for the allergen + placebo group by squares, placebo + RV16 group by open circles, and allergen + RV16 group by closed circles. Allergen exposure resulted in a significant decrease in PC20 (p < 0.001) (random-effects model).

View larger version (23K): [in this window] [in a new window]   Figure 6. Exhaled nitric oxide (NO) (mean exhaled NO in ppb ± SEM). Data are presented for the allergen + placebo group by squares, placebo + RV16 group by open circles, and allergen + RV16 group by closed circles. Allergen exposure resulted in a significant increase in exhaled NO (p < 0.001) (random-effects model).

View larger version (24K): [in this window] [in a new window]   Figure 7. Sputum eosinophils (mean percentage of eosinophils ± SEM) (A) and sputum neutrophils (mean % neutrophils ± SEM) (B). Data are presented for the allergen + placebo group by squares, placebo + RV16 group by open circles, and allergen + RV16 group by closed circles. Allergen exposure and RV16-infection resulted in a significant increase in sputum eosinophils (p < 0.001) and neutrophils (p = 0.01), respectively (random-effects model).

	DISCUSSION

TOP ABSTRACT METHODS RESULTS DISCUSSION REFERENCES

 
In this study, we found that "priming" the airways with repeated low-dose allergen exposure does not result in an aggravated response with regard to airways obstruction and airway inflammation after RV infection in patients with mild asthma. When given alone, allergen exposure and RV16 infection each resulted in a significant decrease in FEV₁. Interestingly, PC₂₀, exhaled NO, and the percentage of sputum eosinophils changed during allergen exposure, whereas sputum neutrophils, neutrophil elastase, and IL-8, and nasal neutrophils and IL-8 increased after RV16 infection. Hence, repeated low-dose allergen exposure and RV16 infection induce distinct inflammatory profiles within the airways in patients with asthma. However, we did not observe any interaction between these two environmental triggers. This indicates that preceding allergen exposure, in the used study design, is not a major determinant of the severity of RV-induced asthma exacerbations in patients with mild asthma.

This is the first study in which the effects of repeated low-dose allergen exposure on the response to a subsequent RV infection in patients with asthma have been investigated. First, our findings during low-dose allergen exposure alone is in line with those of previous studies showing worsening of airway hyperresponsiveness (4, 14, 15), increased exhaled NO (16), and an increased eosinophilic inflammation (4, 15, 16). They also confirm inflammatory changes seen in sputum and nasal lavage after RV infection (9, 22). However, whereas there is good evidence of the reverse interaction, namely of RV infection on subsequent allergen-induced airways obstruction and inflammation (37, 38), we found no evidence that allergen exposure worsens subsequent RV-induced responses. Noticeably, our data are not suggestive of any protective effects of preceding allergen challenge on cold symptoms or nasal inflammation, as reported by Avila and colleagues (39). Therefore, it seems as if the epidemiologic evidence of allergen-virus interactions during asthma exacerbations (5) cannot simply be explained by allergen-induced "priming" of the responses to RV infection by the lower airways.

The lack of interaction between repeated low-dose allergen exposure and subsequent RV16 infection could theoretically be due to method choices. Our design was mainly driven by safety considerations. We selected steroid-naive patients to avoid any confounding effect of antiinflammatory therapy. In addition, the low-dose allergen exposure was meant to be a mild inducer of inflammation, which by itself would not lead to an exacerbation of the disease (16). Nevertheless, this study and our previous one (16) have shown that this model results in definite physiologic and inflammatory changes. However, it cannot be excluded that there could be an interaction between the two interventions using a higher dose of allergen. It also cannot be excluded that the timing of subsequent RV infection may not have been optimal, as there was a 3-day interval between measuring the effects of repeated low-dose allergen and the RV16 inoculation. This may not effectively mimic the real-life situation and could be a possible reason for a lack of interaction between the two interventions. We used an RV16 inoculum that was known to be safe and for that very reason may exhibit limited virulence in vivo when compared with natural occurring common colds (8–10). Although we could not confirm RV-induced worsening of airway hyperresponsiveness (9), we did observe increases in cold scores next to an increase in neutrophil counts in sputum and nasal lavages. Finally, we cannot exclude that our failure to detect interaction between allergen exposure and RV16 infection was due to lack of statistical power. The absence of worsening in hyperresponsiveness after RV alone, in this and other studies (40), could be suggestive of this. However, it appeared that each of the triggers by themselves induced adequate (but distinct) inflammatory profiles within the airways, as required for potential interaction.

