Cellular Infiltrates in Asthma and in Chronic Obstructive Pulmonary Disease 1 , 2 ANNIKA LAITINEN and LAURI A. LAITINEN Introduction According to current definitions, chronic bronchitis is a disease with cough and expectorations for at least 3 months during 2 consecutive yr, and asthma is a disease with reversible airway obstruction. The definitions point out a smooth muscle-related disease in asthma and a mucous- and bronchialgland-associated disease in chronic bronchitis. Very little is known of the morphologic aspects of the airways at the early stages of either disease.So far, both asthma and chronic bronchitis have been defined mainly by clinical observations and respiratory function measurements. However, these lead only to rough definitions of the diseases and leave open the pathophysiologic mechanisms. Classic dogma equates asthma with mucous plugging,bronchialsmooth musclehyperplasia, eosinophilia, and thickening of the epithelial basement membrane. However, these characteristics are those seen in autopsy of lungs obtained from asthmatics dying from status asthmaticus. They do not reflect the changes in the early phase of the disease nor do they relate to disease severity. Developments in bronchoscopic and electron microscopic techniques have been essential in obtaining better knowledge of airway morphology in living patients with asthma, chronic bronchitis, or emphysema. Methods to Investigate the Pathology of Asthma and Chronic Bronchitis Indirect approaches to study airway disease include sputum and bronchoalveolar lavage analysis.Severalinvestigatorshavebeen studying the sputum of asthmatic patients (1-4). Indeed, the presence of respiratory epithelial cells in sputum was one of the first pathologicabnormalities noticed in asthma and was described by Curschmann in 1885 (1). Later, Naylor (2) focused on the tendency of the bronchial epithelium to exfoliate by studying the sputum of asthmatics. He introduced the term Creola body to describe large sheets of exfoliated epithelium that condense to form spherical or elongated masses. Also, inflammatory cells such as eosinophils have been described in the sputum of asthmatics (5). Recent studies have concentrated on measuring immunoglobulins and eosinophil-derivedproteins in the sputum (5, 6). In chronic bronchitis the examination of the sputum is clinically important in the diagnosis of lung cancer and in the identification of bacteria during exacerbations. In either asthma or chronic bronchitis, sputum can be used as a diagnostic tool or it can contribute to the study of the pathophysiologic aspects of the diseases. AM REV RESPIR DIS 1991; 143:1159-1160
Only a few studies examining airway morphology have been conducted in asthma or in chronic obstructive pulmonary disease (COP D) (7-11). The difficulty lies in obtaining representative specimens of the human airways and the lack of quantitative morphologic methods, especially at the electron microscopic level. Asthmatic patients seldom have lung surgery. In chronic bronchitis and emphysema, lung surgery is performed because pulmonary infiltrates appear on chest radiographs or because lung cancer, usually in a late phase of the disease, has been identified. Bronchoscopy as a possible diagnostic tool has not been used in either patient group at an early stage of the disease. Thus, there is no knowledge of the usefulness of airway histology and electron microscopy in establishing a diagnosis. It has even been claimed that in asthma there are no early morphologic changes. Additional information of the bronchial airways may be gained by bronchoalveolar lavage (12). For quantitative studies, the bronchial biopsy specimensshould be taken and processed using a standardized method. An electron microscopic method to study greater areas of airway mucosa in bronchial biopsy specimens has been developed by using Slot grids without copper bars (which can hide parts of areas being examined) by making photomontages of adjacent electron micrographs. The whole thin section being approximately 1 x 1 mm can be photographed and later examined in the photomontage. The numbers of cells in airway epithelium and lamina propria can be quantitated per square millimeter. Electron microscopy allows ultrastructural identification of most of the cells (13, 14). Cellular Infiltrates in Asthma and in COPO Examination of changes in airway epithelium is especially important as it serves as a primary target organ for exogenousirritants such as allergens and tobacco smoke. The structure of the epithelium may be modified by different kinds of mediators derived from the epithelium itself or from invading inflammatory cells. Changes in the wall structure or cellular contents of bronchial blood vessels reflect the inflammatory stimulus elicited via circulatory, nervous, or diffusion mechanisms. While evaluating the stage of the inflammatory process in the airways, it is important to establish the proportion of cells in the epithelium, the lamina propria, and the blood vessels.Changes in epithelial structure and the evaluation of airway edema formation are more difficult to study with quantitative morphologic procedures.
