Abstract
To investigate the relationship between airflow limitation and airway inflammation in smokers, we examined paraffin-embedded bronchial biopsies obtained from 30 smokers: 10 with severe airflow limitation, eight with mild/moderate airflow limitation, and 12 control smokers with normal lung function. Histochemical and immunohistochemical methods were performed to assess the number of inflammatory cells in the subepithelium and the expression of CC chemokines macrophage inflammatory protein (MIP)-1 α and -1 β in the bronchial mucosa. Compared with control smokers, smokers with severe airflow limitation had an increased number of neutrophils (p < 0.02), macrophages (p < 0.03), and NK lymphocytes (p < 0.03) in the subepithelium, and an increased number of MIP-1 α+ epithelial cells (p < 0.02). When all smokers were considered together, the value of FEV1 was inversely correlated with the number of neutrophils (r = − 0.59, p < 0.002), macrophages (r = − 047, p < 0.012), NK-lymphocytes (r = − 0.51, p < 0.006) in the subepithelium, and with the number of MIP-1 α+ epithelial cells (r = − 0.61, p < 0.003). We conclude that in smokers the severity of airflow limitation is correlated with the severity of airway inflammation and that severe airflow limitation is associated with an increased number of neutrophils, macrophages, NK lymphocytes, and MIP-1 α+ cells in the bronchial mucosa.
Cigarette smoking is the major risk factor for the development of chronic obstructive pulmonary disease (COPD). The reason why only 15 to 20% of heavy smokers develop chronic airflow limitation (1, 2) is still unknown. The concept that the site responsible for this airflow limitation is the peripheral airways is well established (3, 4); however, several studies have shown that the large airways are also affected by an inflammatory process in smokers (5-15). We have recently shown that this process is different in smokers who develop chronic airflow limitation and in smokers who do not develop chronic airflow limitation, showing an increased number of T-lymphocytes and macrophages in the former (8). However, the majority of smokers examined in our previous study had a mild degree of airflow limitation, and, to the best of our knowledge, studies of bronchial biopsies have not yet been performed in smokers with severe airflow limitation. Thus, in this report, we aimed to characterize airway inflammation in smokers with a wide range of airflow limitation from none to severe, and to investigate the possible relationship between the severity of airflow limitation and the severity of airway inflammation.
Bronchial biopsies were obtained from 30 smokers whose FEV1 ranged from 14 to 140% predicted. Histochemical and immunohistochemical methods were performed to assess the number of inflammatory cells in the bronchial mucosa and to investigate the associations between distinct types of inflammatory cells and lung function. Because MIP-1α and -1β are cytokines involved in chemotaxis and activation of inflammatory cells (16), the expression of these proteins was assessed in the bronchial mucosa.
We examined 30 smokers in whom the severity of airflow limitation was staged using the criteria of the American Thoracic Society (17) modified according to the ERS-Consensus statement (18): 10 smokers had severe airflow limitation (severe COPD) (FEV1 less than 50% predicted), eight had mild/moderate airflow limitation (mild/moderate COPD) (FEV1 ranged between 50 and 79% predicted), and 12 had normal lung function (control smokers) (FEV1 more than 80% predicted). All subjects with COPD had symptoms of chronic bronchitis. They did not have exacerbations, defined as increased dyspnea associated with a change in quality and quantity of sputum, that would have led them to seek medical attention (19, 20) during the month preceding the study. All subjects had been free of acute upper respiratory tract infections and none had received glucocorticoids or antibiotics within the preceding month. The subjects were nonatopic (i.e., they had negative skin tests for common allegen extracts) and had no past history of asthma or allergic rhinitis.
Each subject underwent interview, chest radiography, ECG, routine blood test, skin tests with common allergen extracts, and pulmonary function tests between 2 and 5 d before bronchoscopy. The study conformed to the Declaration of Helsinki, and informed written consent was obtained from each subject.
