American Journal of Respiratory and Critical Care Medicine

Asthma exacerbations are frequently linked to rhinovirus infections. However, the associated inflammatory pathways are poorly understood, and treatment of exacerbations is often unsatisfactory. In the present study we investigated whether antiinflammatory treatment with inhaled corticosteroids prevents any rhinovirus-induced worsening of lower airway inflammation. To that end, we selected 25 atopic patients with mild asthma who underwent experimental rhinovirus 16 (RV16) infection, while receiving double-blind, placebo-controlled treatment with the inhaled corticosteroid budesonide (800 μ g twice a day) throughout the study period, starting 2 wk before infection. We assessed inflammatory cell numbers in the bronchial mucosa as obtained by bronchial biopsies 2 d before and 6 d after RV16 infection, and analyzed those in relation to cold symptoms, changes in blood leukocyte counts, airway obstruction, and airway hyperresponsiveness. RV16 colds induced an increase in CD3+ cells in the lamina propria (p = 0.03) and tended to decrease the numbers of epithelial eosinophils (p = 0.06) in both groups analyzed as a whole. The T cell accumulation was positively associated with cold symptoms. Budesonide pretreatment improved airway hyperresponsiveness (p = 0.02) and eosinophilic airways inflammation (p = 0.04). Yet it did not significantly affect the RV16-associated changes in the numbers of any of the inflammatory cell types. We conclude that RV16 infection by itself induces only subtle worsening of airway inflammation in asthma, which is not improved (or worsened) by inhaled corticosteroids. The latter finding is in keeping with the limited protection of inhaled corticosteroids against acute asthma exacerbations.

Keywords: asthma; inflammation; rhinoviruses; corticosteroids

Patients with asthma frequently suffer from transient worsening of their disease during respiratory virus infections. Rhinoviruses are most commonly associated with such exacerbations (1, 2). Indeed, experimental rhinovirus infection in patients with asthma worsens asthma symptoms (3, 4), variable airway obstruction (5), and airway hyperresponsiveness to various bronchoconstrictor stimuli (3, 4, 6). This suggests that rhinovirus infection is able to promote airway inflammation in preexisting asthma.

Indeed, there is evidence that rhinovirus infections enhance airway inflammation in asthma, as reflected by an increase in local production of inflammatory mediators such as interleukin (IL)-1β, IL-8, IL-6, and ECP, detectable in nasal lavage (4, 7) and/or in induced sputum (8). In addition, rhinovirus 16 (RV16) infections enhance ICAM-1 expression in the bronchial epithelial layer (9), and promote the infiltration of T cells into the bronchial lamina propria, and of eosinophils into the bronchial epithelium during the acute phase of infection in a sample that combined normal subjects and subjects with asthma (10). The relevance of these observations for development of rhinovirus-induced exacerbations of asthma is, however, still unclear.

Inhaled or oral glucocorticosteroids are the most commonly used antiinflammatory drugs for regular asthma therapy (11). In vitro, glucocorticoids appear to be effective against rhinovirus replication, cytokine release, and ICAM-1 upregulation in cell cultures (12, 13). On the other hand, there is some evidence that glucocorticoids, by suppressing the immune response (14), may hamper viral clearance (15), thereby potentially worsening or protracting the disease. Evidence on the effectiveness of glucocorticoids during proven rhinovirus infections is lacking. Clinical studies in children show little or no benefit of inhaled glucocorticoids in preventing (16) or treating (17-19) acute exacerbations of asthma. In view of the potential role of inhaled glucocorticoids in patient self-management of acute exacerbations, the effectiveness of inhaled glucocorticoids for rhinovirus-associated exacerbations of asthma needs to be investigated.

In the present study we hypothesized that RV16 colds in subjects with asthma induce worsening of the underlying inflammation of the lower airways, which can be prevented (in part) by pretreatment with inhaled glucocorticoids. We conducted a trial in atopic patients with asthma who all underwent experimental RV16 infection, and received double blind, placebo-controlled treatment with inhaled budesonide throughout the study period, starting 2 wk before infection. The primary outcome parameters of the study were the numbers of inflammatory cells in bronchial lamina propria and epithelium before and after RV16 infection, as obtained from bronchial biopsy specimens. The observed changes in these outcome parameters were analyzed in relation to cold score, changes in peripheral blood leukocyte counts (as an indicator of severity of colds), airway obstruction, and airway hyperresponsiveness (as indicators of asthma severity).

Twenty-five nonsmoking atopic steroid-naive adults with asthma with low rhinovirus 16-neutralizing serum titers were recruited (Table 1). The study was approved by the Medical Ethics Committee of the Leiden University Medical Center and all subjects gave their written informed consent. The subjects only used inhaled short-acting β2-agonists on demand. All participants underwent RV16 inoculation on Days 0 and 1. Inhaled budesonide (Turbohaler, 800 μg, twice a day) (BUD) was administered in a randomized, double blind, placebo-controlled (PLAC) fashion for 4 wk, starting 16 d before RV16 inoculation (Day −16). Blood samples were drawn at Days −17, −2, 3, 6, and 28. FEV1 and the provocative concentration of histamine causing a 20% fall in FEV1 (PC20) were recorded at Days −17, −4, 4, and finally at Day 13. Bronchial biopsies were taken at Days −2 and 6.