In a recent study (14)the numbers of mast cells, neutrophils, and eosinophils werequantitated in the airway epithelium in 10 asthmatic patients with variable duration and clinical severity of the disease, in five long-time smokers (15to 50 yr), and in five control subjects. The asthmatic patients differed significantly (p = 0.003)from the long-time smokers and the control subjects with respect to the numbers of mast cells in the bronchial epithelium. The asthmatics and long-time smokers had significantly (p = 0.02) more neutrophils in the airway epithelium than did the control subjects. In the asthmatics, the mast cells in the airway epithelium showed highly degranulated forms containing mainly empty granules. Only a few scroll-type granular substructures were identified in the cells. In patients in the asthma group, large numbers of neutrophils in the airway epithelium occurred only in those with severeasthma and a long duration of the disease. In the long-time smokers, neutrophils were observed in the airway epithelium, but their numbers werelow. In the control group, no neutrophils were detected. Eosinophils occurred only in the airway epithelium of three asthmatic patients, and their number did not differ significantly from the other two groups. These results suggest that there are pathologic differences between patients with COPD, asthmatics, and healthy control subjects. However, in future studies, larger patient groups are needed, and the study area should include blood vessels, smooth muscle, and bronchial glands in the lamina propria. Also, the numbers of other cells such as lymphocytes, plasma cells, macrophages, and fibroblasts should be studied. We conclude that there is a great need for morphologic bronchial biopsy studies, which could give more information of the disease processes in COPD and in asthma. Numbers of inflammatory and other cells should be quantitated in the blood vessels, in the lamina propria, and in the epithelium in asthma and in chronic bronchitis at a clinically early stage of the disease. This could provide information of the differences between the dis-
1 From the Department of Medical and Physiological Chemistry and the Department of Lung Medicine, University of Lund, University Hospital of Lund, Lund, Sweden, and the Department of Electron Microscopy,University of Helsinki, Helsinki, Finland. 2 Correspondence and requests for reprints should be addressed to Professor Lauri A. Laitinen, M.D., Department of Lung Medicine, University Hospital in Lund, S-22185 Lund, Sweden.
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ease processes in these two diseases as well as the mechanisms involved in each disease. With present clinical observations and respiratory physiologic measurements, it is difficult to evaluate the course of the possible inflammatory process in the airways. References 1. Curschmann H. Einige bemerkungen iiber die im bronchialsecret vorkommenden spiralen. Dtsch Arch Klin Med 1985; 36:578-85. 2. Naylor B. The shedding of the mucosa of the bronchial tree in asthma. Thorax 1962; 17:69-72. 3. Sanerkin NG, EvansDMD. The sputum in bronchial asthma: pathognomonic patterns. J Pathol 1965; 89: 535-41. 4. Frigas E, Loegering DA, Solley GO, Farrow GM, Gleich GJ. Elevated levelsof eosinophil granule major basic protein in the sputum of patients with bronchial asthma. Proc Mayo Clin 1981; 56: 345-53. 5. Dor PJ, Ackerman SJ, Gleich GJ. CharcotLeyden crystal protein and eosinophil granule major basic protein in sputum of patients with respiratory diseases. Am Rev Respir Dis 1984; 130: 1072-7. 6. Turnbull LS, Turnbull LW, Crofton LW, Kay AB. Immunoglobulins, complement and arylsulphalase in sputum from chronic bronchitis and other pulmonary diseases.Clin Exp Immunoll978; 32: 226-32. 7. Glynn AA, Michaels L. Bronchial biopsy in chronic bronchitis and asthma. Thorax 1960; 15:142-53. 8. Cutz E, Levison H, Cooper DM. Ultrastructure of airways in children with asthma. Histopathology 1978; 2:407-21. 9. Laitinen LA, Heino M, Laitinen A, Kava T, Haahtela T. Damage of the airway epithelium and bronchial reactivity in patients with asthma. Am Rev Respir Dis 1985; 131:599-606. 10. Lundgren R, Soderberg M, Horstedt, Stenling R. Morphological studies of bronchial mucosal biopsies from asthmatics before and after ten years of treatment with inhaled steroids. Eur Respir J 1988; 1:883-9. 11. Beasley R, Roche WR, Roberts JA, Holgate ST. Cellular events in the bronchi in mild asthma and after bronchial provocation. Am Rev Respir Dis 1989; 139:806-17. 12. Wardlaw AJ, Dunnette S, Gleich GJ, Collins
LAITINEN AND LAITINEN
JV, Kay AB. Eosinophils and mast cells in bronchoalveolar lavage in subjects with mild asthma. Am Rev Respir Dis 1988; 137:62-9. 13. Laitinen A. Ultrastructural organization of intraepithelial nerves in the human airway tract. Thorax 1985; 40:488-92. 14. Laitinen LA. Epithelial damage. In: Malo JL, Hargreave F, Hogg J, eds. Glucocorticoids and mechanisms of asthma. Amsterdam: Excerpta Medica, 1989; 215-29.
