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Mast cells in human bronchi are heterogeneous with respect to granule esterase activity
Respiratory Medicine (1990) 84, 499-501
Mast cells in human bronchi are heterogeneous with respect to granule esterase activity S. LOZEWICZ*, L. GREENWOOD*, A. F. WALLSt, E. GOMEZ*, AND R. J. DAVIES*
*Department of Respiratory Medicine, St Bartholomew's Centrefor ClinicalResearch, St Bartholomew's Hospital, London EC1, tDepartment of Clinical Pharmacology, Universityof Southamptonj Centre Block, Southampton GeneralHospital, Southampton S09 4XY, U.K.
Introduction
A large amount of data indicates that mast cells from different tissues, sites within the same tissue, or different species, may show heterogeneity of morphological, cytochemical, and functional properties (1,2). In the present study, we assessed heterogeneity ofcytochemical properties of" mast cells in human bronchial mucosa by comparing the numbers of cells visualized by three staining methods. Methods
We studied 19 subjects all of whom were nonsmokers. Ten were atopic asthmatic patients (three male) with a mean age of 26.4 yrs (range 21-39). All took inhaled salbutamoi but none required inhaled or oral corticosteroids. The remaining subjects were nine healthy nonatopic volunteers (five male) with a mean age of 24 yrs (range 20-32). Following premedication with intramuscular Omnopon 10-20 mg and scopolamine 0.2-0.4 rag, all subjects underwent fibreoptic bronchoscopy using 4% lignocaine for local anaesthesia. Cup forceps were used to take two biopsies of the mucous membrane from the right upper and right middle lobe carina. Biopsy speci. mens from each subject were fixed in Carnoy's solution, embedded in paraffin wax and sectioned at 4 p.m thickness. ALPHA-NAPHTHOLAS-D CHLOROACETATEESTERASE REACTION(CAER) The method was based closely on that devised by Leder (3) and is described in detail elsewhere (4). TOLUIDINE BLUE STAININGPROCEDURE A 0"5% solution of toluidine blue (BDH) was made up in 0.5 molar hydrochloric acid, and the tissue sections incubated in this solution for 30 min, to demonstrate metachromasia in mast cells and basophils. 0954-6111/90/060499+03 $03.00/0
AVIDIN-BIOTINCOMPLEXIMMUNOPEROXIDASE PROCEDURE WITH ANTI-TRYPTASEMONOCLONAL ANTIBODYAA1 (MAb AA1) This immunohistoehemical technique for identification of mast cells was performed using a method described elsewhere (5). STUDY DESIGN (a) Two adjacent sections of each of" the 19 biopsy specimens were stained for mast cells, one by the CAER and the other with toluidine blue. (b) The biopsy specimens of five of the subjects (two asthmatic, three nonasthmatic) were used to prepare further sections: two adjacent sections were sl~ained with the CAER, and two with M A b AAI. The mean density of mast cells was calculated for each pair of sections. Results
(a) Examination of consecutive sections showed that those mast cells stained by the CAER corresponded to mast cells stained by toluidine blue. The C A E R stain demonstrated significantly fewer mast cells compared with toluidine blue, in both asthmatics and nonasthmatics (Table 1, comparison a). (b) Significantly fewer mast cells '~vere stained by CAER compared with MAb AAI in the five biopsy specimens stained with each of these methods (Table I, comparison b). Discussion
This study demonstrated that within the lower airways in man only a minority of those mast ceils detected on the basis of their metaehromatic granule staining with toluidine blue, or on the basis o f their granule tryptase content, also demonstrated esterase activity using alpha-naphthol AS-D chloroacetate 9 1990BailIi,~reTindall
500
S. L o z e w i c z et al. Table 1 Numbers of mast cells in biopsies of the bronchial mucous membrane. Cell counts
expressed per mm2of tissuesection. Values are given as the median with range in brackets and were analysed by the Wilcoxon matched pairs test (a) or Mann-Whitney U test (b) with P values as follows: ** = <0.01, *-- <0-05
are
Lamina propria Comparison a
Toluidine blue
Nonasthmatics n=9
23.8 (0-120)
**
Asthmatics n = I0
60 (6--196)
*
Comparison b
CAER
n= 5
7'23 (0-145)
Epithelium
CAER
*
Toluidine blue
0
14.9
(0-10.7)
(0-205)
24.4 (0-90)
67.3 (0-506)
MAb AA1
CAER
96"9 (37-200)
0 (0-99)
