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Bronchial biopsy findings in intermittent or "early" asthma
Section I: Inflammatory Determinants of Asthma Severity Bronchial biopsy findings in intermittent or "'early" asthma Lauri A. Laitinen, MD, PhD, Annika Laitinen, MD, PhD, Alan Altraja, MD, Ismo Virtanen, MD, PhD, Mary K/impe, MD, Bo G. Simonsson, MD, PhD, Sven-Erik Karlsson, MD, Lena H&kansson, Per Venge, MD, PhD, and Heinart Sillastu, MD, PhD Helsinki, Finland, Uppsala and Lund, Sweden, and Tartu, Estonia Bronchial biopsy specimens from subjects with intermittent or "early" asthma were compared with specimens taken from healthy subjects. Patients with early asthma included those with seasonal asthma and occupational asthma. There was a small but statistically significant increase in the thickness of the subepithelial extracellular matrix protein tenascin in subjects with seasonal and occupational asthma compared with control subjects. Collagen types IV and VII were increased only in patients with occupational asthma. Eosinophils were the only inflammatory cells that were significantly increased in subjects with seasonal asthma compared with control subjects. These data show that inflammation is present in the airways' of patients with early asthma, and the increase in tenascin expression in the basement membrane zone suggests that structural changes are also initiated at an early stage of" the disease. (J Allergy Clin Immunol 1996;98:$3-6.) Key words: Bronchial biopsy, collagen, early asthma, eosinophils, tenascin
Categorizing asthma according to the degree of severity of the disorder is problematic because of a lack of commonly agreed upon criteria. In a Boston G r o u p report from Finland, 1 patients with asthma were divided into five groups on the basis of given criteria. Most of the patients (60%) were considered to have mild asthma, and one fifth were considered to have severe or very severe asthma (Table I). This distribution involved adults only and is probably different in children. There have been few extensive epidemiologic population studies on the incidence and prevalence of asthma in Finland. Asthma has been From the Department of Medicine, University Central Hospital, Helsinki, Finland, the Department of Anatomy, Institute of Biomedicine, University of Helsinki, Finland, the Departments of Lung Medicine and Clinical Chemistry, University of Uppsala, Sweden, the Department of Lung Medicine, University of Lund, Sweden, and the Department of Puhnonary Medicine, University of Tartu, Estonia. Reprint requests: Lauri A. Laitinen, MD, PhD, Department of Medicine, Helsinki University Central Hospital, Haartmaninkatu 4, FIN-00290 Helsinki, Finland. Copyright © 1996 by- Mosby-Year Book, Inc. 0091-6749/96 $5.00 + 0 1/0/76643
Abbreviations used mAb: Monoclonal antibody PD15FEV~: Provocative dose causing a 15% decrease in FEV 1 TGF-[3: Transforming growth factor-J3
diagnosed in approximately 150,000 Finns, or 3% of the population. A similar percentage of the population receives drug therapy for asthma or receives social security benefits because of asthma. Depending on the methodology, study material, and definition of asthma, the prevalence rate may be up to 5% or 250,000 persons. The diagnosis of asthma can be inaccurate because some persons have only occasional asthma-like symptoms, such as prolonged cough and mild respiratory distress, with no observable impairment on the basis of pulmonary function tests. These symptoms may be associated with stress, respiratory infections, or exposure to allergens and are usually of brief duration. Some persons with these symptoms may develop asthma (Fig. 1). $3
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Laitinen eta[.
