American Journal of Respiratory and Critical Care Medicine

The severity of asthma can be graded from mild intermittent to severe persistent. Airway inflammation is a feature of persistent asthma. We compared several markers of inflammation in mucosal biopsies and bronchoalveolar lavage fluid (BAL fluid) from 12 healthy control subjects, 24 patients with intermittent asthma, and 18 patients with mild-to-moderate persistent asthma. Epithelial shedding, eosinophil (EG2-positive cells), and activated T-cell (UCHL1) counts in biopsies, and ECP levels in BAL fluids were significantly increased in patients with intermittent asthma by comparison with control subjects and this increase was significantly greater for patients with persistent asthma. Alveolar macrophage activation (percentage of hypodense cells) and the thickness of the basement membrane were significantly increased in asthmatic subjects as compared with controls but there was no difference between the two asthmatic groups. Hyaluronic acid levels in BAL fluids were significantly increased in patients with persistent asthma by comparison with control subjects and patients with intermittent asthma. Mast cell numbers (toluidine blue) in biopsies and histamine or levels in BAL fluids were similar in the three groups. This study shows that airways inflammation is present in patients with intermittent asthma but to a lesser extent than in patients with persistent asthma.

The assessment of the severity of asthma is always problematic but clinical scores (1) alone or combined with pulmonary function tests (2) have helped to distinguish between several grades of asthma. Recently, the NHLBI/WHO Global Strategy Management and Prevention of Asthma report (3) has established four grades of the disease: mild intermittent, mild persistent, moderate, and severe asthma depending on symptoms, control of asthma (4), and pulmonary function tests (3).

Most of the studies examining bronchial inflammation have been performed in patients with mild to moderate persistent asthma (5-9) and have shown the involvement of various cell types such as eosinophils (10-12), lymphocytes (11), mast cells (12, 13), macrophages (11, 14) and epithelial cells (15, 16). However, no study has been reported in which the airways inflammation of patients suffering from mild intermittent asthma (as defined by the WHO/NHLBI report [3]) was specifically examined.

We compared the profile and magnitude of airway inflammation in patients with intermittent (as defined in the WHO/ NHLBI report on asthma [3]) and persistent asthma since they may have important consequences for the diagnosis, treatment and prognosis of the disease. All patients underwent the same investigations. Eosinophils, mast cells and T cells were studied in biopsies. Epithelial cell involvement was studied by measuring the percentage of basement membrane covered. The thickness of the basement membrane was also studied. In the BAL fluid, differential cell counts were performed. Eosinophil activation was assessed by the measurement of ECP, mast cell activation by the measurement of histamine and macrophage activation. Finally, hyaluronic acid (HA) was measured to assess turn-over of the extracellular matrix and the extent of tissue damage.

Subjects

We studied 24 asthmatic patients with mild intermittent asthma ranging from 20 to 35 yr of age and 18 asthmatic patients with persistent mild-to-moderate asthma ranging from 19 to 67 yr of age. Asthma was defined as previously described in detail (10). All patients had either a reversible airways obstruction characterized by an increase of at least 12% in the FEV1 value after inhalation of 200 μg of salbutamol or a positive inhalation challenge with carbachol. The severity of asthma was assessed using the Aas score as previously described (1, 10). Patients with intermittent asthma were defined according to the WHO/ NHLBI report on asthma (3) and had an Aas score of 1 or 2 (1). They reported less than one chest tightness episode a week, less than two nights with symptoms a month and periods between exacerbations were completely symptom free, with a normal lung function. In these patients, morning and evening peak flow rates, exacerbations and β2-agonist consumption were monitored every day for the two weeks preceding the bronchoscopy. Patients with persistent asthma had a mild to moderate form of the disease and an Aas score of 2 to 3. Patients with persistent asthma had symptoms more than twice a week and/or they had night symptoms once a week, and they required daily inhalation of β2 agonist. None of the subjects were current or previous smokers. Inhaled corticosteroids or oral corticosteroids had been withdrawn for at least two months prior to the commencement of the study and use of nedocromil sodium or cromoglycate had been stopped for at least two weeks. Patients were studied under steady state conditions and were excluded from the study if they had had a severe exacerbation of asthma requiring a stay in hospital or a pulmonary infection during the month preceding the study. A pulmonary function test (flow volume loop) was carried out the day before bronchoscopy using a Pneumoscreen (E. Jaeger Laboratories, Würzburg, Germany). All subjects underwent identical investigations for allergy testing as previously described in detail (10).