substrate. This suggests that these mast cells are heterogeneous with respect to esterase activity. Since we.~a,ve previously found that equal numbers of mast cells were demonstrated by alcian blue-safranine O, and the CAER in nasal mucosa (6) the results of the present study further suggest that the nature of mast cell heterogeneity differs between upper and lower airways. The CAER technique was assessed by Moloney et al. (7) who examined esterase activity in human leucocytes using alpha-naphthol AS-D chloroacetate substrate and found activity to be present in mast cells, neutrophils, and macrophages but not in basophils, eosinophils, monocytes, lymphocytes, plasma cells, lymphocytes, or red cells. Neutrophils can be distinguished from mast cells by their characteristic nuclear morphology, and macrophages do not stain with the CAER when sections are embedded in paraffin (8), as in the present study. CAER has been shown to stain the same cells as toluidine blue by sequential staining (9,10) and its use has been further validated in studies comparing its mast cell staining properties with other esterase and amidase substrates (11-13), and toluidine blue (14-16). Irani et al. (17) demonstrated that most human airway mast cells contain tryptase (T mast cells) and a minority contain both tryptase and chymotryptic proteinase (TC mast cells). These results may be analogous to those of the present study since the properties of the mast cell enzyme activity responsible for hydrolysis of alpha naphthol ASD chloroacetate in the CAER a r e very similar to those of chymotrypsin (11,18), and the C A E R has been regarded as a stain for
CAER **
0
(0-4)) **
0
(0-67-7) MAb AAI **
190-9 (94-909)
mast cell chymotryptase activity (13,16). It is possible, therefore, that at this site, the minority of airway mast cells demonstrating esterase activity with alphanaphthol AS-D chloroacetate substrate correspond to mast cells containing chymotryptic proteinase (TC cells), whereas those mast cells which did not stain with the CAER correspond to T mast cells. This interpretation is compatible with the recent observation that in nasal polyps, TC mast cells are C A E R positive whereas T mast cells are C A E R negative (19). Acknowledgement
9
We are grateful to Julie Williams for assistance in performing the immunocytochemical procedure with anti-tryptase monoclonal antibody. References
I. Barrett KE, Metcalfe DD. Heterogeneity of mast ceils in the tissues of the respiratory tract and other organ systems. Am Rev Respir Dis 1987; 135:1190-[ 195. 2. BienenstockJ,BefusD, DenburgJ, GotoT, LeeT, Otsuka H, Shanahan F. Comparative aspects of mast cell heterogeneity in differentspecies and sites, hit Arch AIlergy Appl Irnmuno11985; 77: 126-129. 3. Leder LD. Uber die selektive fermentstoffchemische Darstellung yon neutrophilen myeloischen Zellen und Gewebstrastzellen im Paraffinschnitt. Klin Wochenschr 1964; 42: 553. 4. Gomez E, Corrado OJ, Baldwin DL, Swanston AR, Davies RA. Direct 01 vivo evidence for mast ceil degranulation during allergen-induced reactions in man. J Allergy Clin lmmuno11986; 78: 637-645. 5. WaltsAF, JonesDB, Williams JH, ChurchMK, Holgate ST. Immunohistochemical identification of mast ceils in
Mast cells in human bronchi
6. 7.
8.
9.
10.
11. 12.
formaldehyde-fixed tissue using monoclonal antibodies specific to tryptase. J Path 1990 (in press). Gomez E, Corrado O J, Davies RJ. Histochemical and functional characteristics of the human mast cell. lnt Arch Allergy Appl lmmuno11987; 83: 52-56. Moloney WC, Mcpherson K, Fliegelman L. Esterase activity in leukocytes demonstrated by the use of naphthol AS-D chloroacetate substrate, J Histochem Cytochem 1960; 8: 200-207. Sanderson IR, Slavin G, Walker-Smith JA. Density of mucosal mast cells in the lamina propria of the colon and terminal ileum of children. J CI#~ Pathol 1985; 38: 771-773. Newlands GFJ, Huntley JF, Miller HRP. Concomitant detection of mucosal mast cells and eosinophils in the intestines of normal and Nippostrongylus-immune rats. A re-evaluation of histochemical and immunocytochemical techniques. Histochem 1984; gl: 585-589. Huntley JF, Newlands GFJ, Gibson S, Ferguson A, Miller HRP. Histochemical demonstration of chymotrypsin like serine esterases in mueosal mast cells in four species including man. J Clin Pathol 1985; 38: 375-384. Li CY, Lam KW, Yam LT. Esterases in human leukocytes. J Histochem Cytoehem 1973; 21: 1-12. Garrett JR, Osman IA, Smith RE. Selection of a simple protease procedure for identifying mast cells in routinely processed human tissues. J Histochem Cytochem 1987; 35:541-547.