J ALLERGY CLIN IMMUNOL NOVEMBER 1996
No symptoms
TABLE I. Distribution of patients w i t h asthma according to severity of disease
Severity
%
Description
Very severe
2%
Disabling disease; patient spends numerous days in bed; lifethreatening attacks Daily wheezing, poor general condition, disease restricts lifestyle, severe nocturnal symptoms; patient is absent from work for several weeks a year; one in two patients needs hospital treatment Daily symptoms but no significant diurnal variation; patient avoids exercise and has occasional nocturnal symptoms Periodic symptoms; patient reacts to stimulants such as pollen or intense cold; symptoms restrict activity two or three times a week Occasional cough or wheezing that does not cause major impairment; respiratory tract is sensitive to intense cold and infections; allergens cause symptoms of varying degree, depending on exposure
Severe
18%
Moderate
20%
Mild
20%
Very mild
40%
FIG. 1. Asthma and asthmaqike symptoms. (From Koskela K, et al. Asthma Programme in Finland 1994-2004. Report of a Working Group. Clin Exp Allergy 1996;26(suppl):1-24).
W e studied groups of patients w h o m we consider to have intermittent or "early" asthma, including patients with seasonal asthma and occupational asthma. Bronchial biopsy specimens were obtained to study extracellular matrix proteins and inflammatory cells with use of specific antibodies, and the results of patients with seasonal asthma and occupational a s t h m a were c o m p a r e d with those of healthy control subjects.
METHODS Patients Characteristics of patients with seasonal and occupational asthma are presented in Table II. In the patients with seasonal asthma, diagnosis was based on a clinical history of seasonal symptoms during the birch pollen season and skin prick tests that revealed a positive reaction (>3 mm in diameter) for birch pollen (ALK Allergologisk Laboratorium A/S, H~arsholm, Denmark, reagents). Birch pollen-specific serum IgE, measured by a radioallergosorbent test, was present in all patients with seasonal asthma. In patients with occupational asthma, symptoms were associated with exposure to aluminum fluoride (2 patients), welding fumes (2), piperazine (1), methyltetrahydrophthalic anhydride (1), sulfuric acid aerosols (1), or isocyanates (1) lasting from 1 to 48 months (15 -+ 5.5 months, mean +- SEM). Only one patient was allergic to timothy grass, as revealed by skin prick testing. All patients with occupational asthma had bronchial hyperresponsiveness, as tested by methacholine provocation. The provocative concentration causing a reduction of 15% in FEV 1 (PCrsFEV1) ranged from 0.05 to 0.455 mg/ml (0.14 _+ 0.05). None of the 12 control subjects had a history of any respiratory or systemic disease, nor did routine hematologic and serum biochemistry tests show any abnormalities. All control subjects were nonsmokers. None was allergic, on the basis of their histories and negative results of skin prick tests with a panel of t2 commonly used allergens (ALK Allergologisk Laboratorium A/S).
From Koskela K, et al. Asthma Programme in Finland t9942004. Report of a Working Group. Clin Exp Allergy 1996; 26(suppt):1-24.
Lung function was normal in all control subjects. FEV 1 increased less than 15% in a bronchodilator test with 3 × 200 ixg inhaled rimiterol (Pulmadil, 3M Health Care Ltd., Loughborough, United Kingdom). The study was approved by local ethics committees, and written informed consent was obtained from each participant.
Bronchoscopy and bronchial biopsies Bronchoscopic examination and the taking of biopsy specimens conformed to the international guidelines for investigative use of fiberoptic bronchoscopy in patients with asthma. Premedication for all subjects consisted of either atropine (0.5 to 1 mg) or scopolamine (0.3 to 0.4 mg), and droperidol (5 to 10 rag) or morphine hydrochloride (7.5 to 10 rag). Local anesthesia was achieved with the application of lignocaine spray or 2% lignocaine solution to the oropharynx immediately before the procedure, and further anesthesia was achieved by instillation of a 2% lignocaine solution through the bronchoscope into the bronchial tree. Bronchoscopy was performed with the fiberoptic instruments Olympus BF-20 or BT-IT-20D (Olympus Optical Co. Ltd., Tokyo, Japan) in all study groups except the one that included
J ALLERGY CL1N IMMUNOL VOLUME 98, NUMBER 5, PART 2
L a i t i n e n e t al.
