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* From the Department of Internal Medicine (Drs. Ottanelli, Duranti, and Scano), Section of Immunoallergology and Respiratory Diseases, University of Florence, Italy; and Fondazione Don C. Gnocchi (IRCCS) (Drs. Rosi, Romagnoli, Grazzini, Stendardi, and Scano), Pozzolatico, Florence, Italy.
Correspondence to: Giorgio Scano, MD, FCCP, Department of Internal Medicine, Section of Immunoallergology and Respiratory Disease, University of Florence, Viale Morgagni, 85, 50134 Firenze, Italy; e-mail: g.scano{at}dfc.unifi.it
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Abstract |
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 TOP Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Objective: We hypothesized that if the inflammation of the airway wall influences to some extent and in some way the PD in asthma patients, ICSs reverse the effect of airway inflammation on the PD.
Methods: We studied 100 asthma patients who were divided into the following four groups: patients with obstruction who were either ICS-naive (group I) or were treated with ICSs (group II); and nonobstructed patients who were either ICS-naive (group III) or were treated with ICSs (group IV). PD on the visual analog scale (VAS) was assessed during a methacholine-induced FEV1 decrease and specifically was quantified as the VAS slope and score at an FEV1 decrease of 5 to 20%. BHR was assessed in terms of the provocative concentration of methacholine causing a 20% fall in FEV1 (PC20). Eosinophil counts in induced sputum samples also were performed. Regression analysis, univariate analysis of variance, and factor analysis were applied for statistical evaluation.
Results: For a 5 to 20% fall in FEV1 from the lowest point after saline solution induction, VAS score was lowest in group II, slightly higher in group I, slightly higher still in group IV, and the highest in group III. In the patients as a whole, BHR related to PD, but age, clinical score, duration of the disease, and presence of baseline airway obstruction did not. In patients with obstruction who were treated with ICSs, eosinophil counts related to PD negatively. Factor analysis yielded the following four factors that accounted for 70% of the variance in the data: ICS; eosinophil counts; FEV1; and PC20 loaded on separated factors with PD loading on the same factors as PC20. The post hoc analysis carried out dividing the patients into ICS-treated and ICS-naive, showed that in the former group eosinophil counts and BHR proved to be factors negatively associated with PD, while in the latter group eosinophil counts were positively associated with PD.
Conclusions: We have shown that eosinophilic inflammation of the airway wall may increase PD and that the association of eosinophil counts with ICSs may result in lessening the PD.
Key Words: asthma • bronchoconstriction • corticosteroids • dyspnea • eosinophils • hyperresponsiveness • visual analog scale
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Based on the above findings, we hypothesized that if the inflammation of the airway wall to some extent and in some way influences the PD in asthma patients, then ICS therapy may reverse the effect of airway inflammation on PD.
In patients with asthma, Rubinfeld and Pain8 showed that deteriorating airway function may increase the perception of acute exacerbation, and Burdon et al6 showed that patients with airway obstruction had a similar degree of breathlessness as patients without airway obstruction, only at a point when they were considerably more obstructed. Ottanelli et al7 have shown recently a decreased perception of acute exacerbation in patients with initial airflow obstruction compared to those without airway obstruction. The apparent discrepancy between the results of the studies of Ottanelli et al7 and Burdon et al6 is likely to be due to the different levels of induced obstruction, which are greater in the latter study than in the former. Nevertheless, in both cases temporal adaptation, which dictates the high threshold for the perception of acute lung change,6 was considered to be the reason for the different responses in obstructed and nonobstructed patients.
Therefore, the present investigation was carried out in ICS-naive and ICS-treated asthma patients with or without airway obstruction, and with different levels of both BHR and airway inflammation.