Twelve normal healthy volunteers ranging from 21 to 32 yr were used as a control group. Their pulmonary function was within normal range. They were nonallergic and had never suffered from asthma. None of the subjects were current or previous smokers.

The research was carried out with the informed consent of the patients and approval of the Ethics Committee of the Montpellier Hospital.

Investigations

Fiberoptic bronchoscopy. Fiberoptic bronchoscopy was carried out as previously described (10). Two to three biopsies were taken using alligator forceps in the subsegmental bronchi of the left lobe. The BAL was carried out in one of the subsegmental bronchi of the middle lobe by injection of several aliquots of sterile saline (up to a total volume of 0.2 L) reaspirated by gentle syringe suction. Ninety-one percent of the subjects received 0.2 and 3.5% received less than 0.1 L. Immediately after lavage, mucus was removed from the fluid by filtration through a gauze. Cell counts were made on BAL fluid prior to further processing. Then BAL fluid was centrifuged at 400 g for 10 min at +4° C and the supernatant was kept frozen at −80° C until required.

Examination of BAL cells. The BAL cytology was conducted on cytocentrifuged slides (Cytospin, Shandon, UK) and stained by May-Grundwald-Giemsa.

Density fractionation of alveolar macrophages. Density fractionation of AM was performed as previously described in detail (17). Total BAL cells were layered onto a four-step Percoll density gradient with densities of 1.030, 1.040, 1.050 and 1.070 g/ml. The results are shown as percentages i.e., the number of cells found in one of the fractions divided by the total number of cells recovered.

Titration of Mediators in BAL Fluid

Eosinophil cationic protein was measured in BAL fluids using a double antibody radioimmunoassay (Pharmacia Diagnostics AB, Uppsala, Sweden) (10) following the manufacturer's instructions. Levels < 2 μg/L are undetectable.

Hyaluronic acid was measured in BAL fluids using a radioimmunoassay as previously described in detail (18). The detection limit is approximately 4 μg/L.

Histamine was measured in BAL fluids using an enzyme immunoassay incorporating a monoclonal antibody against acylated histamine (Immunotech, Luminy, France). The limit of detectability is 0.2 nM.

Bronchial Biopsies

Hematoxylin and eosin staining. Biopsies were fixed in 10% formaldehyde (pH 7.2) and embedded in paraffin blocks. Six micrometer tissue sections were affixed to microscope slides with Le Page's glue and after deparaffination and rehydration the slides were ready for histology and immunohistochemistry. Hematoxylin and eosin staining (HE) staining was then carried out as previously described (10). The thickness of the basement membrane was measured on biopsies stained by HE. The thickness of the basement membrane was determined as the average of 6 measurements in sections where the epithelium was properly oriented in order to avoid artefacts in measurement. The shedding (epithelial abrasion) was characterized as the percentage of length of basal cells uncovered by columnar cells (16, 19). Toluidine blue staining was carried out using the method report by Pesci and coworkers (13).

Immunohistochemistry. Two monoclonal antibodies were used: antibodies EG2 (Pharmacia Diagnostics AB, Uppsalla, Sweden) specific for a common epitope of a cleaved form of ECP and EPX (20) and antibody specific for a subset of antigen-primed T cells (UCHL1) (14). Immunohistochemistry on bronchial tissue sections was carried out using an alkaline phosphatase linked rabbit anti-mouse antibody as a second antibody. Control slides were treated with an unrelated IgG2 mouse antibody of the same IgG isotype. Bronchial specimens from normal subjects and asthmatic patients were run in the same experimental series since small differences may be seen when experiments are run at different times. All biopsy samples were coded and sections were studied in a blinded fashion. Cellular infiltration of eosinophils, as assessed by cellular staining, was scored by number of cells per square millimeter.