Comments Dr. O'Byrne: Many studies using BAL have shown eosinophils even in subjects with very mild asthma. Could the lack of eosinophils in the electron microscopic studies reflect the fact that the morphologic aspects of the airway are heterogenous and that the biopsies may not reflect changes elsewhere in the airways? Dr. Laitinen: Our patients were stable asthmatics, and the area studied was restricted to the airway epithelium. But we have seen that during exacerbation of asthma veryhigh numbers of eosinophils may occur in the airway epithelium at the lamina propria as well as in blood vessels. A basic question is: Where do the cells in the lavage fluid come from? Dr. Jeffery: When we counted inflammatory cells in the surface epithelium in our bronchial biopsies, wefound that by far the highest proportion were made up of cells with lymphocyte-like structures (between 60 and 80% of inflammatory cells). Was this also your experience? Dr. Laitinen: Wealso found that lymphocytes may form a high proportion of cells observed both in the epithelium and the lamina propria. Dr. Dijkman: You used the rigid bronchoscope and produced large and beautiful biopsies without damage or pinching. Is such material really better than information ob-
tained by sputum, BAL, or trans bronchial biopsies through fiberscopy? Dr. Laitinen: With bronchial biopsies the state of the epithelium and lamina propria can be estimated. Counts of inflammatory and other cells can be made in relation to their location in the epithelium, lamina propria, and blood vessels. In our studies with fiberoptic bronchoscopy, the biopsies obtained were smaller and had more artifacts, and it was difficult to find airway epithelium in the specimens. Sputum and BAL can be used in monitoring the disease activity, but they do not contribute much to the understanding of pathophysiologic mechanisms. Dr. Dijkman: What is your opinion on the role of the basement membrane? Dr. Laitinen: Verylittle is known of the function of the basement membrane or of changes in disease states. Because inflammatory cell counts usually are higher in the lamina propria than in the epithelium, the basement membrane may have a role in restricting cell movements. Dr. Jeffery: I feel it would be helpful to clarify use of the terms "basement membrane" and "subepithelial collagen" that we have heard today. Formally, the term "epithelial basement membrane" as applied to airways (seen by light microscopy in conventional paraffin-embedded sections)encompassed all of what we now distinguish by electron microscopy, as at least two distinct zones: (1) the so-called "true basement membrane" (basal lamina) constituting a distinct 100 nm-thick zone of amorphons, material to which all the overlying epithelial cells attach, and (2) external to this a fibrillary zone of about 8 urn made up of reticular collagen (reticular lamina), which is distinct from the remaining collagen of the airway wall. Dr. Holgate's team, ours, and others have clearly shown that it is the latter that is' thickened in asthma, both in status asthmaticus and in ongoing asymptomatic and symptomatic asthma.
