Clinical Science (1991) 81, 183-188
183
Mast cell tryptase and histamine concentrations in bronchoalveolar lavage fluid from patients with interstitial lung disease A. F. WALLS, A. R. BENNETT, R. C. GODFREY, S. T. HOLGATE
AND
M. K. CHURCH
Immunopharmacology Group, University of Southampton,and *Southampton General Hospital,Southampton,U.K.
(Received 7 August 1990/18 February 1991; accepted 27 February 1991)
SUMMARY 1. Mast cell activation in the lung was investigated by measuring concentrations of mast cell tryptase and histamine in the bronchoalveolar lavage fluid from patients with bronchial carcinoma, sarcoidosis, extrinsic allergic alveolitis or cryptogenic fibrosing alveolitis and from normal subjects. 2. Histamine concentrations in bronchoalveolar lavage fluid supernatants were elevated in the bronchial carcinoma and cryptogenic fibrosing alveolitis groups, and were correlated with the histamine content of the cells recovered. 3. An avidin-biotin-enhanced antigen-capture e.l.i.s.a., using polyclonal rabbit antibody specific for tryptase, and mouse monoclonal antibody AA5, allowed the quantification of tryptase in all samples of bronchoalveolar lavage fluid. Tryptase concentrations were increased in the bronchial carcinoma and extrinsic allergic alveolitis groups and in some of the patients with sarcoidosis, and the levels correlated with mast cell numbers and also with concentrations of albumin. 4. There was no significant correlation between levels of tryptase and histamine, suggesting differences in the rates of metabolism or different cellular sources. 5. The tryptase and histamine concentrations measured suggest that there is continuous d e g r a d a t i o n of mast cells within the normal lung, but that this process is more pronounced in patients with bronchial carcinoma or interstitial lung disease.
Key words: bronchial carcinoma, cryptogenic fibrosing alveolitis, extrinsic allergic alveolitis, histamine, mast cell tryptase, sarcoidosis. Abbreviations: BAL, bronchoalveolar lavage; CFA, cryptogenic fibrosing alveolitis; EAA, extrinsic allergic
Correspondence:Dr Andrew F. Walls, Immunopharmacology Group, University of Southampton, Level F, Centre Block, Southampton General Hospital, Tremona Road, Southampton SO9 4XY, U.K.
alveolitis; PTS, phosphate-buffered 0.05% Tween 20.
saline containing
INTRODUCTION Mast cells are widely distributed throughout the lungs. These cells have been reported to comprise some 2% of the area of the alveolar wall [l], and they are present in large numbers in the bronchial mucosa and submucosa [2] and the bronchioles [3], as well as being found within the lumen of the airways [4, 51. A n increase in mast cell numbers has been found in lung tissue [l, 6, 71 and in bronchoalveolar lavage (BAL) fluid obtained from patients with bronchial carcinoma and with various fibrotic lung diseases [S-121. Mast cells from both the parenchyma and BAL fluid have been demonstrated in vitro to have the potential to release a range of pharmacologically potent mediators of inflammation, including histamine, neutral proteases, prostaglandin D, and leukotriene C, [ l l , 13, 141. However, in many cases the precise pathobiological role of these cells remains poorly understood. In seeking to define their role it will be important to determine the state of mast cell activation in patients with specific lung diseases. Increased levels of histamine have been noted in the BAL fluid of patients with cryptogenic fibrosing alveolitis (CFA) [15-171, sarcoidosis [16, 171 and in some patients with bronchial asthma [18,19]. Histamine, however, is not a discriminating marker of mast cell activation, as it may also be derived from basophils and other cell types. Furthermore, histamine is rapidly metabolized in vivo by histamine N-methyltransferase and diamine oxidase. Recent studies suggest that the measurement of tryptase, a proteolytic enzyme of the mast cell granule, could provide a reliable means for evaluating the degree to which mast cells may participate in disease. Using immunocytochemical techniques this enzyme has been detected in all mast cells, regardless of anatomical site