$5
TABLE II. Patient data (mean ± SEM, range) Seasonal asthma (n= 17) Sex Age (yr) Weight (kg) Height (cm) Duration of asthma (yr) FEV 1 (% of predicted)
6 Female, 11 Male 33.7 + 1.7 (21-44) 75.7 ± 3.0 (56-95) 176 ± 2.3 (160-194) 5.3 + 0.7 (2-12) 96.2 + 2.5 (71-115)
patients with occupational asthma; the Olympus BFXT-20 fiberscope was used with this group. Bronchial biopsy specimens were taken from the right upper and middle lobe bronchi. Care was taken to avoid regions close to carinae and regions previously touched by the bronchoscope. In control subjects, the specimens were taken with Olympus FB-19C forceps; in patients with seasonal asthma, specimens were taken with the Olympus FB-15K; and in patients with occupational asthma, specimens were taken with the Olympus FB-35C forceps.
Immunostaining Sections (5 Fm thick) were cut and fixed in acetone at - 2 0 ° C and stained with use of the indirect immunofluorescence technique. Mouse monoclonal antibodies (mAbs) against tenascin (2), collagen type VII (GIBCO BRL Research Products, Inc., Gaithersburg, Md.) and collagen types III (HEYL, GmbH, Berlin, Germany) and IV (Boehringer Mannheim, GmbH, Germany) were used. The sections were stained with a mouse monoclonal antibody (mAb) to tenascin, 100EB2, which reacts with the fourth and fifth fibronectin-like repeat in the tenascin-C molecule. 2 After washing, the sections were incubated with fluorescein isothiocyanate (FITC)-coupled sheep anti-mouse IgG (Jackson Immunoresearch Laboratories, Inc., West Grove, Pa.). After washing, the sections were mounted and examined under a Leitz Aristoplan fluorescence microscope (Ernst Leitz GmbH, Wetzler, Germany) equipped with appropriate filters. Negative controls were provided by omitting the primary mAb or by replacing it with an irrelevant one of the same isotope. Selected samples were examined by double label immunostaining. The distribution of tenascin was compared with that obtained by the mAb to laminin cd chain. 3 M e a s u r e m e n t of the i m m u n o s t a i n e d area in the b a s e m e n t m e m b r a n e zone After immunostaining, the cross-sectional area of the basement membrane zone from each section was photographed at an original magnification of ?<250. Black and white paper pictures from each specimen were made with a final magnification of ×643. For each photograph the deeper border of the basement membrane zone with continuous immunoreactivity was charted along its maximal length semiautomatically with use of a pointing device; charting was performed on a Compaq Deskpro
Occupational asthma (n=8) 35.8 82.6 182 9.4 81.1
8 Male _+ 2.1 (29-45) +- 3.3 (70-95) ± 2.5 (172-191) ± 2.2 (3-20) -+ 2.1 (69-89)
386 computer (Compaq Computer Corp., Houston, Tex.) from a 42 × 60 inch Kurta IS/THREE digitizing tablet (Kurta Corp., Phoenix, Ariz.). The pointing device was used in a similar manner for the superficial limit of the immunostained area. The image pictures were generated and the data processed by means of the AUTOCAD graphics program (Autodesk, Inc., Sausalito, Calif.). The minimal distances from each superficial point to the point closest in the deeper border of the immunoreactivity zone and their average values and standard deviations were calculated by the program. The average values were considered to be the thickness of the immunostained area of the basement membrane in each specimen. The thicknesses were also counted by dividing the surface area of the stained area with the corresponding length of the measured basement membrane area. When these two measurements were compared, there was no statistical difference between the mean thicknesses obtained by the two methods. With use of the method previously described, we measured expressions of tenascin and collagens III, IV, and VII.