Table 1.  PATIENT CHARACTERISTICS

PatientsSex (M/F )Age (yr)Baseline FEV1 (% predicted  )Baseline PC20 (mg/ml  )Cold ScoreTiter Pre/Post Inoculation§ Viral Culture
Budesonide
1M2473.50.14 41:1/1:1Pos/pos
2* F2186.40.17 21:1/1:8Neg/neg
3M2382.50.29 91:1/1:16Pos/pos
4F2486.10.30 81:1/1:1Pos/neg
5M2395.20.54 31:1/1:8Pos/neg
6M2476.40.54141:1/1:32Pos/neg
7M2381.70.65121:4/1:128Pos/pos
8* F2391.01.28111:2/1:2Pos/neg
9M2476.81.34 81:1/1:256Pos/neg
10M2582.62.13 81:1/1:8Pos/pos
11F2480.83.32 41:1/1:16Neg/neg
12F251035.92 11:1/1:2Pos/pos
Mean ± SEM84.6 ± 2.40.74 ± 0.497 ± 1.2
Placebo
13M2280.90.06 81:1/1:16Pos/neg
14F1980.50.14 81:1/1:4Pos/pos
15F1984.20.30 31:1/1:1Pos/pos
16F2095.70.31171:1/1:8Pos/pos
17* M191010.46101:1/1:1Neg/neg
18M2374.60.52 51:1/1:32Pos/neg
19F2083.90.53101:1/1:1Pos/neg
20F2384.00.65 41:1/1:32Pos/pos
21F2494.21.44 21:1/1:16Neg/neg
22F2096.61.81 51:1/1:1Pos/pos
23* M231032.18 61:2/1:2Neg/neg
24F2095.92.40101:1/1:8Pos/pos
25M2284.14.34 31:1/1:256Pos/pos
Mean ± SEM89.2 ± 2.50.65 ± 0.497 ± 1.1

*Subjects who were excluded from statistical analysis either because RV16 infection could not be confirmed (2, 17, 23) or because a rhinovirus other than RV16 was detected (8).

FEV1 between-group comparison: p = 0.21.

Geometric mean PC20 ± SEM (doubling dose) at Day −4. Between-group comparison: p = 0.79.

§  Titer of neutralizing antibodies against RV16 (serum dilution, 1: . . .) at Day −2/Day 28.

  Tissue culture for rhinovirus at Day 3/Day 6 of nasal lavages.

RV16 inoculation took place according to a previously described protocol (3, 4, 20, 21). The total RV16 dose was 0.6–2.1 × 104 50% tissue culture infective dose (TCID50). Infection was confirmed by at least a 4-fold increase in RV16-neutralizing serum antibody titer and/or by recovery of RV16 from nasal washes (4). Finally, the subjects scored their cold symptoms three times daily (4).

Venous blood leukocyte counts (cells × 109/L) were made by automated blood count analysis (Technicon H1, Technicon, Tarrytown, NY). After assessment of baseline FEV1, PC20 was determined by standardized 2 min tidal breathing challenge tests (4, 22) using doubling concentrations of histamine (0.03–8 mg/ml). A standardized fiberoptic bronchoscopy procedure under local anesthesia (lignocaine 10% and 2% [wt/vol]) (23) was carried out after 6 h of fasting. Premedication consisted of 0.5 mg atropine subcutaneously, 20 mg codeine orally, and 400 μg inhaled salbutamol. The bronchoscope (Pentax Optical Co., Japan, outer diameter 6 mm) was introduced through the mouth. Bronchial biopsies were taken at the (sub) segmental level, using cup forceps (Olympus FB-20C, Tokyo, Japan). Alternate biopsy sites (right or left lung) were randomized over the two visits.

Immunohistochemical staining was performed on 4-μm sections of the formalin-fixed paraffin-embedded biopsies (Table 2). The primary antibodies to CD3, CD4, CD8, EG2, elastase, and AA1 were visualized using the streptavidin-biotin complex (SABC). Biotinylated rabbit anti-mouse antibodies (or swine anti-rabbit antibodies for CD3) were used. Automated cell counting (24) was performed in a blinded fashion on digitized (25) images from a three-chip color camera (KS-400 system, Kontron/Zeiss, The Netherlands). First, the basement membrane was manually delineated. Lamina propria area, defined by the widest possible 125-μm deep zone beneath the basement membrane of at least 86,000 μm2 (excluding BALT, cartilage, and smooth muscle) was automatically determined. The epithelial area above the basement membrane of at least 25,000 μm2 was then determined. Damaged epithelium was not excluded, to avoid bias due to possible virus-induced epithelial damage. The automated cell counting consisted of the following steps: level off background staining, normalize staining intensity, delete noise, fuse stained fragments, delineate stained clusters, and determine the cell count by an algorithm. Cell counts were expressed as cells/0.1 mm2.