Pulmonary function tests included measurements of FEV1 and FEV1/ VC under baseline conditions in all the subjects examined (6200 Autobox Pulmonary Function Laboratory; Sensormedics Corp., Yorba Linda, CA). The predicted normal values used were those from Communitè Europeennè du Carbon et de l'Acier (CECA) (21). In order to assess the reversibility of airway obstruction, the FEV1 measurement in the group of subject with FEV1 ⩽ 80% was repeated 20 min after the inhalation of 0.2 mg of salbutamol. Measurements of residual volume (RV) were performed by the plethysmographic method. Subjects number 1, 2, 11, 12, 19, and 21 (see Table 1) refused to perform plethysmography.
| Age (yr) | Sex (M/F ) | PaO2 (mm Hg) | PaCO2 (mm Hg) | Smoke (pack/yr) | FEV1(% pred ) | FEV1/VC (%) | RV (%) | FEV1 after Salbutamol | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Subject No. | (% base) | |||||||||||||||||
| Severe COPD | ||||||||||||||||||
| 1 | 65 | M | 63 | 91 | 40 | 27 | 41 | — | 4 | |||||||||
| 2 | 49 | M | 71 | 41 | 50 | 14 | 38 | — | 1 | |||||||||
| 3 | 63 | M | 67 | 44 | 22 | 24 | 34 | 173 | 0 | |||||||||
| 4 | 62 | M | 66 | 42 | 47 | 40 | 70 | 201 | 3 | |||||||||
| 5 | 49 | F | 74 | 40 | 30 | 45 | 54 | 157 | 1 | |||||||||
| 6 | 80 | M | 70 | 45 | 10 | 28 | 52 | 180 | 0 | |||||||||
| 7 | 73 | M | 73 | 40 | 75 | 40 | 51 | 167 | 11 | |||||||||
| 8 | 57 | M | 68 | 46 | 37 | 33 | 43 | 163 | 9 | |||||||||
| 9 | 61 | M | 87 | 43 | 80 | 28 | 52 | 259 | 6 | |||||||||
| 10 | 65 | M | 66 | 44 | 40 | 43 | 68 | 294 | 8 | |||||||||
| Mean | 62 | 70 | 48 | 43 | 32†,‡ | 50†,‡ | 199†,‡ | 4 | ||||||||||
| SEM | 3 | 2 | 5 | 7 | 3 | 4 | 18 | 1 | ||||||||||
| Mild/moderate COPD | ||||||||||||||||||
| 11 | 71 | M | 66 | 41 | 25 | 77 | 59 | — | 3 | |||||||||
| 12 | 58 | M | 72 | 36 | 38 | 76 | 75 | — | 2 | |||||||||
| 13 | 70 | F | 69 | 44 | 15 | 61 | 70 | 105 | 5 | |||||||||
| 14 | 62 | M | 77 | 39 | 35 | 65 | 58 | 95 | 3 | |||||||||
| 15 | 65 | M | 60 | 33 | 50 | 58 | 56 | 114 | 0 | |||||||||
| 16 | 63 | F | 94 | 38 | 30 | 63 | 66 | 155 | 9 | |||||||||
| 17 | 69 | M | 81 | 38 | 60 | 79 | 75 | 130 | 3 | |||||||||
| 18 | 70 | M | 84 | 37 | 60 | 66 | 62 | 159 | 4 | |||||||||
| Mean | 66 | 75 | 38 | 39 | 68† | 65† | 126 | 4 | ||||||||||
| SEM | 2 | 4 | 1 | 6 | 3 | 3 | 11 | 1 | ||||||||||
| Control smokers | ||||||||||||||||||
| 19 | 58 | F | 69 | 43 | 24 | 126 | 99 | — | — | |||||||||
| 20 | 67 | M | 70 | 37 | 100 | 112 | 81 | 112 | — | |||||||||
| 21 | 55 | M | 81 | 40 | 80 | 102 | 76 | — | — | |||||||||
| 22 | 54 | M | 83 | 40 | 45 | 105 | 73 | 180 | — | |||||||||
| 23 | 61 | M | 77 | 39 | 40 | 99 | 81 | 110 | — | |||||||||
| 24 | 63 | F | 71 | 41 | 42 | 102 | 76 | 127 | — | |||||||||
| 25 | 51 | F | 75 | 41 | 15 | 140 | 78 | 75 | — | |||||||||
| 26 | 53 | M | 89 | 39 | 50 | 112 | 85 | 64 | — | |||||||||
| 27 | 67 | M | 84 | 38 | 19 | 101 | 81 | 160 | — | |||||||||
| 28 | 59 | M | 76 | 39 | 22 | 121 | 87 | 110 | — | |||||||||
| 29 | 54 | M | 68 | 46 | 40 | 83 | 78 | 88 | — | |||||||||
| 30 | 67 | M | 74 | 40 | 35 | 112 | 81 | 105 | — | |||||||||
| Mean | 59 | 76 | 40 | 43 | 110 | 81 | 113 | — | ||||||||||
| SEM | 2 | 2 | 1 | 7 | 4 | 2 | 11 | — |
All subjects were premedicated intramuscularly with atropine (0.5 mg) and diazepam (10 mg) and orally with dihydrocodein (10 mg). Nares and oropharinx were anesthetized topically with 10% lidocaine before bronchoscopy. Bronchoscopy was performed with a flexible fiberoptic bronchoscope (Pentax FB-18P; Asahi Optical Co. LTD, Tokyo, Japan) in all subjects. Bronchial biopsies were taken through the bronchoscope with standard forceps from the subcarina of a basal segment bronchus of the right lower lobe. From this area two specimens were obtained in each subject.