Quantitation of i n f l a m m a t o r y cells Inflammatory cells were stained with use of mAbs and the alkaline phosphatase antialkaline phosphatase staining method (Dako A/S, Glostrup, Denmark). The quantitation was conducted with use of a computerized system applying the AUTOCAD program, as previously described. Eosinophils, mast cells, macrophages, and T lymphocytes were measured. Statistical analysis The results were analyzed with the Kruskal-Wallis one way analysis of variance by ranks. Where appropriate, the Mann-Whitney U-test, together with correction for multiple comparisons, served to estimate the difference between controls and patient groups. RESULTS A significant increase in tenascin expression was f o u n d in the b a s e m e n t m e m b r a n e o f patients with seasonal (p < 0.05) and occupational asthma (p < 0.001) c o m p a r e d with that of control subjects. O f the collagens studied, collagen types IV and type V I I were increased only in patients with occupational asthma (p < 0.005). Of the inflammatory cells stud-
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Laitinen et al.
ied, the numbers of eosinophils were increased in patients with seasonal asthma (p < 0.001). DISCUSSION
The airways of patients with seasonal and occupational asthma showed an increased accumulation of tenascin in the basement m e m b r a n e area. It is not known if this is a result of upregulated production or decreased breakdown of tenascin. In patients with asthma the tenascin band was mainly restricted to the basement m e m b r a n e zone and was not seen in the epithelium or diffusing into the airway stroma. Increased tenascin expression was seen close to the epithelial cells, which argues in favor of an abnormality in this region. It has been shown that epithelial-stromal interactions are important in the production of tenascin and that both the epithelial and stromal cells may serve as a source of tenascin. 4 While extracellular matrix proteins are important in controlling the development of the lung during early life, the restricted distribution of tenascin in healthy adult airways and other organs and its upregulation in pathologic situations suggest that mechanisms exist to downregulate and upregulate tenascin expression in adult organs. It has been suggested that both transforming growth factor-[3 (TGF-[3) and certain cytokines upregulate tenascin production in cell culture studies. 5 Tenascin and T G F - 6 are both coexpressed during budding morphogenesis of the bronchi 6 and in diffuse inflammatory processes of the lung tissue. 7 On the basis of biopsy studies, bronchial asthma is considered to be an inflammatory disease of the airways. 8 Even the airways of patients with newly detected and mild asthma, who have had asthma for a relatively short time, show changes related to chronic inflammation. 9 These changes are associated with structural changes in the airway epithelium, reflecting epithelial damage such as epithelial shedding and a change from a predominantly ciliated epithelium to a state of goblet cell hyperplasia. Even in early asthma, tenascin production may
J ALLERGY CLIN IMMUNOL NOVEMBER 1996
be associated with alterations in the structure of the airways, a change in the dynamics of the airway epithelium, or a change in the inflammatory process, which are reflecting a remodeling process in the context of airway epithelial damage and airways inflammation in asthma. They may all be associated with an elevated production of TGF-[3, which is known to induce synthesis of tenascin. The accumulation of tenascin may act reciprocally on the developing epithelial cells by inhibiting important receptor contacts with the matrix, thus contributing to epithelial shedding. Tenascin has been shown to interfere with epithelial cell adhesion in vivo. The role of inflammation in increased tenascin production is supported by our finding of increased numbers of eosinophils in patients with seasonal asthma. REFERENCES
1. Koskela K, Haahtela T, Lahdensuo A, Muotka R, Ahonen E, Nurmi T, et al. Asthma Programme in Finland 1994-2004. Report of a Working Group. Clin Exp Allergy 1996;26(suppl 1):1-24. 2. Howeedy AA, Virtanen I, Laitinen L, Gould NS, Koukoulis GK, Gould VE. Differential distribution of tenascin in the normal, hyperplastic and neoplastic breast. Lab Invest 1990; 63:798-806. 3. Engvall E, Davis GE, Dickerson K, Ruoslahti E, Varon S, Manthorpe M. Mapping of domains in human laminin using monoclonal antibodies: localization of the neurite promoting site, J Cell Biol 1986;103:2457-65. 4. Sakakura T, Kusakabe M. Can tenascin be redundant in cancer development? Perspect Dev Neurobiol 1994;120: 1079-81. 5. Linnala A, Kinnula V, Laitinen LA, Lehto V-P, Virtanen I. Transforming growth factor-{3 regulates the expression of fibronectin and tenascin in BEAS 2{3 human bronchial epithelial cells. Am J Respir Cell Mol Biol 1995;13:578-85. 6. McGowan SE. Extracellular matrix and the regulation of lung development and repair. FASEB J 1992;6:2895-904. 7. Kovacs EJ, DiPietro LA. Fibrogenic cytokines and connective tissue production. FASEB J 1994;8:854-61. 8. Bousquet J, Chanez P, Lacoste JY, Barneon G, Ghavanian N, Enander I, et al. Eosinophilic inflammation in asthma. N Engl J Med 1990;323:1033-9. 9. Laitinen LA, Laitinen A, Haahtela T. Airway mucosal inflammation even in patients with newly diagnosed asthma. Am Rev Respir Dis 1993;147:697-704.