Table 2.  ANTIGENS

AntigenMonoclonal AntibodyDilutionAntigen RetrievalSold by
EG2Yes1:200TrypsineKabi Pharmacia*
ElastaseYes1:50Dako
AA1 (tryptase)Yes1:750Dako
CD3No1:400CitrateDako
CD4Yes1:50EDTAThamer Diagnostics
CD8Yes1:400EDTANovocastra§

*Kabi Pharmacia, Woerden, The Netherlands.

Dako, Glostrup, Denmark.

Thamer Diagnostics, Uithoorn, The Netherlands.

§Novocastra, Newcastle upon Tyne, United Kingdom.

Statistical analysis was performed on log-transformed cell counts, after addition of 1 to allow for transformation of zero values (25). The results were expressed as geometric mean ± SEM in doubling cell number (DC), which is the SEM of the logged data, divided by log[2]. Likewise, changes in cell numbers were expressed as doubling cell numbers, being the difference in logged data pairs, divided by log[2]. Paired and unpaired Student's t tests were used where applicable. Relationships between various outcome parameters were investigated using Pearson's correlation test. p Values < 0.05 were considered to be significant.

All 25 patients completed the study. One patient (3) did not undergo the bronchoscopies due to strong subjective discomfort. RV16 infection was confirmed in all RV16-inoculated subjects, except subjects 2, 8, 17, and 23, who were excluded from the statistical analysis of the effect of RV16. All RV16-treated subjects had an anti-RV16 titer serum ⩽ 1:1 before entering the study. However, reassessment just before inoculation of RV16 revealed slightly elevated titers in subjects 7, 8, and 23, coinciding with symptoms of a common cold in subjects 8 and 23, which was confirmed in subject 8 by rhinovirus-positive (RV16-negative) culture of the nasal lavage. Since low levels of neutralizing antibodies per se have not been shown to preclude a symptomatic common cold (4), subject 7 was not excluded from the analysis.

Peripheral Blood Leukocyte Counts

The effects of treatment and RV16 on peripheral blood leukocyte counts are depicted in Figure 1A. In the placebo group the cold score (Table 1) correlated significantly with the increase in the numbers of neutrophils between Days 3 and 6 (r = 0.82, p = 0.003).

Lung Function and Airways Hyperresponsiveness

Before commencement of treatment FEV1 (% predicted) (26) was not significantly different between the groups (mean ± SEM BUD: 84.6 ± 2.4, PLAC: 89.2 ± 2.5, p = 0.21). During the pretreatment period FEV1 tended to increase in the budesonide group (p = 0.07), however, this increase was not significantly different between the groups (p = 0.63). RV16 infection had no significant effect on FEV1 (p = 0.41), and changes were not significantly different between the groups (p = 0.09).

In the budesonide group the PC20 to histamine showed an increase during the pretreatment period (p = 0.005) that was different from placebo (p = 0.02). As a result, there was a (borderline) significant between-group difference in PC20 at Day −4 (p = 0.05). Subsequently, there was no significant effect of RV16 infection on PC20 within either treatment group (PLAC: p = 0.18, BUD: p = 0.65), nor was the effect significantly different between the groups (p = 0.20). PC20 was still higher in the budesonide group as compared with placebo at Day 4 after infection (p = 0.02), but no longer so at Day 13 (p = 0.18) (Figure 1B).

Biopsies: Lamina Propria

The average area of lamina propria per patient that was examined for all stainings and all visits was 266,000 ± 11,000 μm2 (SEM). The descriptive and statistical analyses are presented in Table 3. In both groups there was a similar trend toward an increase in the numbers of CD3+ cells. This increase was significant in the analysis of the pooled data of the two groups (p = 0.03). The increase can be attributed in part to a significant increase in CD8+ cells in the placebo group (p = 0.04). We observed a trend toward a decrease in the number of eosinophils (p = 0.06) only in the placebo group.