Statistical Analysis

Statistical analyses were conducted using nonparametric tests. The Kruskall-Wallis test was used to compare the three groups of subjects. Then, when the Kruskall-Wallis test result was significant, the Mann-Whitney U test with Bonferroni's correction was used to compare the different groups. Since for all inflammatory indices studied, there was no significant difference between atopic and nonatopic patients with persistent asthma, we averaged the results of these two groups for comparison with patients with intermittent asthma and control subjects. For correlations, the Spearman rank test was used.

Characteristics of the Patients

The characteristics of the subjects are presented in Table 1. The median age of mild asthmatic and normal subjects was similar, however, patients with persistent asthma were found to be significantly older (p < 0.01). The pulmonary function of normal subjects was within normal range. Patients with persistent asthma had a mild-to-moderate form of the disease and had a significant decrease in FEV1 values (52 to 106% of the predicted values) in comparison with patients with intermittent asthma (80 to 105% of the predicted values; p = 0.002) and normal subjects (100 to 108% of the predicted values; p = 0.001). The duration of asthma was different in the three asthmatic groups, being significantly increased in atopic patients with persistent asthma (p < 0.02).

Table 1. DEMOGRAPHIC CHARACTERISTICS OF THE SUBJECTS

Control SubjectsIntermittent AsthmaPersistent Asthma
AtopicNonatopic
n1224 9 9
Age, yr   25 (22-34)     24 (20.5–26.5)35 (32–47)42 (30–55)
Sex, % males6058 5544
Duration of asthma, yr     10 (6-13)25 (21-32)       6 (3.5–16)
Allergy, % 092100 0
FEV1, % predicted102 (100–104)98.5 (90–105)76 (67–91)75 (70–78)
PD20FEV1 *      88 (51.5–584)NDND

Definition of abbreviation: ND = not done in patients with FEV1 < 70% of predicted values.

*   Dose of carbachol (μg). Results expressed in medians and 25–75% percentiles in brackets.

Peak flow variability and β2-agonist consumption in patients were carefully monitored in patients with mild intermittent asthma for a period of 2 wk prior to bronchoscopy. Diurnal peak flow variability was always under 10% and β2-agonists were used occasionally (between 0 and 4 times during the previous 2 wk). On the other hand, patients with persistent asthma required β2 agonists daily (0–4) and their daily variation of PEF was ranging from 8 to 42% (median: 15%).

Bronchoalveolar Lavage Cells

With the exception of eosinophils, the number of cells and the differential counts in normal subjects and patients with intermittent and persistent asthma was similar in the three groups (Table 2, Figure 1). The percentage of eosinophils was significantly increased in patients with intermittent asthma and those with mild-to-moderate persistent asthma as compared to normal subjects but there was no significant difference between the two asthmatic groups.

Table 2. RESULTS OF BRONCHOALVEOLAR LAVAGE

Control SubjectsIntermittent AsthmaPersistent Asthmap Value
AtopicNonatopicC/IAC/PA* IA/PA*
BAL cell number    147 (78–178)   151 (85–181)   165 (78–205)    142 (55–201)NSNSNS
BAL eosinophils, %      0 (0–0)     1 (0–2)     1 (1–6.5)         1(0–2.5)0.010.006NS
ECP, μg/L      0 (0–0)     0 (0–2.8)     7 (4.1–22.4)11.4 (4–16.1)0.010.00010.001
Histamine, nM/ml 1.3 (1.1–1.8)0.85 (0.6–6)       1.8 (1.8–2.8) 2.4 (1.2–5.5)NSNSNS
Hyaluronic acid, μg/L      0 (0–10) 0.5 (0–10)    32 (24–37.2)     21 (17–36)NS0.00010.0001
AM fraction 1, %      9 (3.5–14.2)    18 (8.1–31.7)    47 (20.1–58)45.3 (10.6–51.6)0.00020.0003NS
AM fraction 2, % 6.8 (4.2–8.8)    20 (9.7–26.9)11.9 (9–15.1)12.2 (9.4–27.7)0.0010.005NS
AM fraction 3, %22.3 (11–41.2)    16 (8.6–25.8) 9.2 (7.3–17.1)     18 (9–35.4)0.0010.005NS
AM fraction 4, %58.4 (31.2–63.7)    10 (2.1–28.7)     21 (6.3–32.9) 8.7 (2.7–23)0.0040.01NS
AM fraction 5, % 4.2 (2–9.3) 6.3 (0–26.7) 8.3 (2.4–16.2) 4.5 (1.7–9.5)NSNSNS
AM fraction 1 + 2, %   14 (9.5–27.8)47.5 (18.8–72.4)    59 (32.2–74.8)59.3 (32–76)0.00010.0001NS

Definition of abbreviations: C = controls; IA = intermittent asthma; PA = persistent asthma.