Categorizing asthma according to the degree of severity of the disorder is problematic because of a lack of commonly agreed upon criteria. In a Boston G r o u p report from Finland, 1 patients with asthma were divided into five groups on the basis of given criteria. Most of the patients (60%) were considered to have mild asthma, and one fifth were considered to have severe or very severe asthma (Table I). This distribution involved adults only and is probably different in children. There have been few extensive epidemiologic population studies on the incidence and prevalence of asthma in Finland. Asthma has been From the Department of Medicine, University Central Hospital, Helsinki, Finland, the Department of Anatomy, Institute of Biomedicine, University of Helsinki, Finland, the Departments of Lung Medicine and Clinical Chemistry, University of Uppsala, Sweden, the Department of Lung Medicine, University of Lund, Sweden, and the Department of Puhnonary Medicine, University of Tartu, Estonia. Reprint requests: Lauri A. Laitinen, MD, PhD, Department of Medicine, Helsinki University Central Hospital, Haartmaninkatu 4, FIN-00290 Helsinki, Finland. Copyright © 1996 by- Mosby-Year Book, Inc. 0091-6749/96 $5.00 + 0 1/0/76643
Abbreviations used mAb: Monoclonal antibody PD15FEV~: Provocative dose causing a 15% decrease in FEV 1 TGF-[3: Transforming growth factor-J3
diagnosed in approximately 150,000 Finns, or 3% of the population. A similar percentage of the population receives drug therapy for asthma or receives social security benefits because of asthma. Depending on the methodology, study material, and definition of asthma, the prevalence rate may be up to 5% or 250,000 persons. The diagnosis of asthma can be inaccurate because some persons have only occasional asthma-like symptoms, such as prolonged cough and mild respiratory distress, with no observable impairment on the basis of pulmonary function tests. These symptoms may be associated with stress, respiratory infections, or exposure to allergens and are usually of brief duration. Some persons with these symptoms may develop asthma (Fig. 1). $3
S4
Laitinen eta[.
J ALLERGY CLIN IMMUNOL NOVEMBER 1996
No symptoms
TABLE I. Distribution of patients w i t h asthma according to severity of disease
Severity
%
Description
Very severe
2%
Disabling disease; patient spends numerous days in bed; lifethreatening attacks Daily wheezing, poor general condition, disease restricts lifestyle, severe nocturnal symptoms; patient is absent from work for several weeks a year; one in two patients needs hospital treatment Daily symptoms but no significant diurnal variation; patient avoids exercise and has occasional nocturnal symptoms Periodic symptoms; patient reacts to stimulants such as pollen or intense cold; symptoms restrict activity two or three times a week Occasional cough or wheezing that does not cause major impairment; respiratory tract is sensitive to intense cold and infections; allergens cause symptoms of varying degree, depending on exposure
Severe
18%
Moderate
20%
Mild
20%
Very mild
40%
FIG. 1. Asthma and asthmaqike symptoms. (From Koskela K, et al. Asthma Programme in Finland 1994-2004. Report of a Working Group. Clin Exp Allergy 1996;26(suppl):1-24).
W e studied groups of patients w h o m we consider to have intermittent or "early" asthma, including patients with seasonal asthma and occupational asthma. Bronchial biopsy specimens were obtained to study extracellular matrix proteins and inflammatory cells with use of specific antibodies, and the results of patients with seasonal asthma and occupational a s t h m a were c o m p a r e d with those of healthy control subjects.