Table 3.  CELL COUNTS IN LAMINA PROPIA

MarkerGroupPreinoculation: Day −2 (Mean * ± SEM [DC])Postinoculation: Day 6 (Mean * ± SEM [DC])Paired t TestpUnpaired tTest Δp
CD3All70.14 ± 0.18 94.59 ± 0.200.03
BUD55.18 ± 0.22 76.47 ± 0.360.14
[22.9–189.9]§ [29.9–229.1]
PLAC85.35 ± 0.24114.55 ± 0.180.14
[32.9–210.8][70.2–286.6]
p 0.080.180.83
CD4All94.93 ± 0.12100.99 ± 0.130.66
BUD84.10 ± 0.13 88.14 ± 0.210.80
[54.8–197.6][39.6–164.9]
PLAC104.82 ± 0.17114.15 ± 0.140.74
[30.1–245.6][59.7–195.2]
p 0.180.150.98
CD8All53.97 ± 0.18 63.85 ± 0.300.31
BUD47.89 ± 0.30 45.31 ± 0.510.85
[14.2–113.5][12.9–281.1]
PLAC59.52 ± 0.21 86.94 ± 0.310.04
[15.5–120.1][18.1–200.1]
p 0.400.120.19
EG2All 8.69 ± 0.36  6.22 ± 0.340.34
BUD 5.31 ± 0.45  6.69 ± 0.580.71
[0.4–37.1][1.0–59.5]
PLAC13.00 ± 0.50  5.87 ± 0.420.06
[1.0–61.4][0.5–24.1]
p 0.080.790.15
ElastaseAll 6.59 ± 0.30  8.47 ± 0.340.36
BUD10.18 ± 0.22 10.27 ± 0.560.91
[3.4–16.9][2.8–94.6]
PLAC 4.62 ± 0.48  7.26 ± 0.440.30
[0.0–34.7][1.0–56.8]
p 0.050.480.51
AA1All65.54 ± 0.28 82.42 ± 0.270.47
BUD87.65 ± 0.29103.76 ± 0.230.63
[40.5–226.5][57.4–182.2]
PLAC53.10 ± 0.43 68.56 ± 0.450.59
[7.5–178.6][10.0–153.8]
p 0.210.260.86

Definition of abbreviations: All = lumped data of all subjects; BUD = budesonide group; DC = doubling cell numbers; PLAC = placebo group.

*Geometric mean.

Paired t test for comparison of Days −2 and 6 within each group.

Unpaired t test for between-group analysis (budesonide versus placebo group) of changes in cell numbers between Days −2 and 6 (Δ).

§Ranges in brackets.

p Value unpaired t test for between-group analysis (budesonide versus placebo group) at each time point.

In the placebo group cold scores tended to correlate significantly with the increase in the number of CD3+ cells (r = 0.59, p = 0.07) (Figure 2A). In the placebo group, we found that the larger the decrease in the number of lamina propria EG2+ cells, the larger the early phase increase in the number of peripheral blood eosinophils (Day 3: r = −0.74, p = 0.04), and the smaller the worsening of PC20 to histamine was (Day 4: r = −0.70, p = 0.02).

Biopsies: Epithelium

The average area of epithelium per patient that was examined for all stainings and all visits was 90,000 ± 5,000 μm2 (SEM). The descriptive and statistical analyses are presented in Table 4. First, the data show a significantly lower number of EG2+ cells in the budesonide group as compared with placebo after 2 wk treatment (p = 0.04). After RV16 infection, there was a significant increase in the number of CD3+ cells in the budesonide group (p = 0.04), which was not significantly different from the effect in the placebo group (p = 0.41).

Table 4.  CELL COUNTS IN EPITHELIUM

MarkerGroupPreinoculation: Day −2 (Mean * ± SEM [DC])Postinoculation: Day 6 (Mean * ± SEM [DC]Pairedt Test pUnpairedt Test Δ p
CD3All19.88 ± 0.3926.23 ± 0.290.71
BUD13.42 ± 0.3923.07 ± 0.470.04
[3.4–34.1]§ [0.0–103.9]
PLAC26.45 ± 0.5929.06 ± 0.370.74
[6.3–78.1][4.6–59.2]
p 0.220.580.41
CD4All 2.89 ± 0.40 2.99 ± 0.320.71
BUD 3.39 ± 0.47 2.73 ± 0.400.64
[0.0–21.6][0.0–7.6]
PLAC 2.53 ± 0.63 3.24 ± 0.490.42
[0.0–53.4][0.0–31.0]
p 0.610.710.35
CD8All22.64 ± 0.3326.06 ± 0.350.52
BUD24.96 ± 0.3020.86 ± 0.640.74
[3.8–70.1][0.0–64.4]
PLAC20.90 ± 0.5731.83 ± 0.330.19
[0.0–91.9][6.7–84.5]
p 0.710.400.26
EG2All 1.76 ± 0.27 1.27 ± 0.190.06
BUD 1.17 ± 0.15 1.08 ± 0.110.51
[0.0–3.9][0.0–0.8]
PLAC 2.44 ± 0.42 1.46 ± 0.320.08
[0.0–11.8][0.0–7.2]
p 0.040.220.18
ElastaseAll 1.81 ± 0.28 2.47 ± 0.300.33
BUD 1.80 ± 0.52 3.18 ± 0.430.35
[0.0–15.2][0.0–17.0]
PLAC 1.83 ± 0.30 1.98 ± 0.420.74
[0.0–3.2][0.0–8.6]
p 0.970.270.59
AA1All 2.82 ± 0.33 4.75 ± 0.450.11
BUD 4.28 ± 0.51 5.74 ± 0.750.58
[0.0–16.3][0.0–8.0]
PLAC 1.98 ± 0.43 4.01 ± 0.560.07
[0.0–8.1][0.0–15.4]
p 0.110.580.56

Definition of abbreviations: All = lumped data of all subjects; BUD = budesonide group; DC = doubling cell numbers; PLAC = placebo group.