Only a few studies examining airway morphology have been conducted in asthma or in chronic obstructive pulmonary disease (COP D) (7-11). The difficulty lies in obtaining representative specimens of the human airways and the lack of quantitative morphologic methods, especially at the electron microscopic level. Asthmatic patients seldom have lung surgery. In chronic bronchitis and emphysema, lung surgery is performed because pulmonary infiltrates appear on chest radiographs or because lung cancer, usually in a late phase of the disease, has been identified. Bronchoscopy as a possible diagnostic tool has not been used in either patient group at an early stage of the disease. Thus, there is no knowledge of the usefulness of airway histology and electron microscopy in establishing a diagnosis. It has even been claimed that in asthma there are no early morphologic changes. Additional information of the bronchial airways may be gained by bronchoalveolar lavage (12). For quantitative studies, the bronchial biopsy specimensshould be taken and processed using a standardized method. An electron microscopic method to study greater areas of airway mucosa in bronchial biopsy specimens has been developed by using Slot grids without copper bars (which can hide parts of areas being examined) by making photomontages of adjacent electron micrographs. The whole thin section being approximately 1 x 1 mm can be photographed and later examined in the photomontage. The numbers of cells in airway epithelium and lamina propria can be quantitated per square millimeter. Electron microscopy allows ultrastructural identification of most of the cells (13, 14). Cellular Infiltrates in Asthma and in COPO Examination of changes in airway epithelium is especially important as it serves as a primary target organ for exogenousirritants such as allergens and tobacco smoke. The structure of the epithelium may be modified by different kinds of mediators derived from the epithelium itself or from invading inflammatory cells. Changes in the wall structure or cellular contents of bronchial blood vessels reflect the inflammatory stimulus elicited via circulatory, nervous, or diffusion mechanisms. While evaluating the stage of the inflammatory process in the airways, it is important to establish the proportion of cells in the epithelium, the lamina propria, and the blood vessels.Changes in epithelial structure and the evaluation of airway edema formation are more difficult to study with quantitative morphologic procedures.
In a recent study (14)the numbers of mast cells, neutrophils, and eosinophils werequantitated in the airway epithelium in 10 asthmatic patients with variable duration and clinical severity of the disease, in five long-time smokers (15to 50 yr), and in five control subjects. The asthmatic patients differed significantly (p = 0.003)from the long-time smokers and the control subjects with respect to the numbers of mast cells in the bronchial epithelium. The asthmatics and long-time smokers had significantly (p = 0.02) more neutrophils in the airway epithelium than did the control subjects. In the asthmatics, the mast cells in the airway epithelium showed highly degranulated forms containing mainly empty granules. Only a few scroll-type granular substructures were identified in the cells. In patients in the asthma group, large numbers of neutrophils in the airway epithelium occurred only in those with severeasthma and a long duration of the disease. In the long-time smokers, neutrophils were observed in the airway epithelium, but their numbers werelow. In the control group, no neutrophils were detected. Eosinophils occurred only in the airway epithelium of three asthmatic patients, and their number did not differ significantly from the other two groups. These results suggest that there are pathologic differences between patients with COPD, asthmatics, and healthy control subjects. However, in future studies, larger patient groups are needed, and the study area should include blood vessels, smooth muscle, and bronchial glands in the lamina propria. Also, the numbers of other cells such as lymphocytes, plasma cells, macrophages, and fibroblasts should be studied. We conclude that there is a great need for morphologic bronchial biopsy studies, which could give more information of the disease processes in COPD and in asthma. Numbers of inflammatory and other cells should be quantitated in the blood vessels, in the lamina propria, and in the epithelium in asthma and in chronic bronchitis at a clinically early stage of the disease. This could provide information of the differences between the dis-
1 From the Department of Medical and Physiological Chemistry and the Department of Lung Medicine, University of Lund, University Hospital of Lund, Lund, Sweden, and the Department of Electron Microscopy,University of Helsinki, Helsinki, Finland. 2 Correspondence and requests for reprints should be addressed to Professor Lauri A. Laitinen, M.D., Department of Lung Medicine, University Hospital in Lund, S-22185 Lund, Sweden.