Biopsy specimens were gently extracted from the forceps and processed for light microscopy as previously described (8). Briefly, samples were fixed in 4% formaldehyde for 4 h and embedded in paraffin. The best specimen was then oriented and serial sections 4 μm thick were cut. Two sections at an interval of 100 μm were then appropriately stained with histochemical or immunohistochemical methods. Hematoxylin-eosin was used to identify eosinophils, and the following panel of antibodies was used to identify inflammatory cells and MIP-1α and -1β chemokines: antihuman neutrophil elastase (M752; Dako Ltd., Carpenteria, CA) for neutrophils, anti-CD3 antigen (A452; Dako) for total T-lymphocytes, anti-CD4 antigen (M834; Dako) for CD4+ T-lymphocytes, anti-CD8 antigen (M7103; Dako) for CD8+ T-lymphocytes, anti-tryptase (M7052; Dako) for mast cells, anti-CD68 antigen (M814; Dako) for macrophages, anti-NK1 (anti-Leu-7, CD57) antigen (M1014; Dako) for natural killer (NK) lymphocytes, and anti-Macrophage Inflammatory Protein (MIP)-1α and -1β for cells expressing the CC-chemokines MIP-1α and -1β, respectively. The rabbit polyclonal antibodies for identification of MIP-1α and -1β chemokines were kindly provided by Dr. L. Mills (NIH, Bethesda, MD). Sections were pretreated in a microwave oven (20 min) before incubation with anti-CD8 antigen and with 1% trypsin (10 min) before incubation with anti-CD3, -tryptase, and -CD68 antigens. Primary antibodies were revealed as previously described (6, 8). Briefly, antibody binding was demonstrated with the use of an alkaline phosphatase antialkaline phosphatase system (Dako APAAP kit system, K699; Dako) and fast-red substrate. Control slides were included in each staining run using human tonsil as a positive control for immunostaining of inflammatory cells. Cytospin preparations of LPS-stimulated mononuclear cells were used as a positive control in each staining run for immunostaining of MIP-1α and -1β (22) (Figure 4A). Negative control slides included human tonsil immunostained with mouse monoclonal IgG 2α (X943; Dako) or cytospin preparations of unstimulated mononuclear cells immunostained with rabbit anti-MIP-1α and -1β polyclonal antibodies (Figure 4B) or rabbit immunoglobulins from normal rabbit serum.
Fig. 4. (Panel A) Cytospin preparation of LPS-stimulated blood mononuclear cells immunostained for MIP-1α. Arrows indicate MIP-1α-positive cells. Original magnification: ×450. (Panel B) Cytospin preparation of nonstimulated blood mononuclear cells immunostained for MIP-1α. Original magnification: ×450.
[More] [Minimize]Inflammatory cells and cells expressing the CC chemokines MIP-1α and MIP-1β were quantified in the area 100 μm beneath the epithelial basement membrane in several nonoverlapping high power fields until all the available area was covered. The final result, expressed as the number of positive cells per square millimeter, was calculated as the average of all the cellular counts performed in both slides of each biopsy. In the well preserved epithelium, defined by the presence of both basal and columnar cells, the immunoreactivity for MIP-1α and -1β was quantified in basal and columnar epithelial cells, and the final result was expressed as number of basal, columnar, and total (obtained by the sum of basal and columnar) positive cells per millimeter of epithelial length. Light-microscopic analysis was performed at a magnification ×900 for quantification of inflammatory cells and chemokines in the subepithelium. Morphometric measurements of the well-preserved epithelium were performed at a magnification ×400 with a light microscope (Leitz Biomed; Leica Cambridge, UK) connected to a video recorder linked to a computerized image system (Quantimet 500 Image Processing and Analysis System, Software Qwin V0200B; Leica).