*Geometric mean.

Paired t test for comparison of Days −2 and 6 within each group.

Unpaired t test for between-group analysis (budesonide versus placebo group) of changes in cell numbers between Days −2 and 6 (Δ).

§Ranges in brackets.

  p Value unpaired t test for between-group analysis (budesonide versus placebo group) at each time point.

Similar to the relationship in lamina propria, the higher the cold score, the larger the epithelial accumulation of CD3+ cells in the placebo group (r = 0.77, p = 0.02) (Figure 2B). This may not be surprising, as the accumulation of CD3+cells in the epithelium and lamina propria was significantly correlated (r = 0.66, p = 0.04). Moreover, accumulation of CD8+ cells (in the placebo group) or CD4+ cells (in the budesonide group) was associated with decreased worsening of PC20 during the RV16 cold (PLAC: r = 0.71, p = 0.02, BUD: r = 0.67, p = 0.047) (Figures 3A and 3B). Finally, the rise in numbers of circulating monocytes (a CD4+ cell) in the early phase of the cold (Day 3) was significantly, but inversely related to accumulation of epithelial CD4+ cells (r = −0.74, p = 0.03) in the placebo group.

In this study we demonstrated that an experimental RV16 infection in subjects with asthma is associated with subtle inflammatory changes within the bronchial wall. Six days after RV16 infection we observed an accumulation of T cells, particularly cytotoxic T cells, accompanied by a trend toward lowering of eosinophil numbers in lamina propria and epithelium. Two weeks treatment with inhaled budesonide improved airway hyperresponsiveness and lowered the numbers of eosinophils in biopsies, and this improvement was maintained after subsequent RV16 infection. There were no significant effects of budesonide on the RV16-associated accumulation of any of the inflammatory cell types. Our results suggest the rhinovirus infection alone may not be sufficient to provoke the physiological and inflammatory events observed during “spontaneous” exacerbation of asthma. This precludes definite conclusions as to the effectiveness of inhaled corticosteroids against rhinovirus-induced exacerbations of asthma.

This is the first study to describe the effects of placebo-controlled (pre)treatment with inhaled glucocorticoids on lower airways inflammation, as induced by experimental RV16 colds in subjects with asthma. The observed RV16-associated infiltration by T cells into the lamina propria confirms the results of a previous study on RV16-induced airways inflammation by Fraenkel and coworkers (10). The absence of an increase in numbers of activated eosinophils is in keeping with findings in normal and atopic subjects after natural colds (27), but is in apparent contrast to the observed increase in EG2+ cells in the epithelium in the previously mentioned study (10). Methodological differences such as patient selection (atopic subjects with asthma only versus a mixed sample of normal subjects and subjects with asthma) and study design (biopsies taken 6 d versus 4 d after inoculation) may have contributed in part to the different outcomes in these two studies.

The lack of significant effects of budesonide on airway obstruction, airway hyperresponsiveness, or cellular infiltration into the bronchial mucosa after the rhinovirus infection seems to be in keeping with clinical studies, showing a lack of effectiveness to protect against or treat acute exacerbations of asthma in children (16-19). However, in view of the mildness of the current responses to RV16 infection, it remains to be determined whether this also holds true for more severe virus-associated exacerbations of asthma. Reassuringly, neither did we observe any detrimental effects of inhaled steroids on any of the parameters, in terms of severity of response and recovery time, which a priori could not be excluded, based on potentially impaired viral clearance (14, 15).

The results of this study were obtained after carefully considering study design and subjects selection, while using validated methods for administering RV16 (3, 20) and recording lung function and airway responsiveness (22). By using an automated system to perform biopsy cell counting we were able to analyze relatively large amounts of tissue for each staining (25), in a standardized and highly reproducible way (24). We included only steroid-naive patients who had mild persistent asthma, who were eligible for regular treatment with inhaled steroids, according to treatment guidelines (11). However, the dosage used (800 μg twice a day) was higher than the recommended dose for mild persistent asthma (11), to ensure the optimal protection that can be achieved in a relatively short pretreatment period (28). The decrease in airway hyperresponsiveness and the reduced eosinophil counts in the bronchial mucosa of the budesonide-treated subjects after the 2 wk pretreatment period fit in with the results of previous studies on the effects of inhaled corticosteroids (29, 30), and indicate that the pretreatment with inhaled corticosteroids was adequate to produce distinct antiinflammatory effects (28). Whether more prolonged pretreatment aimed at reduction of the lymphocytic infiltrate and airway remodeling (23) would be more effective for preventing virus-induced airways inflammation remains to be investigated.

The present data do not seem to be affected by lack of statistical power, as the power allowed detection of a 2-fold difference in eosinophil numbers between the groups after 2 wk budesonide treatment, and also of the rhinovirus-associated within-group changes in cell numbers of similar or smaller magnitude (25). The various correlations between biopsy cell counts and clinical/physiological outcome parameters indicate that even variation resulting in nonsignificant changes may not just be random noise, but can indeed have biological relevance. As the present sample size has previously been shown to allow detection of the effect of placebo-controlled treatment with inhaled steroids on bronchial inflammatory cell counts (29), we speculate that any effects of budesonide treatment on rhinovirus-induced airways inflammation are likely to be smaller than those observed during treatment with inhaled steroids alone.