How can these results be explained? When developing our hypothesis, we postulated that ongoing allergic inflammation would facilitate or augment virus-induced proinflammatory responses in the airways. There are multiple potential pathways for this, which we did not specifically explore in this study. First, allergen-induced IL-4 release could impair viral clearance by a switch in CD8+ T-cell cytokine profile (IFN- to IL-5) (21) and/or a reduction in cytotoxic activity (20, 41). Although exhaled NO may reflect allergic inflammation in asthmatic airways (42), allergen-induced impaired synthesis of protective nitric oxide might disturb defensive response against viral infection (43). Second, allergen-induced cytokines increase the expression of the major RV receptor intercellular adhesion molecule-1 (18). Third, combined eosinophilic and neutrophilic airway inflammation may enhance eosinophilic degranulation through neutrophil protease activity (25). In this proof of concept study, we have not attempted to investigate these putative mechanisms specifically because this would have required invasive measurements, such as bronchial biopsies. Apparently, none of these mechanisms are leading to manifest interaction with regard to the clinical and physiologic responses in patients with mild asthma. However, it cannot be excluded that these interactive pathways might have been balanced by potential protective mechanisms between allergens and virus infections, such as expression of antiinflammatory mediators (e.g., IFN- and IL-10), local production of NO, or antiviral effects of eosinophil products (39, 44).

Nevertheless, clinical and epidemiologic data (5, 24) favor additive or synergistic responses to allergens and virus infections in asthma. It can be envisaged that such an interaction during asthma exacerbations can also be dependent on specific host characteristics or more severe degrees of the disease. First, patients with asthma who are susceptible for RV-induced asthma exacerbations could have relatively high IgE levels. Duff and colleagues (45) have shown that high IgE levels together with RV infections are synergistic risk factors for wheezing with colds. Furthermore, very recent data indicate that high levels of total serum IgE are also predictive of lower respiratory tract symptoms after experimental rhinovirus infection in patients with asthma (46). Second, host factors such as an altered balance between proinflammatory IL-1ß and antiinflammatory IL-1ra could also influence virus-induced inflammation and symptoms (47–49). Finally, diminished antiviral activity could also occur in patients with a decreased production of IFN- (50). Therefore, phenotypic characteristics need to be investigated in the future search for the determinants of severe asthma exacerbations. Obviously, there are ethical restraints to the induction of exacerbations by using experimental infections in patients with more severe asthma so that most of the information should be derived from observational studies. Eventually, this may characterize the patients who are particularly vulnerable to RV infections and may allow the development of novel strategies to prevent/treat exacerbations in asthma.

	Acknowledgments

 
The authors sincerely thank all of the volunteers in the study and K. Grünberg (Department of Pathology, VU Medical Center, Amsterdam, The Netherlands) and J.E. Gern (Department of Pediatrics, University of Wisconsin Hospital, Madison, WI) for their expertise and advice. The authors acknowledge the expertise and technical assistance of H. van der Veen, M.C. Timmers, S.P.G. Manesse-Lazeroms, and A.C. van der Linden of the Department of Pulmonology and the technicians and E.P.A. de Klerk of the Department of Virology of the Leiden University Medical Center.

	FOOTNOTES

 
Supported by the Netherlands Asthma Foundation (Grant 98.06).

This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Conflict of Interest Statement: J.d.K. has no declared conflict of interest; C.E.E. has no declared conflict of interest; J.K.S. has no declared conflict of interest; J.A.S. has no declared conflict of interest; C.J.G.vZ-v.D.H. has no declared conflict of interest; C.R.D. has no declared conflict of interest; K.F.R. has participated as a speaker in scientific meetings and has been a member of advisory boards for AstraZeneca, AltanaPharma, Boehringer, Pfizer, GSK, MSD, Novartis and Schering Plough. The Department of Pulmonology received research grants from AltanaPharma ($202,616), Novartis ($90,640), Bayer ($61,762), AstraZeneca ($103,155) and GSK ($299,495) in the years 2000 until 2002; P.S.H. and P.J.S. are staff members in the Department of Pulmonology that received research grants from AltanaPharma ($202,616), Novartis ($90,640), Bayer ($61,762), AstraZeneca ($103,155) and GSK ($299,495) in the years 2000 until 2002.

Received in original form December 23, 2002; accepted in final form July 24, 2003

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