METHODS Patients Characteristics of patients with seasonal and occupational asthma are presented in Table II. In the patients with seasonal asthma, diagnosis was based on a clinical history of seasonal symptoms during the birch pollen season and skin prick tests that revealed a positive reaction (>3 mm in diameter) for birch pollen (ALK Allergologisk Laboratorium A/S, H~arsholm, Denmark, reagents). Birch pollen-specific serum IgE, measured by a radioallergosorbent test, was present in all patients with seasonal asthma. In patients with occupational asthma, symptoms were associated with exposure to aluminum fluoride (2 patients), welding fumes (2), piperazine (1), methyltetrahydrophthalic anhydride (1), sulfuric acid aerosols (1), or isocyanates (1) lasting from 1 to 48 months (15 -+ 5.5 months, mean +- SEM). Only one patient was allergic to timothy grass, as revealed by skin prick testing. All patients with occupational asthma had bronchial hyperresponsiveness, as tested by methacholine provocation. The provocative concentration causing a reduction of 15% in FEV 1 (PCrsFEV1) ranged from 0.05 to 0.455 mg/ml (0.14 _+ 0.05). None of the 12 control subjects had a history of any respiratory or systemic disease, nor did routine hematologic and serum biochemistry tests show any abnormalities. All control subjects were nonsmokers. None was allergic, on the basis of their histories and negative results of skin prick tests with a panel of t2 commonly used allergens (ALK Allergologisk Laboratorium A/S).
From Koskela K, et al. Asthma Programme in Finland t9942004. Report of a Working Group. Clin Exp Allergy 1996; 26(suppt):1-24.
Lung function was normal in all control subjects. FEV 1 increased less than 15% in a bronchodilator test with 3 × 200 ixg inhaled rimiterol (Pulmadil, 3M Health Care Ltd., Loughborough, United Kingdom). The study was approved by local ethics committees, and written informed consent was obtained from each participant.
Bronchoscopy and bronchial biopsies Bronchoscopic examination and the taking of biopsy specimens conformed to the international guidelines for investigative use of fiberoptic bronchoscopy in patients with asthma. Premedication for all subjects consisted of either atropine (0.5 to 1 mg) or scopolamine (0.3 to 0.4 mg), and droperidol (5 to 10 rag) or morphine hydrochloride (7.5 to 10 rag). Local anesthesia was achieved with the application of lignocaine spray or 2% lignocaine solution to the oropharynx immediately before the procedure, and further anesthesia was achieved by instillation of a 2% lignocaine solution through the bronchoscope into the bronchial tree. Bronchoscopy was performed with the fiberoptic instruments Olympus BF-20 or BT-IT-20D (Olympus Optical Co. Ltd., Tokyo, Japan) in all study groups except the one that included
J ALLERGY CL1N IMMUNOL VOLUME 98, NUMBER 5, PART 2
L a i t i n e n e t al.
$5
TABLE II. Patient data (mean ± SEM, range) Seasonal asthma (n= 17) Sex Age (yr) Weight (kg) Height (cm) Duration of asthma (yr) FEV 1 (% of predicted)
6 Female, 11 Male 33.7 + 1.7 (21-44) 75.7 ± 3.0 (56-95) 176 ± 2.3 (160-194) 5.3 + 0.7 (2-12) 96.2 + 2.5 (71-115)
patients with occupational asthma; the Olympus BFXT-20 fiberscope was used with this group. Bronchial biopsy specimens were taken from the right upper and middle lobe bronchi. Care was taken to avoid regions close to carinae and regions previously touched by the bronchoscope. In control subjects, the specimens were taken with Olympus FB-19C forceps; in patients with seasonal asthma, specimens were taken with the Olympus FB-15K; and in patients with occupational asthma, specimens were taken with the Olympus FB-35C forceps.