*  For statistical analysis patients with atopic and nonatopic persistent asthma were averaged. Results expressed in medians and 25–75% percentiles. Statistical analysis: Mann-Whitney U test with Bonferroni's correction.

Density Fractionation of Alveolar Macrophages

The proportional number of hypodense cells recovered from the first and second fractions of the gradient was significantly greater in patients with either intermittent or persistent asthma than in normal subjects whereas no significant difference was found between the two groups of asthmatic patients (Table and Figure 1). There was no correlation between the severity of asthma assessed by the Aas score or FEV1 values and the numbers of cells recovered from the first two density fractions. The percentage of asthmatic patients with increased hypodense AM (fractions 1 and 2) was similar for intermittent (74% of the patients) and persistent asthma (75%).

Mediator Levels in Bronchoalveolar Lavage Fluid

Results of mediator levels are presented in Figure 1 and Table . ECP levels were detectable in eight of 24 patients with intermittent asthma and in all patients with persistent asthma, but not in the normal subjects. Median ECP levels significantly increased from normal subjects to intermittent asthma and persistent asthma. There was a significant correlation between the severity of asthma assessed by FEV1 and ECP levels in asthmatics (Rho = 0.50, p < 0.002).

The concentration of HA in the BAL fluids was similar in normal subjects and patients with intermittent asthma but increased significantly from intermittent to persistent asthma. Very few patients with intermittent asthma had detectable levels of HA in BAL fluids. There was a significant correlation between the severity of asthma assessed by FEV1 and HA levels in asthmatics (Rho = 0.53, p < 0.001).

Histamine levels were highly variable. They were increased in patients with persistent asthma but not significantly when the Bonferroni's correction was applied (Table ).

Histology of Bronchial Biopsies

Epithelium. There were striking differences in the epithelium between the three groups of subjects (Table 3). Shedding was absent or moderate in most normal subjects whereas all patients with persistent asthma exhibited a severe shedding. Patients with intermittent asthma presented an intermediate shedding pattern (Figure 2).

Table 3. RESULTS OF BRONCHIAL BIOPSIES

Control SubjectsIntermittent AsthmaPersistent Asthmap Value
AtopicNonatopicC/IAC/PA* IA/PA*
Epithelial shedding10 (5-10)   50 (31–80)   100 (95–100)   100 (95–100)0.00010.00010.004
BM size, μm 7 (6-9)9.5 (7-11)10.7 (8–12.5)11.3 (8-15)NS0.025NS
Mast cells in submucosa 8 (4-11)    9 (5-12)     8 (4–11.5) 8.5 (5-12)NSNSNS
EG2 in submucosa 0 (0–0)1.5 (0.5–3.5)     9 (7-21)    15 (10-21)0.0030.0010.001
UCHL1 in submucosa 0 (0–9.5)   30 (20–40)   145 (121–216)   250 (286–369)0.00010.00010.0001

Definition of abbreviations: C = controls; IA = intermittent asthma; PA = persistent asthma; BM = basement membrane.

*   For statistical analysis atopic and nonatopic patients were averaged. Results expressed in medians and 25–75% percentiles. Statistical analysis: Mann-Whitney U test with Bonferroni's correction. Epithelial shedding: % of length of basal cells denudated. Mast cells, EG2 and UCHL1 in submucosa: number of cells/mm2.

Basement membrane. Basement membrane thickness was similar in both asthmatic groups and significantly increased in comparison with control subjects (Table ).

Toluidine blue staining. There was no difference in the number of cells stained by toluidine blue in the three groups (Table ).