In the present study, RV16 inoculation resulted in successful infection in 21 of 25 patients. Yet in contrast to previous studies by others (20) and ourselves (4), the RV16 colds in the present study were not associated with a significant increase in airway hyperresponsiveness to histamine. The severity of infection, as reflected by cold scores and rise in numbers of circulating neutrophils, has been shown to be linked to the rhinovirus-associated enhancement of airways hyperresponsiveness (4), such that only the severest colds lead to a significant decrease in PC20. The cold scores tended to be lower than those described in a previous study (4), and we speculate that this might explain the observed lack of increase in airway hyperresponsiveness. Possibly, unidentified virus- or host-associated factors might explain this variation. Yet the present data extend previous findings (4) by showing that the severity of cold symptoms significantly correlated not only with the rise in numbers of circulating neutrophils but also with the accumulation of T cells in the bronchial mucosa. This indicates that mild rhinovirus infections may exacerbate some aspects of airway inflammation, even in the absence of marked clinical worsening of asthma.

We observed RV16-associated accumulation of CD3+ cells in the groups as a whole, and of CD8+ cells in the lamina propria in the placebo group in particular. These findings are in keeping with an MHC class I restricted cytotoxic T cell response. Such an immune response is considered to be most efficient for viral clearance and recovery (31-33), and adds to previous evidence of rhinoviral infection of the bronchial tissues themselves (34, 35). Indeed, the increase in epithelial CD8+ cell infiltration was associated with improvement rather than worsening of airway hyperresponsiveness in the placebo group. In the budesonide-treated subjects, however, it was the migration of CD4+ cells that appeared to be associated with changes in airway hyperresponsiveness. Based on the observations that the average numbers of CD4+ cells exceed CD3+ cell numbers in the lamina propria, and that accumulation of CD4+ cells correlates significantly with the shift in numbers of circulating monocytes, it seems likely that the CD4+ cell infiltrate represents both T helper cells and monocytes. Therefore, additional studies are required in order to assess the relative contribution of monocytes and T helper cells in an MHC class II restricted response to rhinovirus-induced airway pathology, particularly in relation to ICAM-1 expression and to glucocorticoid therapy.

We did not observe an effect of budesonide treatment on rhinovirus-induced cellular infiltration of the bronchial mucosa. The interaction of glucocorticoids, rhinovirus infection, and underlying allergic airways inflammation is likely to be complex. For example, glucocorticosteroids down-regulate cytokine expression (36). However, some cytokines (IL-4, IFN-γ) counteract the glucocorticoid-induced inhibition of the effects of IL-1β (37, 38), which is a pivotal cytokine in the rhinovirus-induced immune response (39, 40). Moreover, all three factors may affect monocyte responsiveness in various ways (38, 41, 42). Based on the present results one could argue that glucocorticoids reduce allergic airway inflammation, possibly even during a rhinovirus infection, whereas they do not seem to have a detrimental effect on the antiviral immune response.

In summary, our data demonstrate that rhinovirus colds per se have rather limited effects on bronchial inflammation. Experimental rhinovirus infection seems to promote the accumulation of T cells, particularly cytotoxic T cells, in the bronchial mucosa. Treatment with inhaled steroids improves airway hyperresponsiveness and eosinophilic airway inflammation. However, inhaled corticosteroid treatment does not appear to affect the inflammatory changes associated with rhinovirus infections. The present findings indicate that rhinovirus infection by itself may not be sufficiently deleterious to induce the clinical and inflammatory worsening as are observed during spontaneous exacerbations. This may require the presence of cofactors, such as ongoing allergen exposure. Our data suggest that the merits of prophylaxis with inhaled steroids lie in improvement of the baseline condition of patients with asthma, while their effectiveness for preventing and treating severe rhinovirus-induced exacerbations of asthma remains to be established.

The authors wish to thank AstraZeneca and the Department of Clinical Pharmacy and Toxicology of the LUMC for providing the study medication, the laboratory for Clinical Hematology for performing the blood analysis, and the pulmonologists and technicians for skillfully performing the bronchoscopies.

Supported by the Netherlands Asthma Foundation (Grant 93.17) and AstraZeneca, The Netherlands.

