Original Paper Int Arch Allergy Immunol 1992;98:233-238
a Department of Clinical Chemistry, University Hospital, Uppsala; b Department of Human Pharmacology, Astra Draco AB, Lund, Sweden
Key Words Chemotaxis Allergy Human Eosinophils Lung Allergic alveolitis
Increased Eosinophil Chemotactic Activity in Bronchial Washes Obtained from Patients with Asymptomatic Allergic Alveolitis
Abstract The chemotactic activities of eosinophils and neutrophils were measured in bronchial washes obtained from 6 symptom-free patients with previous extrin sic allergic alveolitis and 9 healthy volunteers. An increased chemotactic activ ity was found in the washes obtained from the patients, and the recovery of eo sinophils was correlated with the eosinophil chemotactic activity, suggesting a causal relationship. Chromatography of the fluids obtained suggested that low molecular weight compounds were principally responsible for the increased chemotactic activity. A mild disturbance of the integrity of the alveolocapillary barrier could to some extent add to the explanation of the increased chemo tactic activity in the washes obtained from the patients.
Introduction Allergic alveolitis is a hypersensitivity pneumonitis in which sensitized subjects develop a hypersensitivity re sponse. The acute form of the disease is characterized by respiratory and systemic symptoms, occuring 6-12 h after antigen exposure, while the chronic form of the disease has an insidious onset of symptoms. Exposed, but asymp tomatic subjects may have increased circulating antibody levels [1] and lymphocytic alveolitis as signs of cell-medi ated response [2]. Although the mediators responsible for the genesis of the clinical disease are unknown, the pres ence of suppressor T cells and granulocyte chemotactic factors in lavage fluid [3] may suggest a causal involvement of these cells in the disease process.
We have previously found signs of increased eosinophil involvement, as reflected by lavage eosinophilia, in asymp tomatic patients who previously had suffered from extrin sic allergic alveolitis [4], Production of chemotactic factors that attract cells is considered to be one essential mecha nism for the accumulation of inflammatory cells. In the present study, we intended to elucidate further the eosi nophil involvement by assessing the eosinophil chemotac tic activity in bronchial washes (BWs) obtained from these asymptomatic subjects. We choose to assess the chemo tactic activity in the BWs and not in the bronchoalveolar lavages (BALs), since the dilution of the epithelial lining fluid, by the injected saline, was larger and tended to vary more in the BALs as compared with the BWs.
Correspondence to: Dr. Birgitta Schmekel Department o f Human Pharmacology Astra Draco AB, Box 34 S—211 00 Lund (Sweden)
© 1992 S. Karger AG, Base! 1018-2438/92/0983-0227 $ 2.75/0
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 11:11:16 PM
Lena H3 leansson* Birgitta Schmekel* - b
Dwell time, min Recovery volume, ml Total cell count, x 10'1
Patients (n = 6)
Controls (n = 9)
median
range
median
range
1.4 17.5 3.0
1.1-1.8 13-25 1.4-6.13
1.7 14 2.6
1.1-2.7 5-20 0.97-4.1
Materials and Methods Subjects Six nonsmoking patients with a history of extrinsic allergic alveo litis were studied. Their mean age was 48.2 years (range 39-56 years), they had all been exposed to either bird proteins or Aspergillus fumigatus, and they had developed typical symptoms and signs of extrinsic allergic alveolitis. The diagnosis had been established 2-15 years be fore this study based on a typical case history, chest radiographic findings consistent with the disease, and presence of precipitating an tibodies in peripheral blood. The antigens were eliminated in all cases as soon as the diagnosis was established. At the time of the study all patients were free of symptoms since more than 1 year, they received no medication, and physical examination as well as chest radiographs were normal. Nine healthy male volunteers with a mean age of 37.1 years (range 29-48 years) served as controls. None had ever smoked tobacco or had any history of allergy or lung disease. Chest radiographs and lung volumes were normal. Informed consent was obtained from patients and volunteers, and the study was approved by the Ethical Research Committee and the Faculty of Medicine, University of Lund. Bronchoalveolar Lavage After premedication with morphine and scopolamine and top ically applied lidocaine (Xylocaine®; Astra, Sweden), the fiberoptic bronchoscope (Olympus BF 1 T 10) was introduced via the nasal route into the lower airways. BW and BAL were performed as previ ously documented in detail [4], An initial BW of 50 ml was infused and immediately aspirated, followed by two 50-ml aliquots constitut ing BAL. Results on BAL are not reported here, due to the fact that there is a higher degree of dilution of the epithelial lining fluid by sa line in the larger BALs, and this affects the possibilities to measure chemotactic activités with a high precision. The dwell time of the wash fluids was defined by the time passed between the start of in fusion of the fluid and the end of the aspiration. The aspirated wash fluids were collected in siliconized glass containers kept on ice and centrifuged at 4°C at 200 g for 10 min. The cell pellets were then re suspended in RPMI 1640 (Gibco, UK) supplemented with 5% fetal calf serum and gentamicin (50 pg/ml) and transported to the lab oratory for differential counts. The cell-free supernatants were kept frozen at -70 °C until the analyzes were performed.