A. F. Walls et al.
184
[20-231, but is present in negligible quantities in basophils [24] and is wholly absent in all other cell types investigated. Elevated concentrations of human mast cell tryptase have been detected in the plasma of patients with systemic mastocytosis, during anaphylaxis [25, 261, and after allergen challenge in skin blister fluid [27, 281, nasal lavage fluid [29] and BAL fluid [30]. In the present study we have determined the levels of both tryptase and histamine in the BAL fluid of normal subjects and in patients with bronchial carcinoma, sarcoidosis, extrinsic allergic alveolitis (EAA) or CFA, and we have compared the levels with the numbers of mast cells recovered. Our findings suggest that there is a constant degranulation of mast cells in the lung, and that the extent of mast cell activation is increased in patients with these lung diseases. MATERIALS AND METHODS Subjects
All subjects were undergoing bronchoscopy for diagnostic purposes. Their participation in this study was approved by the Southampton University and Hospital Ethical Committee, and all gave informed consent. Diagnoses were based on clinical history, roentgenographic evidence and histological investigation of bronchial biopsy tissue. Patient details are given in Table 1. No abnormality was revealed in the ‘normal’ group, and in the subsequent 6 months there was no clinical manifestation of lung disease. Most of this group had presented with an isolated haemoptisis. Six of the patients with EAA were exposed to budgerigars and had a precipitin reaction consistent with bird fancier’s lung, while the remaining patient had farmer’s lung. For the purposes of this study, ‘non-smokers’ were defined as those who had not smoked in the preceding 12 months.
Lignocaine (2%) was employed to anaesthetize the upper airways, and the bronchoscope was wedged into a basal subsegment of the lung. Two 60 ml portions of prewarmed saline (150 mmol/l NaCI) were instilled, and after two breaths by the patient were gently aspirated (maximum negative pressure of 100 mmHg) into a polypropylene collection vessel maintained in ice. Preparation of fluid and cells
Within 5 min of collection, BAL fluid was passed through a 0.9 mm gauge sieve and centrifuged (400 g for 10 min, at 4°C). The supernatant was immediately transferred to a - 80°C freezer, where it was stored in 1.0 ml portions. Cells were resuspended in Hanks minimal essential medium (Gibco, Paisley, Renfrewshire, Scotland, U.K.) supplemented with 2% (v/v) foetal calf serum (Gibco) and counted in an Improved Neubauer Haemocytometer. BAL fluid samples with more than l o 5 erythrocytes/ml were excluded from this study. Cytocentrifuge preparations were made with approximately 3 x los BAL leucocytes (60 g for 5 min) and air-dried overnight before staining. Portions of the cell suspension (about l o hcells/ml) were stored at - 80°C for subsequent measurement of histamine. Cell staining
Mast cells were enumerated by staining Carnoy’s-fixed cytocentrifuge preparations with 0.5% Toluidine Blue in 0.5 mol/l hydrochloric acid for 30 min as described previously [6]. The percentages of metachromatically staining cells were estimated by a standard field-counting procedure. The equivalent of 5000-40 000 nucleated cells were counted blind by a single observer. Determination of albumin concentration
BAL
On the day of bronchoscopy, patients did not consume food or fluids before the procedure. Premedication was 0.6 mg of intramuscular atropine and 10-15 mg of intramuscular papaveretum 30 min before bronchoscopy.
Rocket immunoelectrophoresis [31] was employed to measure albumin levels in BAL fluid using the method described previously [32]. Portions (3 p l ) of BAL fluid or various dilutions of a purified albumin standard (Sigma, Poole, Dorset, U.K.) were electrophoresed (4 V/cm, 20 h) into agarose gel containing rabbit antiserum specific for
Table 1. Clinical data and details of BAL fluid recovery Mean values and the range are shown. Statistical significance (Mann-Whitney U-test): *P< 0.05, **P
183
Mast cell tryptase and histamine concentrations in bronchoalveolar lavage fluid from patients with interstitial lung disease A. F. WALLS, A. R. BENNETT, R. C. GODFREY, S. T. HOLGATE
AND
M. K. CHURCH
Immunopharmacology Group, University of Southampton,and *Southampton General Hospital,Southampton,U.K.