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Clinical Scores
Degree of asthma severity was assessed by a modified version of
the asthma severity score proposed by Brooks et al.18
Possible scores ranged from 0 to 20. The asthma severity score was
based on the following: (1) the frequency of attacks of wheezing and/or
chest tightness that occurred during an average day, scored from 0 to 4
(0, none; 1, two attacks per year; 2, monthly attacks; 3, weekly
attacks; 4, daily attacks); (2) the frequency of asthma attacks that
awoke the patient at night, scored from 0 to 4 (0, none; 1, two attacks
per year; 2, monthly attacks; 3, weekly attacks; 4, daily attacks); (3)
chronic exertional dyspnea (from the modified Medical Research Council
dyspnea scale,19
scored from 0 [no breathlessness] to 4
[breathlessness during dressing or undressing]); (4) treatment
required to control asthma, scored from 0 to 4 (0, none or
ß2-agonist bronchodilators received 1 or 2
times per year; 1, bronchodilators received intermittently only; 2,
bronchodilators received daily; 3, bronchodilators received in
association with ICSs; 4, oral corticosteroids received); and (5)
frequency of cough, scored from 0 to 4 (0, no cough; 1, two attacks per
year; 2, monthly episodes; 3, weekly episodes; 4, daily
episodes).
Measurements
Lung Function:
Baseline pulmonary function testing was
performed by measuring static and dynamic lung volumes with a
water-sealed spirometer (Pulmonet Godart; SensorMedics Corp; Yorba
Linda, CA), as previously reported.20
The normal values
for lung volumes are those proposed by the European Community for Coal
and Steel.21
Bronchial Challenge:
Each patient was administered a
methacholine aerosol inhalation test according to a standardized tidal
breathing procedure.22
Increasing concentrations of
methacholine-chlorhydrate in normal phosphate-buffered saline
solution (prepared by the University Hospital Pharmacy) were inhaled
from a nebulizer (model 646; DeVilbiss Co; Somerset, PA), resulting in
an output of 0.13 mL/min. With this method, 4 mL of solution was placed
in the nebulizer, and inhalation continued during tidal breathing for 2
min. The methacholine solution was stored at 4°C and was nebulized at
room temperature. Normal phosphate-buffered saline solution was inhaled
first, followed at 5-min intervals by methacholine in twofold
increasing concentrations from 0.015 to 8 mg/mL. The test was withheld
at the PC20. FEV1 was measured 1 to 1.5 min
after the inhalation of each concentration of methacholine. Two
measurements of FEV1 were performed, and the best value was
used. The PC20 was noted from the log dose-response curve.
The log cumulative dose of methacholine also was calculated for each
patient.
Dyspnea:
Dyspnea was described to the patients as
nonspecific discomfort associated with the act of breathing. This was
chosen as a global expression to encompass all uncomfortable stimuli
and their associated sensations.23
The PD during
methacholine-induced bronchoconstriction was evaluated by administering
each patient a visual analog scale (VAS) before the first inhalation
and just after the inhalation of each concentration of methacholine and
before FEV1 measurement.5
6
11
24
The VAS
scale is a horizontal 10-cm line labeled "no breathlessness at all"
at the left end and "the worst breathlessness ever experienced" at
the right end. The subjects were asked to place a vertical mark on the
horizontal line. The VAS dyspnea score was expressed as the distance of
the mark from the left end of the VAS in millimeters.24
We
also recommended that patients rate only dyspnea and that they ignore
other sensations such as irritating cough, throat irritation, headache,
or pharyngitis. During the test, the subjects were blinded to their
lung function responses.