Immunostaining Sections (5 Fm thick) were cut and fixed in acetone at - 2 0 ° C and stained with use of the indirect immunofluorescence technique. Mouse monoclonal antibodies (mAbs) against tenascin (2), collagen type VII (GIBCO BRL Research Products, Inc., Gaithersburg, Md.) and collagen types III (HEYL, GmbH, Berlin, Germany) and IV (Boehringer Mannheim, GmbH, Germany) were used. The sections were stained with a mouse monoclonal antibody (mAb) to tenascin, 100EB2, which reacts with the fourth and fifth fibronectin-like repeat in the tenascin-C molecule. 2 After washing, the sections were incubated with fluorescein isothiocyanate (FITC)-coupled sheep anti-mouse IgG (Jackson Immunoresearch Laboratories, Inc., West Grove, Pa.). After washing, the sections were mounted and examined under a Leitz Aristoplan fluorescence microscope (Ernst Leitz GmbH, Wetzler, Germany) equipped with appropriate filters. Negative controls were provided by omitting the primary mAb or by replacing it with an irrelevant one of the same isotope. Selected samples were examined by double label immunostaining. The distribution of tenascin was compared with that obtained by the mAb to laminin cd chain. 3 M e a s u r e m e n t of the i m m u n o s t a i n e d area in the b a s e m e n t m e m b r a n e zone After immunostaining, the cross-sectional area of the basement membrane zone from each section was photographed at an original magnification of ?<250. Black and white paper pictures from each specimen were made with a final magnification of ×643. For each photograph the deeper border of the basement membrane zone with continuous immunoreactivity was charted along its maximal length semiautomatically with use of a pointing device; charting was performed on a Compaq Deskpro
Occupational asthma (n=8) 35.8 82.6 182 9.4 81.1
8 Male _+ 2.1 (29-45) +- 3.3 (70-95) ± 2.5 (172-191) ± 2.2 (3-20) -+ 2.1 (69-89)
386 computer (Compaq Computer Corp., Houston, Tex.) from a 42 × 60 inch Kurta IS/THREE digitizing tablet (Kurta Corp., Phoenix, Ariz.). The pointing device was used in a similar manner for the superficial limit of the immunostained area. The image pictures were generated and the data processed by means of the AUTOCAD graphics program (Autodesk, Inc., Sausalito, Calif.). The minimal distances from each superficial point to the point closest in the deeper border of the immunoreactivity zone and their average values and standard deviations were calculated by the program. The average values were considered to be the thickness of the immunostained area of the basement membrane in each specimen. The thicknesses were also counted by dividing the surface area of the stained area with the corresponding length of the measured basement membrane area. When these two measurements were compared, there was no statistical difference between the mean thicknesses obtained by the two methods. With use of the method previously described, we measured expressions of tenascin and collagens III, IV, and VII.
Quantitation of i n f l a m m a t o r y cells Inflammatory cells were stained with use of mAbs and the alkaline phosphatase antialkaline phosphatase staining method (Dako A/S, Glostrup, Denmark). The quantitation was conducted with use of a computerized system applying the AUTOCAD program, as previously described. Eosinophils, mast cells, macrophages, and T lymphocytes were measured. Statistical analysis The results were analyzed with the Kruskal-Wallis one way analysis of variance by ranks. Where appropriate, the Mann-Whitney U-test, together with correction for multiple comparisons, served to estimate the difference between controls and patient groups. RESULTS A significant increase in tenascin expression was f o u n d in the b a s e m e n t m e m b r a n e o f patients with seasonal (p < 0.05) and occupational asthma (p < 0.001) c o m p a r e d with that of control subjects. O f the collagens studied, collagen types IV and type V I I were increased only in patients with occupational asthma (p < 0.005). Of the inflammatory cells stud-
S6
Laitinen et al.