(Received 7 August 1990/18 February 1991; accepted 27 February 1991)
SUMMARY 1. Mast cell activation in the lung was investigated by measuring concentrations of mast cell tryptase and histamine in the bronchoalveolar lavage fluid from patients with bronchial carcinoma, sarcoidosis, extrinsic allergic alveolitis or cryptogenic fibrosing alveolitis and from normal subjects. 2. Histamine concentrations in bronchoalveolar lavage fluid supernatants were elevated in the bronchial carcinoma and cryptogenic fibrosing alveolitis groups, and were correlated with the histamine content of the cells recovered. 3. An avidin-biotin-enhanced antigen-capture e.l.i.s.a., using polyclonal rabbit antibody specific for tryptase, and mouse monoclonal antibody AA5, allowed the quantification of tryptase in all samples of bronchoalveolar lavage fluid. Tryptase concentrations were increased in the bronchial carcinoma and extrinsic allergic alveolitis groups and in some of the patients with sarcoidosis, and the levels correlated with mast cell numbers and also with concentrations of albumin. 4. There was no significant correlation between levels of tryptase and histamine, suggesting differences in the rates of metabolism or different cellular sources. 5. The tryptase and histamine concentrations measured suggest that there is continuous d e g r a d a t i o n of mast cells within the normal lung, but that this process is more pronounced in patients with bronchial carcinoma or interstitial lung disease.
Key words: bronchial carcinoma, cryptogenic fibrosing alveolitis, extrinsic allergic alveolitis, histamine, mast cell tryptase, sarcoidosis. Abbreviations: BAL, bronchoalveolar lavage; CFA, cryptogenic fibrosing alveolitis; EAA, extrinsic allergic
Correspondence:Dr Andrew F. Walls, Immunopharmacology Group, University of Southampton, Level F, Centre Block, Southampton General Hospital, Tremona Road, Southampton SO9 4XY, U.K.
alveolitis; PTS, phosphate-buffered 0.05% Tween 20.
saline containing
INTRODUCTION Mast cells are widely distributed throughout the lungs. These cells have been reported to comprise some 2% of the area of the alveolar wall [l], and they are present in large numbers in the bronchial mucosa and submucosa [2] and the bronchioles [3], as well as being found within the lumen of the airways [4, 51. A n increase in mast cell numbers has been found in lung tissue [l, 6, 71 and in bronchoalveolar lavage (BAL) fluid obtained from patients with bronchial carcinoma and with various fibrotic lung diseases [S-121. Mast cells from both the parenchyma and BAL fluid have been demonstrated in vitro to have the potential to release a range of pharmacologically potent mediators of inflammation, including histamine, neutral proteases, prostaglandin D, and leukotriene C, [ l l , 13, 141. However, in many cases the precise pathobiological role of these cells remains poorly understood. In seeking to define their role it will be important to determine the state of mast cell activation in patients with specific lung diseases. Increased levels of histamine have been noted in the BAL fluid of patients with cryptogenic fibrosing alveolitis (CFA) [15-171, sarcoidosis [16, 171 and in some patients with bronchial asthma [18,19]. Histamine, however, is not a discriminating marker of mast cell activation, as it may also be derived from basophils and other cell types. Furthermore, histamine is rapidly metabolized in vivo by histamine N-methyltransferase and diamine oxidase. Recent studies suggest that the measurement of tryptase, a proteolytic enzyme of the mast cell granule, could provide a reliable means for evaluating the degree to which mast cells may participate in disease. Using immunocytochemical techniques this enzyme has been detected in all mast cells, regardless of anatomical site