Group data were expressed as mean ± standard error, or as median and range when appropriate. Differences between groups were analyzed using analysis of variance (ANOVA) for functional data, and the Kruskal-Wallis test for morphologic and morphometric data. When the differences were significant, the Kruskal-Wallis test was followed by the Mann-Whitney U test for comparison between groups. Correlation coefficients were calculated using Spearman's rank method. Probability values of p < 0.05 were considered as significant. The coefficient of repeatability, as described by Bland and Altman (23), was used to compare measurements performed on the two sections of the same biopsy. At least three replicate measurements of morphometric parameters were performed by the same observer (ADS), and the intraobserver reproducibility was assessed with the coefficient of variation (CV) for repeated measurements. The mean CV for three repeated measurements performed by the same observer ranged from 4 to 12% for the inflammatory cells and chemokine-positive cells studied. The coefficients of repeatability for measurements performed on the two sections of each biopsy were 40, 33, 35, 18, 68, 60, 11, 10, 18, and 15 cells/mm2 for CD3, CD4, CD8, mast cells, neutrophils, macrophages (CD68), NK lymphocytes, eosinophils, MIP-1α, and MIP-1β positive cells, respectively; and 40, 22, 17, and 41, 29 and 16 cells/mm for MIP-1α+ and MIP-1β+ total, basal, and columnar epithelial cells, respectively.
The characteristics of subjects are reported in Table 1. The three groups of subjects examined were similar with regard to age, sex, and smoking history. As expected from the selection criteria, the values of FEV1 (% predicted) and FEV1/VC (%) were significantly different in the three groups of smokers examined. Residual Volume (RV%) was significantly higher in subjects with severe COPD when compared with both subjects with mild/moderate COPD and control smokers. The mean response to bronchodilator was 4 ± 1% baseline in both severe and mild/moderate COPD.
Bronchoscopy with endobronchial biopsy was performed successfully and was well tolerated by all subjects. In Subjects 6, 10, 15, 26 and 28 in Table 1, morphometric analysis of epithelium could not be performed because of complete epithelial denudation.
Subepithelium. In the subepithelium, subjects with severe COPD had a greater number of neutrophils, macrophages, and NK lymphocytes than did control smokers (Table 2 and Figures 1 and 5A). The number of neutrophils was also significantly higher in mild/moderate COPD than in control smokers while the number of macrophages and NK-lymphocytes did not differ significantly between these two groups (Figure 1). The number of neutrophils, macrophages, and NK-lymphocytes was not significantly different when comparing severe with mild/moderate COPD. The number of CD3, CD4 and CD8 T-lymphocytes, mast cells, eosinophils as well as the number of MIP-1α and -1β+ cells were not significantly different in the three groups of smokers examined (Table 2).
| Severe COPD | Mild/ Moderate COPD | Control Smokers | p Value (Kruskal–Wallis) | |||||
|---|---|---|---|---|---|---|---|---|
| Subepithelium, cells/mm2 | ||||||||
| CD3 | 159 (19–496) | 83 (20–274) | 198 (46–399) | 0.17 | ||||
| CD4 | 98 (20–398) | 104 (17–223) | 72 (36–157) | 0.90 | ||||
| CD8 | 65 (0–183) | 50 (9–211) | 74 (11–216) | 0.75 | ||||
| Mast cells | 108 (6–193) | 41 (8–112) | 67 (5–150) | 0.19 | ||||
| Neutrophils | 144 (61–807) | 115 (73–428) | 60 (26–166) | 0.017 | ||||
| Macrophages | 172 (49–479) | 74 (47–227) | 63 (25–259) | 0.044 | ||||
| NK lymphocytes | 51 (4–131) | 17 (7–43) | 16 (3–32) | 0.049 | ||||
| Eosinophils | 8 (0–29) | 0 (0–20) | 4 (0–20) | 0.09 | ||||
| MIP–1α | 67 (32–199) | 74 (23–127) | 44 (10–107) | 0.17 | ||||
| MIP–1β | 45 (23–83) | 45 (12–80) | 43 (20–76) | 0.97 | ||||
| Epithelium, cells/mm | ||||||||
| MIP–1α+ total | 172 (117–235) | 116 (77–167) | 99 (46–174) | 0.014 | ||||
| MIP–1α+ basal | 106 (68–180) | 80 (50–109) | 64 (26–144) | 0.068 | ||||
| MIP–1α+ columnar | 65 (27–109) | 39 (14–87) | 29 (9–66) | 0.032 | ||||
| MIP–1β+ total | 189 (72–242) | 168 (37–227) | 122 (66–241) | 0.42 | ||||
| MIP–1β+ basal | 132 (62–204) | 126 (32–160) | 88 (48–183) | 0.45 | ||||
| MIP–1β+ columnar | 52 (10–95) | 37 (5–93) | 38 (15–76) | 0.46 |
Fig. 1. Individual counts for neutrophils, macrophages, and NK lymphocytes in the subepithelium of subjects with severe COPD, mild/moderate COPD, and control smokers. The results are expressed as the number of positive cells per mm2 of subepithelium. Horizontal bars represent median values.