ied, the numbers of eosinophils were increased in patients with seasonal asthma (p < 0.001). DISCUSSION
The airways of patients with seasonal and occupational asthma showed an increased accumulation of tenascin in the basement m e m b r a n e area. It is not known if this is a result of upregulated production or decreased breakdown of tenascin. In patients with asthma the tenascin band was mainly restricted to the basement m e m b r a n e zone and was not seen in the epithelium or diffusing into the airway stroma. Increased tenascin expression was seen close to the epithelial cells, which argues in favor of an abnormality in this region. It has been shown that epithelial-stromal interactions are important in the production of tenascin and that both the epithelial and stromal cells may serve as a source of tenascin. 4 While extracellular matrix proteins are important in controlling the development of the lung during early life, the restricted distribution of tenascin in healthy adult airways and other organs and its upregulation in pathologic situations suggest that mechanisms exist to downregulate and upregulate tenascin expression in adult organs. It has been suggested that both transforming growth factor-[3 (TGF-[3) and certain cytokines upregulate tenascin production in cell culture studies. 5 Tenascin and T G F - 6 are both coexpressed during budding morphogenesis of the bronchi 6 and in diffuse inflammatory processes of the lung tissue. 7 On the basis of biopsy studies, bronchial asthma is considered to be an inflammatory disease of the airways. 8 Even the airways of patients with newly detected and mild asthma, who have had asthma for a relatively short time, show changes related to chronic inflammation. 9 These changes are associated with structural changes in the airway epithelium, reflecting epithelial damage such as epithelial shedding and a change from a predominantly ciliated epithelium to a state of goblet cell hyperplasia. Even in early asthma, tenascin production may
J ALLERGY CLIN IMMUNOL NOVEMBER 1996
be associated with alterations in the structure of the airways, a change in the dynamics of the airway epithelium, or a change in the inflammatory process, which are reflecting a remodeling process in the context of airway epithelial damage and airways inflammation in asthma. They may all be associated with an elevated production of TGF-[3, which is known to induce synthesis of tenascin. The accumulation of tenascin may act reciprocally on the developing epithelial cells by inhibiting important receptor contacts with the matrix, thus contributing to epithelial shedding. Tenascin has been shown to interfere with epithelial cell adhesion in vivo. The role of inflammation in increased tenascin production is supported by our finding of increased numbers of eosinophils in patients with seasonal asthma. REFERENCES
1. Koskela K, Haahtela T, Lahdensuo A, Muotka R, Ahonen E, Nurmi T, et al. Asthma Programme in Finland 1994-2004. Report of a Working Group. Clin Exp Allergy 1996;26(suppl 1):1-24. 2. Howeedy AA, Virtanen I, Laitinen L, Gould NS, Koukoulis GK, Gould VE. Differential distribution of tenascin in the normal, hyperplastic and neoplastic breast. Lab Invest 1990; 63:798-806. 3. Engvall E, Davis GE, Dickerson K, Ruoslahti E, Varon S, Manthorpe M. Mapping of domains in human laminin using monoclonal antibodies: localization of the neurite promoting site, J Cell Biol 1986;103:2457-65. 4. Sakakura T, Kusakabe M. Can tenascin be redundant in cancer development? Perspect Dev Neurobiol 1994;120: 1079-81. 5. Linnala A, Kinnula V, Laitinen LA, Lehto V-P, Virtanen I. Transforming growth factor-{3 regulates the expression of fibronectin and tenascin in BEAS 2{3 human bronchial epithelial cells. Am J Respir Cell Mol Biol 1995;13:578-85. 6. McGowan SE. Extracellular matrix and the regulation of lung development and repair. FASEB J 1992;6:2895-904. 7. Kovacs EJ, DiPietro LA. Fibrogenic cytokines and connective tissue production. FASEB J 1994;8:854-61. 8. Bousquet J, Chanez P, Lacoste JY, Barneon G, Ghavanian N, Enander I, et al. Eosinophilic inflammation in asthma. N Engl J Med 1990;323:1033-9. 9. Laitinen LA, Laitinen A, Haahtela T. Airway mucosal inflammation even in patients with newly diagnosed asthma. Am Rev Respir Dis 1993;147:697-704.