501
13. Caughey GH, Viro NF, Calonico LD, McDonald DM, Lazarus SC, Gold WM. Chymase and tryptase in dog mastocytoma cells: asynchronous expression as revealed by enzyme cytoehemical staining. J Histochem Cytochem 1988; 36: I053-1060. 14. Wong E, Morgan EW, MacDonald DM. The chloroacetate esterase reaction for mast cells in dermatopathology: a comparison with metaehromatic staining methods. Acta Derma Venereol (Stock) 1982; 62: 431-434. 15. King SJ, Reilly K, Dawes J, Miller HRP. The presence in blood of both glycosaminoglycan and mucosal mast cell protease following systemic anaphylaxis in the rat. Int Arch Allergy Appl Immuno11985;76: 286-288. 16. Osborne OL, SommerhoffCP, Nadel JA, Mcdonald DM. Histochemicalcomparison ofmast cells obtained from the airways of mongrel dogs and Basenji-greyhound dogs by bronchoalveolar lavage. Am Rev Respir Dis 1989; 140: 749-755. 17. Irani AA, Schechter NM, Craig SS, DeBlois G, Schwartz LB. Two types of" human mast cells that have distinct neutral protease compositions. Proc Nat1 Acad Sei USA 1986; 83: 4464-4468. 18. Benditt EP, Arase M. An enzyme in mast cells with properties like chymotrypsin. J Exp Med 1959; 110: 451-460. 19. Kawabori S, Denburg J, Irani A-M A, Schwartz LB, Dolovich J. Characteristics of mast cells in nasal polyps. J Allergy Clin lmmuno11990; 85:253 (abstract).
Mast cells in human bronchi are heterogeneous with respect to granule esterase activity S. LOZEWICZ*, L. GREENWOOD*, A. F. WALLSt, E. GOMEZ*, AND R. J. DAVIES*
*Department of Respiratory Medicine, St Bartholomew's Centrefor ClinicalResearch, St Bartholomew's Hospital, London EC1, tDepartment of Clinical Pharmacology, Universityof Southamptonj Centre Block, Southampton GeneralHospital, Southampton S09 4XY, U.K.
Introduction
A large amount of data indicates that mast cells from different tissues, sites within the same tissue, or different species, may show heterogeneity of morphological, cytochemical, and functional properties (1,2). In the present study, we assessed heterogeneity ofcytochemical properties of" mast cells in human bronchial mucosa by comparing the numbers of cells visualized by three staining methods. Methods
We studied 19 subjects all of whom were nonsmokers. Ten were atopic asthmatic patients (three male) with a mean age of 26.4 yrs (range 21-39). All took inhaled salbutamoi but none required inhaled or oral corticosteroids. The remaining subjects were nine healthy nonatopic volunteers (five male) with a mean age of 24 yrs (range 20-32). Following premedication with intramuscular Omnopon 10-20 mg and scopolamine 0.2-0.4 rag, all subjects underwent fibreoptic bronchoscopy using 4% lignocaine for local anaesthesia. Cup forceps were used to take two biopsies of the mucous membrane from the right upper and right middle lobe carina. Biopsy speci. mens from each subject were fixed in Carnoy's solution, embedded in paraffin wax and sectioned at 4 p.m thickness. ALPHA-NAPHTHOLAS-D CHLOROACETATEESTERASE REACTION(CAER) The method was based closely on that devised by Leder (3) and is described in detail elsewhere (4). TOLUIDINE BLUE STAININGPROCEDURE A 0"5% solution of toluidine blue (BDH) was made up in 0.5 molar hydrochloric acid, and the tissue sections incubated in this solution for 30 min, to demonstrate metachromasia in mast cells and basophils. 0954-6111/90/060499+03 $03.00/0
AVIDIN-BIOTINCOMPLEXIMMUNOPEROXIDASE PROCEDURE WITH ANTI-TRYPTASEMONOCLONAL ANTIBODYAA1 (MAb AA1) This immunohistoehemical technique for identification of mast cells was performed using a method described elsewhere (5). STUDY DESIGN (a) Two adjacent sections of each of" the 19 biopsy specimens were stained for mast cells, one by the CAER and the other with toluidine blue. (b) The biopsy specimens of five of the subjects (two asthmatic, three nonasthmatic) were used to prepare further sections: two adjacent sections were sl~ained with the CAER, and two with M A b AAI. The mean density of mast cells was calculated for each pair of sections. Results
(a) Examination of consecutive sections showed that those mast cells stained by the CAER corresponded to mast cells stained by toluidine blue. The C A E R stain demonstrated significantly fewer mast cells compared with toluidine blue, in both asthmatics and nonasthmatics (Table 1, comparison a). (b) Significantly fewer mast cells '~vere stained by CAER compared with MAb AAI in the five biopsy specimens stained with each of these methods (Table I, comparison b). Discussion