Immunohistochemistry of Bronchial Biopsies

There was a trend for an increased number in UCHL-1 positive cells in biopsies of nonatopic patients with persistent asthma by comparison with atopic patients with persistent asthma but the difference was not significant. For both EG2 and UCHL-1 positive cells, there was a significant increase in their numbers in the order: normal < intermittent < persistent (Table , Figure 2). For EG2 immunostaining, there was some extracellular immunostaining observed in samples from three patients with intermittent asthma and in 14 of 18 patients with persistent asthma. There was a significant correlation between the severity of asthma assessed by FEV1 levels and the numbers of UCHL-1- or EG2-positive cell in asthmatics (Rho = 0.53 and 0.50, respectively, p < 0.001 and p < 0.002).

One of the most critical points in asthma research is the characterization of the relationship between the clinical patterns of the disease and the histopathological profiles of the underlying airways inflammation. In this study, we put forward the hypothesis that there might be differences in the inflammatory processes associated with intermittent and persistent asthma. In particular, the aim of this study was to examine in BAL fluids and biopsies the number of cells such as eosinophils, mast cells, T-cells and macrophages which appear to be the most pertinent in asthmatic inflammation and to quantitate their activation products in the BAL fluids. This study is, to our knowledge, the first to examine patients with intermittent asthma (as defined by the WHO/NHLBI report) in detail and to compare the extent of inflammation with that of patients with a persistent form of the disease.

The clinical characterization of asthmatic patients is often difficult, but care was taken to ensure that it was particularly precise in this study. The clinical characteristics of patients with intermittent asthma are as proposed in the WHO/NHLBI document and in particular, the prn β2-agonist consumption and peak flow variability were carefully checked. Patients with intermittent asthma had stable asthma (no circadian variability of PEFR and minimal need for β2-agonists prn) but had a demonstrable airways hyperreactivity using carbachol. None of the patients studied were receiving anti-inflammatory drugs when studied. Patients with persistent asthma had a longer duration of asthma when they are atopic and this result might have been expected since atopic manifestations occur early in life whereas non-atopic asthma is often a feature of adulthood. These age differences are, however, unlikely to cause artefacts in the present study.

The results of this study show a heterogeneous inflammatory process in intermittent asthma, the profile and magnitude of which present both similarities and differences compared with persistent asthma, depending on the cell types and the mediators assessed, as well as the sample (BAL or biopsy) studied.

Eosinophils were found to be increased in numbers in the biopsies and BAL of all patients with persistent asthma. In addition, ECP levels were detectable in the BAL fluids showing that all patients with persistent asthma presented with an eosinophilic inflammation, confirming the results obtained in previous studies using the same methodology (10, 18). On the other hand, while most patients with intermittent asthma had an increased number of eosinophils in BAL, the levels of ECP in BAL fluids were lower than in patients with persistent asthma. In addition, in bronchial biopsies the numbers of EG2-positive cells in biopsies and the magnitude of degranulation was always reduced in comparison with patients with persistent asthma. The presence of an eosinophilic inflammation seems to be of importance since this feature is inconsistently observed in non-asthmatic atopic patients (6, 21, 22) and absent in patients with chronic cough (23). Furthermore, these results indicate that although eosinophils are recruited in intermittent asthma, they are less activated than in persistent asthma. The different patterns of eosinophilic activation found in persistent as compared with intermittent asthma, might be important consequences on the integrity of the bronchial mucosa.

Epithelial shedding was greater in patients with persistent asthma than in those with intermittent asthma, suggesting a possible relationship between eosinophilic activation and epithelium damage. Epithelial cells are often shed even in mild forms of the disease (15, 24) and the degree of bronchial hyperreactivity was found to correlate with the denudation of the epithelial layer (15). However, in patients with a mild form of the disease it is not clear whether mechanical denudation of the epithelium is an artefact of the biopsy procedure itself since this has been observed in normal subjects (25). In this study, patients with intermittent asthma exhibited significantly more shedding than normal subjects but significantly less shedding than patients with persistent asthma and these findings accord with previous data on mild asthmatics (26). However, we were unable to determine in these subjects whether epithelial loss was partly due to the biopsy procedure or was occurring in vivo (5, 15). Furthermore, we did not find a significant correlation between the epithelium shedding and the clinical severity of asthma or the bronchial hyperreactivity, probably because most patients analyzed had a mild form of the disease and we did not include normal subjects in the analysis.