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ease processes in these two diseases as well as the mechanisms involved in each disease. With present clinical observations and respiratory physiologic measurements, it is difficult to evaluate the course of the possible inflammatory process in the airways. References 1. Curschmann H. Einige bemerkungen iiber die im bronchialsecret vorkommenden spiralen. Dtsch Arch Klin Med 1985; 36:578-85. 2. Naylor B. The shedding of the mucosa of the bronchial tree in asthma. Thorax 1962; 17:69-72. 3. Sanerkin NG, EvansDMD. The sputum in bronchial asthma: pathognomonic patterns. J Pathol 1965; 89: 535-41. 4. Frigas E, Loegering DA, Solley GO, Farrow GM, Gleich GJ. Elevated levelsof eosinophil granule major basic protein in the sputum of patients with bronchial asthma. Proc Mayo Clin 1981; 56: 345-53. 5. Dor PJ, Ackerman SJ, Gleich GJ. CharcotLeyden crystal protein and eosinophil granule major basic protein in sputum of patients with respiratory diseases. Am Rev Respir Dis 1984; 130: 1072-7. 6. Turnbull LS, Turnbull LW, Crofton LW, Kay AB. Immunoglobulins, complement and arylsulphalase in sputum from chronic bronchitis and other pulmonary diseases.Clin Exp Immunoll978; 32: 226-32. 7. Glynn AA, Michaels L. Bronchial biopsy in chronic bronchitis and asthma. Thorax 1960; 15:142-53. 8. Cutz E, Levison H, Cooper DM. Ultrastructure of airways in children with asthma. Histopathology 1978; 2:407-21. 9. Laitinen LA, Heino M, Laitinen A, Kava T, Haahtela T. Damage of the airway epithelium and bronchial reactivity in patients with asthma. Am Rev Respir Dis 1985; 131:599-606. 10. Lundgren R, Soderberg M, Horstedt, Stenling R. Morphological studies of bronchial mucosal biopsies from asthmatics before and after ten years of treatment with inhaled steroids. Eur Respir J 1988; 1:883-9. 11. Beasley R, Roche WR, Roberts JA, Holgate ST. Cellular events in the bronchi in mild asthma and after bronchial provocation. Am Rev Respir Dis 1989; 139:806-17. 12. Wardlaw AJ, Dunnette S, Gleich GJ, Collins
LAITINEN AND LAITINEN
JV, Kay AB. Eosinophils and mast cells in bronchoalveolar lavage in subjects with mild asthma. Am Rev Respir Dis 1988; 137:62-9. 13. Laitinen A. Ultrastructural organization of intraepithelial nerves in the human airway tract. Thorax 1985; 40:488-92. 14. Laitinen LA. Epithelial damage. In: Malo JL, Hargreave F, Hogg J, eds. Glucocorticoids and mechanisms of asthma. Amsterdam: Excerpta Medica, 1989; 215-29.
Comments Dr. O'Byrne: Many studies using BAL have shown eosinophils even in subjects with very mild asthma. Could the lack of eosinophils in the electron microscopic studies reflect the fact that the morphologic aspects of the airway are heterogenous and that the biopsies may not reflect changes elsewhere in the airways? Dr. Laitinen: Our patients were stable asthmatics, and the area studied was restricted to the airway epithelium. But we have seen that during exacerbation of asthma veryhigh numbers of eosinophils may occur in the airway epithelium at the lamina propria as well as in blood vessels. A basic question is: Where do the cells in the lavage fluid come from? Dr. Jeffery: When we counted inflammatory cells in the surface epithelium in our bronchial biopsies, wefound that by far the highest proportion were made up of cells with lymphocyte-like structures (between 60 and 80% of inflammatory cells). Was this also your experience? Dr. Laitinen: Wealso found that lymphocytes may form a high proportion of cells observed both in the epithelium and the lamina propria. Dr. Dijkman: You used the rigid bronchoscope and produced large and beautiful biopsies without damage or pinching. Is such material really better than information ob-
tained by sputum, BAL, or trans bronchial biopsies through fiberscopy? Dr. Laitinen: With bronchial biopsies the state of the epithelium and lamina propria can be estimated. Counts of inflammatory and other cells can be made in relation to their location in the epithelium, lamina propria, and blood vessels. In our studies with fiberoptic bronchoscopy, the biopsies obtained were smaller and had more artifacts, and it was difficult to find airway epithelium in the specimens. Sputum and BAL can be used in monitoring the disease activity, but they do not contribute much to the understanding of pathophysiologic mechanisms. Dr. Dijkman: What is your opinion on the role of the basement membrane? Dr. Laitinen: Verylittle is known of the function of the basement membrane or of changes in disease states. Because inflammatory cell counts usually are higher in the lamina propria than in the epithelium, the basement membrane may have a role in restricting cell movements. Dr. Jeffery: I feel it would be helpful to clarify use of the terms "basement membrane" and "subepithelial collagen" that we have heard today. Formally, the term "epithelial basement membrane" as applied to airways (seen by light microscopy in conventional paraffin-embedded sections)encompassed all of what we now distinguish by electron microscopy, as at least two distinct zones: (1) the so-called "true basement membrane" (basal lamina) constituting a distinct 100 nm-thick zone of amorphons, material to which all the overlying epithelial cells attach, and (2) external to this a fibrillary zone of about 8 urn made up of reticular collagen (reticular lamina), which is distinct from the remaining collagen of the airway wall. Dr. Holgate's team, ours, and others have clearly shown that it is the latter that is' thickened in asthma, both in status asthmaticus and in ongoing asymptomatic and symptomatic asthma.