Induction of Sputum:
The induction of sputum was performed
according to the method of Ronchi et al.25
Briefly, 10 min
after fenoterol inhalation (200 µg), hypertonic saline solution was
nebulized with an ultrasonic nebulizer (Fisoneb; Fisons Corp;
Rochester, NY) and was inhaled for 5-min periods up to 20 min. The
concentration of saline solution was increased at intervals of 10 min
from 3 to 4%. FEV1 was measured every 5 min during
inhalation. The sputum induction procedure did not cause troublesome
symptoms, and the FEV1 did not decrease by > 20% in any
patient. Every 5 min, patients were asked to try to cough sputum into a
Petri dish and to collect saliva in a separate container. Plugs free of
salivary contamination were suspended in a dithiothreitol solution
(0.1%) and were incubated for 30 min at 37°C for slide making. Cells
were centrifuged at 400g for 10 min and then were
resuspended in saline solution. Three sputum slides then were prepared
for cytologic examination by cytocentrifugation. Cells were air dried
and stained with May-Grunwald-Giemsa stain. Cell differentials were
determined by counting 200 nonsquamous cells on each sputum slide. All
of the sputum samples were collected from patients as induced sputum.
Ninety-one patients were able to produce adequate sputum samples to
perform the differential cell count.
Protocol
Spirometry and sputum induction were performed on day 1.
Twenty-four hours later, each patient underwent a methacholine
bronchoprovocation test.
Data Analysis
The PC20 was logarithmically transformed
for statistical analysis. Linear regression analysis for
parametric and nonparametric statistics was applied when appropriate to
assess the relationship between the dyspnea score and the reduction in
FEV1.
PD was assessed as slopes and intercepts of changes in VAS on FEV1 decreases as the percentage of the predicted value and as PD at 5 to 20% of the lowest decrease in FEV1 after saline solution inhalation. The PD20 was defined as the perception of dyspnea when FEV1 decreased by 20% from that after inhalation of saline solution.
The differences between the four subgroups were evaluated by using analysis of variance and univariate analysis of variance for repeated measures (ie, split plot)26 when appropriate. The Bonferroni test was used for comparisons between subgroups.
Factor analysis was used to determine the dimensions underlying the pattern of interrelationships.27 Factor analysis is a statistical technique applied to a single set of variables to discover which sets of variables form coherent subsets that are relatively independent of one another. Variables that are correlated with one another, which are also largely independent of other subsets of variables, are combined into factors. The purpose of the analysis is to obtain a small number of factors that account for most of the variability in the variables. Accordingly, it is important to recognize that factor analysis does not merely regroup variables that are highly correlated with one another. In the final solution, correlations are obtained among all variables entered in the analysis and the virtual factors. Each of the original variables employed in the analysis are said to load on the factors, to a greater or lesser extent, based on the magnitude of the obtained correlations between each variable and the factor. In general, each variable loads (ie, is correlated most highly with) a single factor. Factors are labeled, conveniently, in terms of the pattern of these loadings.
Thus, the purpose of using factor analysis in the present study was to determine whether, in clinically stable asthma patients, PD, respiratory function, BHR, and airway inflammation would reduce to similar or different factors. To determine the number of factors we should extract, we employed a combination of methods.
The possibility of performing the factor analysis was tested by means of Bartlett’s test of sphericity. Details of the method have been thoroughly discussed elsewhere.28
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Results |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 analysis) for age,
gender, atopy, and clinical score. Groups I and III also were matched
for duration of the disease, and groups II and IV were matched for
duration of the disease and the daily dosage of ICSs. In groups
II and IV, the duration of the disease was significantly higher than
that in groups I and III.
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= 0.25). Age, the
duration of the disease, clinical scores, and
FEV1 did not significantly relate to the slopes
or to PD20 (p = 0.19 to 0.95). While in the
patients as a whole no relationship was found between eosinophil counts
and either slopes or PD20, a negative
relationship (exponential model,
y = exp[a + bx])
in obstructed ICS-treated patients (group II) was found between
eosinophil counts and VAS slopes (Fig 2
). This relationship was not significant in the other subgroups.
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2, 103.005; degrees of freedom, 21; p = 0.000). Four
interpretable factors accounted for 70% of the variance in the data;
the correlations with the original variables obtained for each rotated
factor (factor loadings) are displayed in Table 4
. The results did not change when eosinophil counts were expressed as
the total number of cells present. As shown in the table,
FEV1 and age loaded on factor I, ICSs and duration of
disease loaded on factor II, slope VAS and PC20 loaded on
factor III, eosinophil counts and PC20 were loaded on
factor IV.