1. Nicholson KG, Kent J, Ireland DCRespiratory viruses and exacerbations of asthma in adults. BMJ3071993982986
2. Johnston SL, Pattemore PK, Sanderson G, Smith S, Lampe F, Josephs L, Symington P, O'Toole S, Myint SH, Tyrrell DA, et al.. Community study of role of viral infections in exacerbations of asthma in 9–11 year old children. BMJ310199512251229
3. Cheung D, Dick EC, Timmers MC, de Klerk EP, Spaan WJ, Sterk PJRhinovirus inhalation causes long-lasting excessive airway narrowing in response to methacholine in asthmatic subjects in vivo. Am J Respir Crit Care Med152199514901496
4. Grünberg K, Timmers MC, Smits HH, De Klerk EPA, Dick EC, Spaan WJM, Hiemstra PS, Sterk PJEffects of experimental rhinovirus 16 colds on airway hyperresponsiveness to histamine and interleukin-8 in nasal lavage in asthmatic subjects in vivo. Clin Exp Allergy2719973645
5. Grünberg K, Timmers MC, De Klerk EPA, Dick EC, Sterk PJExperimental rhinovirus 16 infection causes variable airways obstruction in atopic asthmatic subjects. Am J Respir Crit Care Med160199913751380
6. Gern JE, Calhoun WJ, Swenson CA, Shen G, Busse WWRhinovirus infection preferentially increases lower airway responsiveness in allergic subjects. Am J Respir Crit Care Med155199718721876
7. Naclerio RM, Proud D, Lichtenstein LM, Kagey-Sobotka A, Hendley JO, Sorrentino J, Gwaltney JMKinins are generated during experimental rhinovirus colds. J Infect Dis1571988133142
8. Grünberg K, Smits HH, Timmers MC, De Klerk EPA, Dolhain RJEM, Dick EC, Hiemstra PS, Sterk PJExperimental rhinovirus 16 infection: effects on cell differentials and soluble markers in sputum in asthmatic subjects. Am J Respir Crit Care Med1561997609616
9. Grünberg K, Sharon RF, Hiltermann TJN, Brahim JJ, Dick EC, Sterk PJ, Van Krieken JHJMExperimental rhinovirus-16 infection increases intercellular adhesion molecule-1 expression in bronchial epithelium of asthmatics regardless of inhaled steroid treatment. Clin Exp Allergy30200010151023
10. Fraenkel DJ, Bardin PG, Sanderson G, Lampe F, Johnston SL, Holgate STLower airways inflammation during rhinovirus colds in normal and in asthmatic subjects. Am J Respir Crit Care Med1511995879886
11. National Institutes of Health, National Heart LaBI. Global initiative for asthma. Global strategy for asthma management and prevention. NHLBI/WHO workshop report, 1995; Publication number 95-3659.
12. Papi A, Papadopoulos NG, Degitz K, Holgate ST, Johnston SLCorticosteroids inhibit rhinovirus-induced intercellular adhesion molecule-1 up-regulation and promotor activation on respiratory epithelial cells. J Allergy Clin Immunol1052000318326
13. Suzuki T, Yamaya M, Sekizawa K, Yamada N, Nakayama K, Ishizuka S, Kamanaka M, Morimoto T, Numazaki Y, Sasaki HEffects of dexamethasone on rhinovirus infection in cultured human tracheal epithelial cells. Am J Physiol Lung Cell Mol Physiol2782000L560L571
14. Oehling AG, Akdis CA, Schapowal A, Blaser K, Schmitz M, Simon HUSuppression of the immune system by oral glucocorticoid therapy in bronchial asthma. Allergy521997144154
15. Gustafson LM, Proud D, Hendley JO, Hayden FG, Gwaltney JMOral prednisone therapy in experimental rhinovirus infections. J Allergy Clin Immunol97199610091014
16. Doull IJM, Lampe FC, Smith S, Schreiber J, Freezer NJ, Holgate STEffect of inhaled corticosteroids on episodes of wheezing associated with viral infection in school age children: randomized double blind placebo controlled trial. BMJ3151997858862
17. Svedmyr J, Nyberg E, Asbrink-Nilsson E, Hedlin GIntermittent treatment with inhaled steroids for deterioration of asthma due to upper respiratory tract infections. Acta Paediatr841995884888
18. Garrett J, Williams S, Wong C, Holdaway DTreatment of acute asthmatic exacerbations with increased dose of inhaled steroid. Arch Dis Child7919981217
19. Schuh S, Reisman J, Alshehri M, Dupuis A, Corey M, Arseneault R, Alothman G, Tennis O, Canny GA comparison of inhaled fluticasone and oral prednisone for children with severe acute asthma. N Engl J Med3432000689694
20. Lemanske RF, Dick EC, Swenson CA, Vrtis RF, Busse WWRhinovirus upper respiratory infection increases airway hyperreactivity and late asthmatic reactions. J Clin Invest831989110
21. Gwaltney JM, Hendley O, Hayden FG, McIntosh K, Hollinger FB, Melnick JL, Turner RBUpdated recommendations for safety-testing of viral inocula used in volunteer experiments on rhinovirus colds. Prog Med Virol391992256263
22. Sterk PJ, Fabbri LM, Quanjer PH, Cockcroft DW, O'Byrne PM, Anderson SD, Juniper EF, Malo JLAirway responsiveness. Standardized challenge testing with pharmacological, physical and sensitizing stimuli in adults. Eur Respir J Suppl1619935383