234
Hâkansson/Schmekcl
Gel Filtration o f BWs Gel filtration of BWs was performed on Sephacryl S-200 Super fine (Pharmacia LKB Biotechnology, Uppsala, Sweden) at 4 °C. Two milliliters of freshly frozen BW was applied to the column (2.6x90 cm). Gey’s solution with 0.1 mol/1 c-amino-caproic acid per liter was used as eluent. The elution rate was 10 ml/h and the fraction volume 2.5 ml. Measurement o f Chemotactic Activity in BWs The migration of eosinophils and neutrophils was measured using a modified Boyden chamber technique as previously described [5], Granulocytes were isolated from apparently healthy blood donors by dextran sedimentation [6] and diluted to a concentration of 1.5 x 109/1 in Gey’s solution with human serum albumin (2 g/1). The samples of BWs were diluted 1:4, 1:8, and 1:16. The migration of eosinophils and neutrophils towards the diluted BW samples was measured ac cording to the leading-front method as previously described [7], The chemotactic activity (units) of the samples was calculated as the maxi mal migration (pm/h) exceeding that towards Gey’s buffer, multi plied by the actual dilution of the sample. Measurement o f Chemotactic Activity in Sephacryl S-200 Fractions The chemotactic activity in fractions of BWs separated on Sepha cryl was measured according to the same principle as described for BWs. The fraction samples were diluted 1:3,1:6, and 1:12 if needed. The chemotactic activity was measured in every third fraction. Measurements o f Soluble Factors in BWs The concentrations of eosinophil cationic protein (ECP), and myeloperoxidase (MPO) were measured by means of radioimmu noassay [8,9]. The concentrations of albumin and urea were analyzed according to previously published methods [10,11], The total amounts of the substances were calculated by multiplying the concentration and the volume of the aspirated wash fluid. Analyses o f Cellular Composition in BWs Differential counts were performed on cytocentrifuge prepara tions (Cytospin 2; Shandon, UK) at 500 rpm for 10 min. The prep arations were stained with May-Griinwald-Giemsa, and differential counts were done by counting 400 cells on two separate cytospin preparations. Statistical Analysis Data are expressed as median values and ranges. The Wilcoxon rank sum test was used to evaluate differences between groups of pa rameters and the Spearman rank correlation test to evaluate correla tions between parameters, p values < 0.05 were considered significant.
Results Recovery volumes, dwell times, and total cell recovery of the BW fluid were similar in patients and controls (table 1). The variability in cell recovery was large, whether the differentials were expressed as percentage counts or as to tal number of the cells (table 2). Significantly increased levels of eosinophils were noted in BWs obtained from pa tients as compared with controls.
Chemotactic Activity in Allergic Alveolitis
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 11:11:16 PM
Table 1. Dwell times and recoveries of fluid and cells of BWs obtained from 6 symptom-free patients who previously had extrinsic allergic alveolitis and 9 healthy controls
Table 2. Cell recoveries in BWs obtained from asymptomatic subjects who previously had extrinsic allergic alveolitis and from healthy controls
Percentage counts Patients (n = 6) Controls (n = 9) Total counts, x 106 Patients (n = 6) Controls (n = 9)
Alveolar macrophages
Lymphocytes
Neutrophils
Eosinophils
median
range
median
median
median
60.5 74
21.841.8- 82
1.21 1.71
0.77-3.35 0.73-2.36
79.8
16.2 7.4
0.32 0.18
range
0.5-72.6 0.6-10.3
0.03-4.07 0.06-0.30
10.4 8.1
0.21 0.21
range
1.9-30.8 1.6-54.3
0.03-1.88 0.02-2.17
1.5 0.3
0.03 0.003
range
0.6-23** 0-0.3
0.009-1.41** 0-0.01
** p
a Department of Clinical Chemistry, University Hospital, Uppsala; b Department of Human Pharmacology, Astra Draco AB, Lund, Sweden
Key Words Chemotaxis Allergy Human Eosinophils Lung Allergic alveolitis
Increased Eosinophil Chemotactic Activity in Bronchial Washes Obtained from Patients with Asymptomatic Allergic Alveolitis
Abstract The chemotactic activities of eosinophils and neutrophils were measured in bronchial washes obtained from 6 symptom-free patients with previous extrin sic allergic alveolitis and 9 healthy volunteers. An increased chemotactic activ ity was found in the washes obtained from the patients, and the recovery of eo sinophils was correlated with the eosinophil chemotactic activity, suggesting a causal relationship. Chromatography of the fluids obtained suggested that low molecular weight compounds were principally responsible for the increased chemotactic activity. A mild disturbance of the integrity of the alveolocapillary barrier could to some extent add to the explanation of the increased chemo tactic activity in the washes obtained from the patients.