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2 test
carried out between subgroups. We obtained similar results for each
subgroup, and the analysis was comparable to the analysis of the entire
group of patients. In fact, the factor analysis yielded four factors
with similar factor loadings, accounting for 79.5 of the variance in
one subgroup and 78.3 in the other.
Post Hoc Analyses: Additional analysis was conducted by
dividing patients into two subgroups on the basis of ICS therapy, with
the same variables included as in the primary analysis.
In the post hoc analysis, ICS-naive patients were younger
(p < 0.05) with no differences in eosinophil counts (p > 0.05),
pulmonary function (p > 0.05), or PC20
(p > 0.05) but with different PD (p < 0.05 to p < 0.01). In
ICS-naive patients and ICS-treated patients, four and three factors,
respectively, accounted for a similar percentage of the variance.
Eosinophil counts were associated with PD, positively in ICS-naive
patients and negatively in ICS-treated patients.
PC20 was either independent loading in ICS-naive
patients or associated loading with PD in ICS-treated patients (Table 5
). In both groups, the duration of the disease was an independent
loading on factor III.
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Discussion |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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These data are in line with studies on the effect of eosinophil inflammation and ICSs11 12 13 14 on PD. Eosinophil airway inflammation has been proposed as a determinant of breathlessness13 14 29 via mechanisms affecting either the mechanical pathways that control breathlessness5 30 or the afferent nerves involved in PD.13 in’t Veen et al14 have suggested that an ongoing airway inflammation characterized by eosinophilic airway infiltration, despite treatment with inhaled ICSs, was associated with a reduced perception of airflow obstruction. They believe the influence of both the severity of asthma and the use of ICSs to be the most plausible explanation for the PD/eosinophil relationship. In this regard, however, it must be mentioned that the same effect of ICSs on dyspnea has been noted in studies dealing with the following different types of asthma: inadequately controlled by inhaled bronchodilator alone12 ; severely brittle14 ; and moderate, as in the present study. Consistent with the above data, Higgs and Laszlo12 were able to find a reduction in the symptoms of asthma with beclomethasone (BCM) therapy without any improvement in the results of airflow tests, as shown by the fact that the slopes of Borg on peak expiratory flow were less steep when patients received BCM than when they received either cromoglycate or theophylline. However, as opposed to the results of in’t Veen et al,14 Higgs and Laszlo12 maintained that this effect could be due to BCM-induced reduction in airway inflammation.12 At variance, other investigators11 13 have reported on the positive role of ICSs on PD. Roisman et al13 found a negative correlation between bradykinin-induced PD and the magnitude of eosinophilic inflammation in airway mucosa, but, at variance with in’t Veen et al,14 the negative relationship appears to be held by subjects who were not receiving ICSs. In contrast, subjects receiving ICSs had lower eosinophil infiltration and greater PD than those not using those agents. Boulet et al11 have confirmed a higher symptom score for a given degree of fall in expiratory flows in patients using ICSs than patients using only bronchodilators on demand. It has been suggested13 that the asthmatic inflammatory process with its eosinophilic airway inflammation, which is present even in the mildest cases of asthma,15 25 could reduce PD and is possibly due to damage to the sensory receptors in the airways. Accordingly, the increase in PD with inhaled ICS therapy may be inferred to be a result of the reduction of the inflammatory process and the restoration of the integrity of the airway epithelium.13
In light of these discrepancies, to substantiate our data we tried to break down the relative potential contributions of eosinophil counts and ICSs to PD by applying factor analysis, which overcomes the problem of overlapping between variables. Some variables indeed share high percentages of their variation with other variables. This is the case with FEV1.31 Insight also may be gained from variables that do not appear to be significantly related to others in a standard analysis, which was the case in the lack of association between VAS score and eosinophil counts in subgroups other than obstructed ICS-treated patients. For the above reasons, factor analysis can be used successfully as a data reduction technique in a clinical study of patients with multiple interrelated outcome measures that are used to characterize the disease process.