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Fig. 5. (Panel A) Photomicrography showing the subepithelium of a smoker with severe airflow limitation immunostained for NK lymphocytes (arrows). Original magnification: ×650. (Panel B) Photomicrography showing the bronchial epithelium of a smoker with severe airflow limitation immunostained for MIP-1α. Original magnification: ×650.
[More] [Minimize]Epithelium. In the epithelium, the number of MIP-1α+ epithelial cells was significantly greater in severe COPD as compared to both control smokers and mild/moderate COPD (Table 2 and Figures 2 and 5B). No statistical difference was observed in the number of MIP-1α+ epithelial cells between subjects with mild/moderate COPD and control smokers (Table 2 and Figure 2). When basal and columnar epithelial cells were considered separately, the number of MIP-1α+ basal and columnar cells in subjects with severe COPD was significantly greater than in control smokers, but they did not differ from subjects with mild/moderate COPD (Figure 2). No statistical differences were observed in the number of MIP-1α+ basal and columnar epithelial cells between subjects with mild/moderate COPD and control smokers (Table 2 and Figure 2). The number of total, basal and columnar epithelial cells positively stained for MIP-1β was similar in the three groups of smokers examined (Table 2).
Fig. 2. Individual values for MIP-1α immunoreactivity of total, basal, and columnar epithelial cells in subjects with severe COPD, mild/moderate COPD, and control smokers. The results are expressed as the number of positive cells per mm of epithelium. Horizontal bars represent median values.
[More] [Minimize]Correlations. When all subjects were considered together (n = 30), the number of neutrophils, macrophages, NK-lymphocytes in the subepithelium and the number of MIP-1α+ total epithelial cells (n = 25) were inversely correlated with the FEV1 values (Figure 3). When control smokers were excluded from the analysis, these correlations were maintained (n = 18 or n = 15 for epithelial cells) (neutrophils: r = −0.55, p < 0.025; macrophages: r = −0.56,: p < 0.025; NK-lymphocytes r = −0.46, p < 0.05; MIP-1α+ total epithelial cells: r = −0.68, p < 0.015). Furthermore, the number of MIP-1α+ total epithelial cells was positively correlated with the number of macrophages (r = 0.42, p < 0.04), neutrophils (r = 0.66, p < 0.0015), and NK lymphocytes (r = 0.51, p < 0.015) in the subepithelium.
Fig. 3. Regression analysis between FEV1% predicted values and numbers of neutrophils, macrophages, NK lymphocytes, and MIP-1α+ epithelial cells in the bronchial mucosa of subjects with severe COPD, mild/moderate COPD, and control smokers (Spearman's rank correlation).
[More] [Minimize]This study has shown that the severity of airflow limitation is correlated with the severity of airway inflammation in smokers. In addition, severe airflow limitation is associated with an increased number of neutrophils, macrophages, NK lymphocytes, and MIP-1α+ epithelial cells in the bronchial mucosa.
We took advantage of the presence of a wide range of airflow limitations in the population of smokers examined to investigate possible correlations between clinical and cellular parameters. Although we are well aware that correlations do not imply cause-effect relationship (24), we believe that the significant correlations observed in the overall population of smokers between the severity of airflow limitation and the number of neutrophils, macrophages, NK lymphocytes, and MIP-1α+ epithelial cells support a possible role for these cells and chemokines in the pathogenesis of COPD.