This study demonstrated that within the lower airways in man only a minority of those mast ceils detected on the basis of their metaehromatic granule staining with toluidine blue, or on the basis o f their granule tryptase content, also demonstrated esterase activity using alpha-naphthol AS-D chloroacetate 9 1990BailIi,~reTindall
500
S. L o z e w i c z et al. Table 1 Numbers of mast cells in biopsies of the bronchial mucous membrane. Cell counts
expressed per mm2of tissuesection. Values are given as the median with range in brackets and were analysed by the Wilcoxon matched pairs test (a) or Mann-Whitney U test (b) with P values as follows: ** = <0.01, *-- <0-05
are
Lamina propria Comparison a
Toluidine blue
Nonasthmatics n=9
23.8 (0-120)
**
Asthmatics n = I0
60 (6--196)
*
Comparison b
CAER
n= 5
7'23 (0-145)
Epithelium
CAER
*
Toluidine blue
0
14.9
(0-10.7)
(0-205)
24.4 (0-90)
67.3 (0-506)
MAb AA1
CAER
96"9 (37-200)
0 (0-99)
substrate. This suggests that these mast cells are heterogeneous with respect to esterase activity. Since we.~a,ve previously found that equal numbers of mast cells were demonstrated by alcian blue-safranine O, and the CAER in nasal mucosa (6) the results of the present study further suggest that the nature of mast cell heterogeneity differs between upper and lower airways. The CAER technique was assessed by Moloney et al. (7) who examined esterase activity in human leucocytes using alpha-naphthol AS-D chloroacetate substrate and found activity to be present in mast cells, neutrophils, and macrophages but not in basophils, eosinophils, monocytes, lymphocytes, plasma cells, lymphocytes, or red cells. Neutrophils can be distinguished from mast cells by their characteristic nuclear morphology, and macrophages do not stain with the CAER when sections are embedded in paraffin (8), as in the present study. CAER has been shown to stain the same cells as toluidine blue by sequential staining (9,10) and its use has been further validated in studies comparing its mast cell staining properties with other esterase and amidase substrates (11-13), and toluidine blue (14-16). Irani et al. (17) demonstrated that most human airway mast cells contain tryptase (T mast cells) and a minority contain both tryptase and chymotryptic proteinase (TC mast cells). These results may be analogous to those of the present study since the properties of the mast cell enzyme activity responsible for hydrolysis of alpha naphthol ASD chloroacetate in the CAER a r e very similar to those of chymotrypsin (11,18), and the C A E R has been regarded as a stain for
CAER **
0
(0-4)) **
0
(0-67-7) MAb AAI **
190-9 (94-909)
mast cell chymotryptase activity (13,16). It is possible, therefore, that at this site, the minority of airway mast cells demonstrating esterase activity with alphanaphthol AS-D chloroacetate substrate correspond to mast cells containing chymotryptic proteinase (TC cells), whereas those mast cells which did not stain with the CAER correspond to T mast cells. This interpretation is compatible with the recent observation that in nasal polyps, TC mast cells are C A E R positive whereas T mast cells are C A E R negative (19). Acknowledgement
9
We are grateful to Julie Williams for assistance in performing the immunocytochemical procedure with anti-tryptase monoclonal antibody. References
I. Barrett KE, Metcalfe DD. Heterogeneity of mast ceils in the tissues of the respiratory tract and other organ systems. Am Rev Respir Dis 1987; 135:1190-[ 195. 2. BienenstockJ,BefusD, DenburgJ, GotoT, LeeT, Otsuka H, Shanahan F. Comparative aspects of mast cell heterogeneity in differentspecies and sites, hit Arch AIlergy Appl Irnmuno11985; 77: 126-129. 3. Leder LD. Uber die selektive fermentstoffchemische Darstellung yon neutrophilen myeloischen Zellen und Gewebstrastzellen im Paraffinschnitt. Klin Wochenschr 1964; 42: 553. 4. Gomez E, Corrado OJ, Baldwin DL, Swanston AR, Davies RA. Direct 01 vivo evidence for mast ceil degranulation during allergen-induced reactions in man. J Allergy Clin lmmuno11986; 78: 637-645. 5. WaltsAF, JonesDB, Williams JH, ChurchMK, Holgate ST. Immunohistochemical identification of mast ceils in
Mast cells in human bronchi
6. 7.