Accordingly, we first sought to determine whether the clinical characteristics of the disease, such as PD, airway obstruction, BHR, eosinophilic bronchial inflammation, or anti-inflammatory treatment, would reduce to similar or different factors in patients in stable condition with chronic asthma. As shown by the analysis, measurements of airway obstruction (factor I), ICS treatment (factor II), BHR (factor III), and eosinophil counts and PD (factor IV) remained important dimensions in the assessment of patients with chronic asthma. Furthermore, to better define the potential effect of the association of ICSs with eosinophil counts on PD, in the post hoc analysis ICS-naive patients were separated from ICS-treated patients. The data indicate that eosinophil counts were associated with PD, positively in the former group and negatively in the latter group. The positive association of eosinophil counts with PD in ICS-naive patients is consistent with the data by in’t Veen et al.14 The inverse association of eosinophil counts with PD in ICS-treated patients, also reported by the same authors,14 implies that the ICS-eosinophil blend may result in deteriorating PDs.
However, it is worth noting that in the present study, as in other studies, airway eosinophil inflammation was assessed by measuring eosinophil counts in induced sputum samples. This might explain the differences with another report13 that assessed the inflammatory cell population in the asthmatic bronchial mucosa. One could note that in the studies by in’t Veen et al14 and Roisman et al,13 unlike in our study, airway eosinophil inflammation did not significantly relate to the dyspnea sensitivity associated with methacholine-induced bronchoconstriction. Discrepancies between their and our results may simply be due to differences in the number of patients and their clinical characteristics.
Attempts to relate PD to BHR have shown that patients with high responsiveness to histamine perceive less bronchoconstriction than less responsive subjects.6 However, the relationship between PC20 and breathlessness was weak in the study by Burdon et al,6 and further studies on the same topic9 11 12 did not confirm those results. Our data, indicating that PD related to BHR in the patients as a group, are only in part in line with the study by Burdon et al6 in which the effect of ICS therapy was not taken into account. In the present study, indeed, PD was associated with BHR in ICS-treated patients but was independent of BHR in ICS-naive patients. Thus, whether they were obstructed or nonobstructed, ICS-treated and ICS-naive patients had similar PC20 values, which casts some doubts on the association of BHR with PD found in the data in Table 4 . The present data therefore do not permit us to share the effects of ICSs and BHR on PD.
As found in previous articles both by us7 and others,32 in neither group reported on in the present study was baseline airway obstruction associated with PD or PC20.
Significant individual variability in PD was observed, with some patients showing reduced ventilatory function without dyspnea and others evidently dyspneic with minimal ventilatory impairment. Although the explanation of this is likely to be complex, one might consider that dyspnea has an important affective dimension and often evokes emotional and behavioral responses that can vary among patients.33 Some subjects are more fearful than others, leading to anxiety and an exaggerated response; others are fearless and have an insufficient response.
That ICSs may have a direct effect at the level of the CNS in asthma patients has not been excluded.12 14 In this regard, it has been postulated that a central effect34 of ICSs on symptom severity via the inhibition of adrenocorticotropic hormone secretion35 results in a reduced sensory acuity in human beings.36 Our data seem to be in favor of an associated effect of eosinophil counts with ICSs rather than an effect of ICSs, per se, on PD.
In summary, we have shown that eosinophilic inflammation of the airway wall may increase PD and that the association of eosinophil counts with ICS may result in lessening the PD. It is worthwhile noting the importance of this information for patients in whom reduced awareness of symptoms could delay the assumption of rescue treatment.
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Footnotes |
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This work was supported by grants from the Ministero dell’Università e della Ricerca Scientifica e Tecnologica, Italy, and by the Fondazione Don C. Gnocchi IRCCS.
Received for publication October 9, 2000. Accepted for publication April 6, 2001.
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