A. F. Walls et al.
184
[20-231, but is present in negligible quantities in basophils [24] and is wholly absent in all other cell types investigated. Elevated concentrations of human mast cell tryptase have been detected in the plasma of patients with systemic mastocytosis, during anaphylaxis [25, 261, and after allergen challenge in skin blister fluid [27, 281, nasal lavage fluid [29] and BAL fluid [30]. In the present study we have determined the levels of both tryptase and histamine in the BAL fluid of normal subjects and in patients with bronchial carcinoma, sarcoidosis, extrinsic allergic alveolitis (EAA) or CFA, and we have compared the levels with the numbers of mast cells recovered. Our findings suggest that there is a constant degranulation of mast cells in the lung, and that the extent of mast cell activation is increased in patients with these lung diseases. MATERIALS AND METHODS Subjects
All subjects were undergoing bronchoscopy for diagnostic purposes. Their participation in this study was approved by the Southampton University and Hospital Ethical Committee, and all gave informed consent. Diagnoses were based on clinical history, roentgenographic evidence and histological investigation of bronchial biopsy tissue. Patient details are given in Table 1. No abnormality was revealed in the ‘normal’ group, and in the subsequent 6 months there was no clinical manifestation of lung disease. Most of this group had presented with an isolated haemoptisis. Six of the patients with EAA were exposed to budgerigars and had a precipitin reaction consistent with bird fancier’s lung, while the remaining patient had farmer’s lung. For the purposes of this study, ‘non-smokers’ were defined as those who had not smoked in the preceding 12 months.
Lignocaine (2%) was employed to anaesthetize the upper airways, and the bronchoscope was wedged into a basal subsegment of the lung. Two 60 ml portions of prewarmed saline (150 mmol/l NaCI) were instilled, and after two breaths by the patient were gently aspirated (maximum negative pressure of 100 mmHg) into a polypropylene collection vessel maintained in ice. Preparation of fluid and cells
Within 5 min of collection, BAL fluid was passed through a 0.9 mm gauge sieve and centrifuged (400 g for 10 min, at 4°C). The supernatant was immediately transferred to a - 80°C freezer, where it was stored in 1.0 ml portions. Cells were resuspended in Hanks minimal essential medium (Gibco, Paisley, Renfrewshire, Scotland, U.K.) supplemented with 2% (v/v) foetal calf serum (Gibco) and counted in an Improved Neubauer Haemocytometer. BAL fluid samples with more than l o 5 erythrocytes/ml were excluded from this study. Cytocentrifuge preparations were made with approximately 3 x los BAL leucocytes (60 g for 5 min) and air-dried overnight before staining. Portions of the cell suspension (about l o hcells/ml) were stored at - 80°C for subsequent measurement of histamine. Cell staining
Mast cells were enumerated by staining Carnoy’s-fixed cytocentrifuge preparations with 0.5% Toluidine Blue in 0.5 mol/l hydrochloric acid for 30 min as described previously [6]. The percentages of metachromatically staining cells were estimated by a standard field-counting procedure. The equivalent of 5000-40 000 nucleated cells were counted blind by a single observer. Determination of albumin concentration
BAL
On the day of bronchoscopy, patients did not consume food or fluids before the procedure. Premedication was 0.6 mg of intramuscular atropine and 10-15 mg of intramuscular papaveretum 30 min before bronchoscopy.
Rocket immunoelectrophoresis [31] was employed to measure albumin levels in BAL fluid using the method described previously [32]. Portions (3 p l ) of BAL fluid or various dilutions of a purified albumin standard (Sigma, Poole, Dorset, U.K.) were electrophoresed (4 V/cm, 20 h) into agarose gel containing rabbit antiserum specific for
Table 1. Clinical data and details of BAL fluid recovery Mean values and the range are shown. Statistical significance (Mann-Whitney U-test): *P< 0.05, **P