At variance with FEV1 values, no correlations between the number of inflammatory cells and lung hyperinflation (RV values) were observed. These results suggest that cellular infiltrate found in the upper airways may not be associated with lung hyperinflation caused by emphysema.
Caution must be exercised with regard to the implication of this study. The data presented do not raise doubt that the site responsible for airflow limitation in smokers is the peripheral airways (3), but they show that the large airways are also affected by an inflammatory process, and that the severity of this inflammatory process increases with the severity of airflow limitation.
The finding of an increased number of macrophages in the subepithelium of smokers with severe airflow limitation confirms and extends previous findings (5, 7-9, 11) by showing that the presence of this cell is a characteristic feature not only in mild but also in severe COPD. Along with the increase in the number of macrophages, we found an increased number of NK lymphocytes in smokers with severe COPD. NK lymphocytes are a distinct population of large-granular lymphocytes with specialized cytotoxic functions that act as a first line of defense against transformed or virus-infected cells (25, 26). In the present study the increased number of NK lymphocytes in the subepithelium was associated with an increased epithelial expression of MIP-1α. It is possible that an excessive recruitment of NK lymphocytes, upregulated by MIP-1α, may occur in response to repeated bouts of viral or bacterial infections (27), which are a frequent occurrence in smokers with COPD. The correlation observed in the present study between MIP-1α positive epithelial cells and inflammatory cells is also supported by in vitro studies demonstrating CC chemokine-induced migration and activation of mononuclear cells and granulocytes (16, 28, 29).
Previous studies reported a predominance of neutrophils in the airway lumen but not in the subepithelium of subjects with mild airflow limitation (20, 30, 31). The present study shows that, as the severity of airflow limitation increases, the number of neutrophils in the subepithelium also increases, suggesting a role for this cell in the progression of the disease. This hypothesis is supported by the recent observation that a prominent neutrophilia is present in the bronchial glands and in the bronchial epithelium of smokers who develop chronic airflow obstruction as compared with smokers with normal lung function (13, 32).
An increased number of neutrophils in the submucosa has also been reported during exacerbations of chronic bronchitis (19). In our study, we carefully selected patients who were in a stable condition by excluding subjects with an exacerbation of the disease, though the presence of subclinical viral infections could not be excluded. The view that our patients with COPD were in a stable condition is supported by the fact that the number of eosinophils in the subepithelium lay in the same range as that of the control smokers at variance with chronic bronchitics during exacerbations, where the number of eosinophils was 30-fold that of chronic bronchitics in baseline conditions (19). These observations, together with our findings of a neutrophilia in a stable condition, support a role for these cells when COPD becomes severe.
There are interesting recent observations by Wenzel and colleagues (33) of possible relevance to our present report. These investigators consistently showed significantly higher numbers of neutrophils in the airways of patients with severe asthma when compared with those with mild asthma and with normal control subjects. These findings, even though probably biased by the glucocorticoid treatment of patients with severe asthma, invite speculation that in asthma, as in COPD, when the disease becomes severe, a prominent airway neutrophilia is present. The precise role of neutrophils in the development of the structural changes characteristic of the two well distinguished diseases still remains to be investigated.
The fact that our study had a relatively small number of subjects while featuring a relatively large number of comparisons raises the risk of statistical artifacts. Therefore, even though statistical significances are sustained by tests for nonparametric data, our results need to be confirmed in a wider population of smokers.
We conclude that in smokers the severity of airflow limitation is correlated with the severity of airway inflammation, and that severe airflow limitation is associated with an increased number of neutrophils, macrophages, NK lymphocytes, and MIP-1α+ cells in the bronchial mucosa.
The writers thank Mrs. Rosemary Allpress and Nadia Novello for revising and typing the manuscript, and Isabella Gnemmi, Mariella Colombo, Massimo Sacchi, and Ada Patriarca for technical assistance.
Supported by Salvatore Maugeri Foundation, IRCCS, “Ricerca Corrente” Grant. Grant ENFUMOSA on Severe Asthma (Contract BMH4-CT96-1471), and a Special Grant to L.M.F. from the Arcispedale Sant'Anna, Ferrara.
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