Introduction Allergic alveolitis is a hypersensitivity pneumonitis in which sensitized subjects develop a hypersensitivity re sponse. The acute form of the disease is characterized by respiratory and systemic symptoms, occuring 6-12 h after antigen exposure, while the chronic form of the disease has an insidious onset of symptoms. Exposed, but asymp tomatic subjects may have increased circulating antibody levels [1] and lymphocytic alveolitis as signs of cell-medi ated response [2]. Although the mediators responsible for the genesis of the clinical disease are unknown, the pres ence of suppressor T cells and granulocyte chemotactic factors in lavage fluid [3] may suggest a causal involvement of these cells in the disease process.
We have previously found signs of increased eosinophil involvement, as reflected by lavage eosinophilia, in asymp tomatic patients who previously had suffered from extrin sic allergic alveolitis [4], Production of chemotactic factors that attract cells is considered to be one essential mecha nism for the accumulation of inflammatory cells. In the present study, we intended to elucidate further the eosi nophil involvement by assessing the eosinophil chemotac tic activity in bronchial washes (BWs) obtained from these asymptomatic subjects. We choose to assess the chemo tactic activity in the BWs and not in the bronchoalveolar lavages (BALs), since the dilution of the epithelial lining fluid, by the injected saline, was larger and tended to vary more in the BALs as compared with the BWs.
Correspondence to: Dr. Birgitta Schmekel Department o f Human Pharmacology Astra Draco AB, Box 34 S—211 00 Lund (Sweden)
© 1992 S. Karger AG, Base! 1018-2438/92/0983-0227 $ 2.75/0
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 11:11:16 PM
Lena H3 leansson* Birgitta Schmekel* - b
Dwell time, min Recovery volume, ml Total cell count, x 10'1
Patients (n = 6)
Controls (n = 9)
median
range
median
range
1.4 17.5 3.0
1.1-1.8 13-25 1.4-6.13
1.7 14 2.6
1.1-2.7 5-20 0.97-4.1
Materials and Methods Subjects Six nonsmoking patients with a history of extrinsic allergic alveo litis were studied. Their mean age was 48.2 years (range 39-56 years), they had all been exposed to either bird proteins or Aspergillus fumigatus, and they had developed typical symptoms and signs of extrinsic allergic alveolitis. The diagnosis had been established 2-15 years be fore this study based on a typical case history, chest radiographic findings consistent with the disease, and presence of precipitating an tibodies in peripheral blood. The antigens were eliminated in all cases as soon as the diagnosis was established. At the time of the study all patients were free of symptoms since more than 1 year, they received no medication, and physical examination as well as chest radiographs were normal. Nine healthy male volunteers with a mean age of 37.1 years (range 29-48 years) served as controls. None had ever smoked tobacco or had any history of allergy or lung disease. Chest radiographs and lung volumes were normal. Informed consent was obtained from patients and volunteers, and the study was approved by the Ethical Research Committee and the Faculty of Medicine, University of Lund. Bronchoalveolar Lavage After premedication with morphine and scopolamine and top ically applied lidocaine (Xylocaine®; Astra, Sweden), the fiberoptic bronchoscope (Olympus BF 1 T 10) was introduced via the nasal route into the lower airways. BW and BAL were performed as previ ously documented in detail [4], An initial BW of 50 ml was infused and immediately aspirated, followed by two 50-ml aliquots constitut ing BAL. Results on BAL are not reported here, due to the fact that there is a higher degree of dilution of the epithelial lining fluid by sa line in the larger BALs, and this affects the possibilities to measure chemotactic activités with a high precision. The dwell time of the wash fluids was defined by the time passed between the start of in fusion of the fluid and the end of the aspiration. The aspirated wash fluids were collected in siliconized glass containers kept on ice and centrifuged at 4°C at 200 g for 10 min. The cell pellets were then re suspended in RPMI 1640 (Gibco, UK) supplemented with 5% fetal calf serum and gentamicin (50 pg/ml) and transported to the lab oratory for differential counts. The cell-free supernatants were kept frozen at -70 °C until the analyzes were performed.