23. Sont JK, Willems LN, Bel EH, van Krieken JH, Vandenbroucke JP, Sterk PJClinical control and histopathologic outcome of asthma when using airway hyperresponsiveness as an additional guide to long-term treatment. The AMPUL Study Group. Am J Respir Crit Care Med159199910431051
24. Sont JK, Grünberg K, Sterk PJ, Van Krieken JHJMAutomated assessment of inflammatory cell counts in bronchial biopsy specimens: repeatability and agreement with interactive point counting. Eur Respir J121998195s
25. Sont JK, Willems LNA, Evertse CE, Hooijer R, Neeskens P, Sterk PJ, Van Krieken JHJMRepeatability of measures of inflammatory cell number in bronchial biopsies in atopic asthma. Eur Respir J10199826022608
26. Quanjer PH, Tammeling GJ, Cotes JE, Pedersen OF, Peslin R, Yernault JCLung volumes and forced ventilatory flows. Report Working Party Standardization of Lung Function Tests, European Community for Steel and Coal. Official Statement of the European Respiratory Society. Eur Respir J Suppl161993540
27. Trigg CJ, Nicholson KG, Wang JH, Ireland DC, Jordan S, Duddle JM, Hamilton S, Davies RJBronchial inflammation and the common cold: a comparison of atopic and non-atopic individuals. Clin Exp Allergy261996665676
28. Gauvreau GM, Doctor J, Watson RM, Jordana M, O'Byrne PMEffects of inhaled budesonide on allergen-induced airway responses and airway inflammation. Am J Respir Crit Care Med154199612671271
29. Trigg CJPlacebo-controlled immunopathologic study of four months of inhaled corticosteroids in asthma. Am J Respir Crit Care Med15019941722
30. Pederson B, Dahl R, Karlstrom R, Peterson CGB, Venge PEosinophil and neutrophil activity in asthma in a one-year trial with inhaled budesonide. Am J Respir Crit Care Med153199615191529
31. Graham MB, Braciale VL, Braciale TJInfluenza virus-specific CD4+ T helper type 2 T lymphocytes do not promote recovery from experimental virus infection. J Exp Med180199412731282
32. Tang Y-W, Graham BSAnti-IL-4 treatment at immunization modulates cytokine expression, reduces illness, and increases cytotoxic T lymphocyte activity in mice challenged with respiratory syncytial virus. J Clin Invest94199419531958
33. Subauste MC, Jacoby DB, Richards SM, Proud DInfection of a human respiratory epithelial cell line with rhinovirus. Induction of cytokine release and modulation of susceptibility to infection by cytokine exposure. J Clin Invest961995549557
34. Gern JE, Galagan DM, Jarjour NN, Dick EC, Busse WWDetection of rhinovirus RNA in lower airway cells during experimentally induced infection. Am J Respir Crit Care Med155199711591161
35. Papadopoulos NG, Bates PJ, Bardin PG, Papi A, Leir SH, Fraenkel DJ, Meyer J, Lackie PM, Sanderson G, Holgate ST, et al.. Rhinoviruses infect the lower airways. J Infect Dis181200018751884
36. Barnes PJAnti-inflammatory actions of glucocorticoids: molecular mechanisms. Clin Sci941998557572
37. Chizzolini C, Chicheportiche R, Burger D, Dayer JMHuman Th1 cells preferentially induce interleukin (IL)-1beta while Th2 cells induce IL-1 receptor antagonist production upon cell/cell contact with monocytes. Eur J Immunol271997171177
38. Kovalovsky D, Paez PM, Sauer J, Perez CC, Nahmod VE, Stalla GK, Holsboer F, Arzt EThe Th1 and Th2 cytokines IFN-gamma and IL-4 antagonize the inhibition of monocyte IL-1 receptor antagonist by glucocorticoids: involvement of IL-1. Eur J Immunol28199820752085
39. Proud D, Gwaltney JM, Hendley JO, Dinarello CA, Gillis S, Schleimer RPIncreased levels of interleukin-1 are detected in nasal secretions of volunteers during experimental rhinovirus colds. J Infect Dis169199410071013
40. Gern JE, Dick EC, Ming Lee W, Murray S, Meyer K, Handzel ZT, Busse WW. Rhinovirus enters but does not replicate inside monocytes and airway macrophages. J Immunol 1996;156:621–627.
41. Gern JE, Joseph B, Galagan DM, Borcherding WR, Dick ECRhinovirus inhibits antigen-specific T cell proliferation through an intercellular adhesion molecule-1 dependant mechanism. J Infect Dis174199611431150
42. Stöckl J, Vetr H, Majdic O, Zlabinger G, Kuechler E, Knapp WHuman major group rhinoviruses downmodulate the accessory function of monocytes by inducing IL-10. J Clin Invest1041999957965
Correspondence and requests for reprints should be addressed to Prof. P. J. Sterk, M.D., Lung Function Laboratory, C2-P, Leiden University Medical Center (LUMC), Albinusdreef 2/P.O. Box 9600, NL-2300 RC Leiden, The Netherlands. E-mail:

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