8.
9.
10.
11. 12.
formaldehyde-fixed tissue using monoclonal antibodies specific to tryptase. J Path 1990 (in press). Gomez E, Corrado O J, Davies RJ. Histochemical and functional characteristics of the human mast cell. lnt Arch Allergy Appl lmmuno11987; 83: 52-56. Moloney WC, Mcpherson K, Fliegelman L. Esterase activity in leukocytes demonstrated by the use of naphthol AS-D chloroacetate substrate, J Histochem Cytochem 1960; 8: 200-207. Sanderson IR, Slavin G, Walker-Smith JA. Density of mucosal mast cells in the lamina propria of the colon and terminal ileum of children. J CI#~ Pathol 1985; 38: 771-773. Newlands GFJ, Huntley JF, Miller HRP. Concomitant detection of mucosal mast cells and eosinophils in the intestines of normal and Nippostrongylus-immune rats. A re-evaluation of histochemical and immunocytochemical techniques. Histochem 1984; gl: 585-589. Huntley JF, Newlands GFJ, Gibson S, Ferguson A, Miller HRP. Histochemical demonstration of chymotrypsin like serine esterases in mueosal mast cells in four species including man. J Clin Pathol 1985; 38: 375-384. Li CY, Lam KW, Yam LT. Esterases in human leukocytes. J Histochem Cytoehem 1973; 21: 1-12. Garrett JR, Osman IA, Smith RE. Selection of a simple protease procedure for identifying mast cells in routinely processed human tissues. J Histochem Cytochem 1987; 35:541-547.
501
13. Caughey GH, Viro NF, Calonico LD, McDonald DM, Lazarus SC, Gold WM. Chymase and tryptase in dog mastocytoma cells: asynchronous expression as revealed by enzyme cytoehemical staining. J Histochem Cytochem 1988; 36: I053-1060. 14. Wong E, Morgan EW, MacDonald DM. The chloroacetate esterase reaction for mast cells in dermatopathology: a comparison with metaehromatic staining methods. Acta Derma Venereol (Stock) 1982; 62: 431-434. 15. King SJ, Reilly K, Dawes J, Miller HRP. The presence in blood of both glycosaminoglycan and mucosal mast cell protease following systemic anaphylaxis in the rat. Int Arch Allergy Appl Immuno11985;76: 286-288. 16. Osborne OL, SommerhoffCP, Nadel JA, Mcdonald DM. Histochemicalcomparison ofmast cells obtained from the airways of mongrel dogs and Basenji-greyhound dogs by bronchoalveolar lavage. Am Rev Respir Dis 1989; 140: 749-755. 17. Irani AA, Schechter NM, Craig SS, DeBlois G, Schwartz LB. Two types of" human mast cells that have distinct neutral protease compositions. Proc Nat1 Acad Sei USA 1986; 83: 4464-4468. 18. Benditt EP, Arase M. An enzyme in mast cells with properties like chymotrypsin. J Exp Med 1959; 110: 451-460. 19. Kawabori S, Denburg J, Irani A-M A, Schwartz LB, Dolovich J. Characteristics of mast cells in nasal polyps. J Allergy Clin lmmuno11990; 85:253 (abstract).