234
Hâkansson/Schmekcl
Gel Filtration o f BWs Gel filtration of BWs was performed on Sephacryl S-200 Super fine (Pharmacia LKB Biotechnology, Uppsala, Sweden) at 4 °C. Two milliliters of freshly frozen BW was applied to the column (2.6x90 cm). Gey’s solution with 0.1 mol/1 c-amino-caproic acid per liter was used as eluent. The elution rate was 10 ml/h and the fraction volume 2.5 ml. Measurement o f Chemotactic Activity in BWs The migration of eosinophils and neutrophils was measured using a modified Boyden chamber technique as previously described [5], Granulocytes were isolated from apparently healthy blood donors by dextran sedimentation [6] and diluted to a concentration of 1.5 x 109/1 in Gey’s solution with human serum albumin (2 g/1). The samples of BWs were diluted 1:4, 1:8, and 1:16. The migration of eosinophils and neutrophils towards the diluted BW samples was measured ac cording to the leading-front method as previously described [7], The chemotactic activity (units) of the samples was calculated as the maxi mal migration (pm/h) exceeding that towards Gey’s buffer, multi plied by the actual dilution of the sample. Measurement o f Chemotactic Activity in Sephacryl S-200 Fractions The chemotactic activity in fractions of BWs separated on Sepha cryl was measured according to the same principle as described for BWs. The fraction samples were diluted 1:3,1:6, and 1:12 if needed. The chemotactic activity was measured in every third fraction. Measurements o f Soluble Factors in BWs The concentrations of eosinophil cationic protein (ECP), and myeloperoxidase (MPO) were measured by means of radioimmu noassay [8,9]. The concentrations of albumin and urea were analyzed according to previously published methods [10,11], The total amounts of the substances were calculated by multiplying the concentration and the volume of the aspirated wash fluid. Analyses o f Cellular Composition in BWs Differential counts were performed on cytocentrifuge prepara tions (Cytospin 2; Shandon, UK) at 500 rpm for 10 min. The prep arations were stained with May-Griinwald-Giemsa, and differential counts were done by counting 400 cells on two separate cytospin preparations. Statistical Analysis Data are expressed as median values and ranges. The Wilcoxon rank sum test was used to evaluate differences between groups of pa rameters and the Spearman rank correlation test to evaluate correla tions between parameters, p values < 0.05 were considered significant.
Results Recovery volumes, dwell times, and total cell recovery of the BW fluid were similar in patients and controls (table 1). The variability in cell recovery was large, whether the differentials were expressed as percentage counts or as to tal number of the cells (table 2). Significantly increased levels of eosinophils were noted in BWs obtained from pa tients as compared with controls.
Chemotactic Activity in Allergic Alveolitis
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/6/2018 11:11:16 PM
Table 1. Dwell times and recoveries of fluid and cells of BWs obtained from 6 symptom-free patients who previously had extrinsic allergic alveolitis and 9 healthy controls
Table 2. Cell recoveries in BWs obtained from asymptomatic subjects who previously had extrinsic allergic alveolitis and from healthy controls
Percentage counts Patients (n = 6) Controls (n = 9) Total counts, x 106 Patients (n = 6) Controls (n = 9)
Alveolar macrophages
Lymphocytes
Neutrophils
Eosinophils
median
range
median
median
median
60.5 74
21.841.8- 82
1.21 1.71
0.77-3.35 0.73-2.36
79.8
16.2 7.4
0.32 0.18
range
0.5-72.6 0.6-10.3
0.03-4.07 0.06-0.30
10.4 8.1
0.21 0.21
range
1.9-30.8 1.6-54.3
0.03-1.88 0.02-2.17
1.5 0.3
0.03 0.003
range
0.6-23** 0-0.3
0.009-1.41** 0-0.01
** p