In the present study we have also found the presence of a subgroup of primed T-cells (CD45RO+ cells) in biopsies in almost all patients with persistent asthma but only in 42% of patients with intermittent asthma. Even in mild asthma, T-cells have been found in increased numbers (22) and in an activated state in the airways (27). However, since we did not study other T-cell markers or the cytokines released by these cells, it is not possible to determine whether or not their state of activation differs between the groups of patients studied. The increased number of primed T-cells in the mucosa of patients with persistent asthma might play a role in the development of airway inflammation, particularly with regard to eosinophilic inflammation. T-cells are involved in the regulation of eosinophilopoiesis through the secretion of cytokines. Thus, the increased eosinophilic inflammation characterizing persistent asthma might be linked to the presence of increased numbers of primed T-lymphocytes in the bronchial mucosa. The relationships between atopy and asthma are not clearly defined at the bronchial level. Most of the studies failed to find a different pattern of airways inflammation (10, 11, 17, 18). Moreover, in the present study, although UCHL1-positive cells were in greater numbers in non-atopic patients, we did not find any significant difference in the inflammatory markers assessed in mucosal biopsies and BAL fluids obtained from atopic or non-atopic patients.

We also studied the activation of alveolar macrophages in BAL fluids by assessing their cellular density (17). We observed that most patients with persistent or intermittent asthma had an increase in hypodense macrophages and we failed to find any significant difference between these two groups. This may be due, as also occurs after allergen challenge (28-30), to the rapid activation of alveolar macrophages even in the mildest forms of the disease. These data for BAL fluid together with those showing increased numbers in biopsies from patients with persistent or even mild asthma (14, 31) indicate that macrophages are important cells in the pathogenesis of asthma and that they are characterized by a high state of activation after the early phases of the clinical evolution of the disease.

In addition to eosinophils, lymphocytes and macrophages, we also studied mast cells since in asthma their number and their state of activation is increased (5, 9, 13, 32, 33). Moreover, the degree of bronchial hyperreactivity was found to correlate with mast cell products (7, 22, 33). Our results show that in patients with intermittent asthma mast cells may not be important since their numbers in biopsies and histamine levels in the BAL fluids are not increased. Taken together these data do not highlight an important role of mast cells in stable intermittent asthma. Mast cell involvement might have been observed if tryptase had been measured or if immunohistochemistry using an anti-tryptase antibody had been studied. However, since in other studies mast cells involvement has been evaluated using the same procedures, we feel that the results are of value and sufficiently clear.

Since airway inflammation is often associated to histopathological features of airway remodelling, we also evaluated in this study the thickness of the basement membrane as well as the turnover of the ECM by measuring the level of HA. We also examined a relationship between the increased thickness of the basement membrane and the clinical severity of asthma. In this study, we found that the basement membrane size was significantly increased in patients with intermittent or persistent asthma in comparison with control subjects, but there was no difference between the two asthmatic groups. These data suggest that airways remodeling occurs even in patients suffering from very mild forms of the disease and that such a structural alteration is present also in the early phases of airway inflammation in asthma. Finally in BAL fluids, HA was only found to be increased in patients with persistent asthma. The significance of this finding is not clear since HA is released by fibroblasts and this increase may therefore be considered as an hyperreactive state of these cells. It may however be considered that increased HA levels in persistent asthma are related to tissue damage and extracellular matrix remodelling. The presence of HA may be considered as a marker for an initial phase of the extracellular matrix remodeling following tissue damage. HA can contribute to repair processes by facilitating cell migration and the stimulation of collagen production (34). Thus, the increased levels of HA measured in patients with persistent asthma may suggest that airway remodelling processes are more active than in intermittent asthma. This is supported by previous evidence showing that CT-scan abnormalities due to airway remodelling such as bronchiectasis, bronchial wall thickening, sequellar line shadows and emphysema were significantly more prevalent in severe asthma than in mild asthma. Most of these abnormalities were likely to be related to bronchial destruction (35).

Taken altogether the results of the present study confirm that airways inflammation occurs at a very early stage of the disease even in patients who present with intermittent asthma. However, in these patients inflammation is found at a lower grade than in patients with persistent asthma.

Supported by INSERM (France) and CNR (Italy).

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Correspondence and requests for reprints should be addressed to Jean Bousquet, M.D., Hôpital Arnaud de Villeneuve, 34295-Montpellier-Cedex 5, France. E-mail:

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