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Immunohistochemical classification of the localization of laminin in the thickened bronchial epithelial basement membrane of deceased bronchial asthma patients
Vol. 97 (2003) 688 ^ 694
Immunohistochemical classi¢cation of the localization of laminin in the thickened bronchial epithelial basement membrane of deceased bronchial asthma patients A. SAOTOME*, N. KANAIw,T. NAGAI*,T.YASHIROz, AND S.TOKUDOME* *Department of Legal Medicine, Dokkyo University School of Medicine, 880, Kitakobayashi, Mibu, Tochigi 321- 0293, Japan; wDepartment of Pathology, Jichi Medical School, Tochigi, Japan; zDepartment of Anatomy, Jichi Medical School, Tochigi, Japan Abstract To ascertain histological changes in the basal lamina of the bronchial epithelial basement membrane in patients with severe bronchial asthma, an immunohistochemical study was conducted in 43 patients who died of bronchial asthma. Antibodies againstlaminin, a component ofthe lamina lucida, were utilized.The results revealed various patterns for immunoreactivity to laminin in the thickened basement membrane layer.We were able to classify these reactivities into four patterns. In Pattern A, laminin reactions branched vertically in relation to the thickened basement membrane layer.In Pattern B, laminin reactions formed lines along the lower margin of the thickened basement membrane layer. In Pattern C, laminin reactions formed lines along the upper margin of the thickened basement membrane layer. Finally, in Pattern D, no laminin reactions were observed.In addition, relationships between immunohistological characteristics of laminin and findings such as epithelial cell shedding, basal cell proliferation and basement membrane layer thickening were investigated.In many Pattern A patients, epithelial cell shedding was observed, but goblet cell hyperplasia and basal cell proliferation were barely detectable.Conversely, in numerous Pattern D patients, epithelial cell shedding was barely seen, butgobletcellhyperplasia and basal cellproliferation were marked.Hence,Patterns A and D were on opposite ends of the spectrum of morphological characteristics associated with severe bronchial asthma. In Patterns B and C, laminin reactions formedlines along the lower and upper margin ofthe thickened basement membranelayer, respectively. However, no marked differences existed in epithelial cell shedding and basement membrane layer thickening. The present study is thus the firstto clarify that laminin reactions in the thickened basement membrane layer vary, and this feature is unique to the bronchi of patients with severe bronchial asthma. r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1053/rmed.2003.1502, available online at http://www.sciencedirect.com
Keywords immunohistochemistry; laminin; severe bronchial asthma; epithelial basement membrane.
INTRODUCTION The principal components of the basement membrane are glycoproteins such as collagen type IV, glycosaminoglycan, laminin, ¢bronectin and entactin (1^5). Under electron microscopy, the basement membrane has three layers: lamina lucida, lamina densa and lamina reticularis, in order from the cell side (5^7). Laminin, the principal component of the lamina lucida, is secreted from endothelial and epithelial cells, and binds strongly with collagen type IV (8 ^10) to play an important role in the Received 28 May 2002; accepted in revised form 4 November 2002 Corresponding author should be addressed to author. Dr. Shogo Tokudome, Department of Legal Medicine, Dokkyo University School of Medicine, 880, Kitakobayashi, Mibu, Tochigi 321- 0293, Japan. Fax: +81-282- 86 -7678
adhesion, di¡erentiation and migration of these cells (4,9,10).When injury causes epithelial cells to detach, cells are regenerated along the existing basement membrane. Since a lack of the basement membrane hinders cell regeneration, this membrane is considered to act as an anchor for cell proliferation (8). For a long time, basement membrane layer thickening has been considered one of the typical morphological changes in the bronchi of patients with bronchial asthma (11). This thickening is seen as a uniform amorphic thick layer under light microscopy. Nonetheless, electron microscopy reveals thickening of the lamina reticularis, while the basal lamina, consisting of the lamina lucida and lamina densa, does not thicken (12,13). Many studies have been conducted to investigate histological changes in the lamina reticularis (12^22), but
IMMUNOHISTOCHEMICAL CLASSIFICATION OF THE LOCALIZATION OF LAMININ
few studies have investigated the basal lamina (12,13),and virtually no studies have examined the basal lamina of patients with severe bronchial asthma. The present study immunohistochemically investigated laminin, the principal component of the lamina lucida, to ascertain histological changes in the thickened basement membrane layer in the lungs of individuals who died of severe bronchial asthma. The results demonstrated that immunoreactivity to laminin in the basement membrane layer was not uniform in all patients, showing a great degree of variation.
MATERIALS AND METHODS Subjects were 43 patients who died of bronchial asthma and were autopsied at theTokyo Medical Examiner’s Of¢ce during about 10 years since 1989. The subject group comprised 30 men and 13 women, ranging in age from 22 to 74 years (mean: 50 years) at time of death. In every patient, autopsy revealed lung hyperin£ation and mucus plug in the bronchial lumen, and pathological ¢ndings revealed severe bronchial lesions. No other lethal abnormalities were observed in other organs. Although the duration of bronchial asthma could not be ascertained, inspection reports yielded information about the health status of most patients at the time of death. The general health of two patients was ¢ne; 2 patients were sick but not receiving medical care; 1 patient had received medical care in the past, but not at the time of death; and 36 patients were receiving treatments. Health status at time of death was unknown for 2 patients. For controls, 20 patients younger than 65 years, who were autopsied within 24 h of death at our department, were subjected to the same set of tests. These control patients had not died of respiratory organ injuries such as carbon monoxide poisoning, burns, asphyxiation caused by neck compression, or drowning, and they did not display pulmonary abnormalities. As a general rule, a 10% formaldehyde solution was injected via the bronchi into the lungs at a low pressure for ¢xation. However, this could not be performed in some patients. After ¢xation for 2^ 4 days, a total of 15 specimens were collected from three locations in each left and right lung, and created an incision perpendicular to the longitudinal axis of the airway. Each formalin-¢xed specimens were embedded in para⁄n and stored at room temperature. Then, 3^ 4 mm sections were prepared from each specimens, depara⁄nized, and subjected to immunohistochemical staining using antilaminin antibodies. Since changes associated with bronchial asthma have been reported in the segmental and subsegmental bronchi (23^25), the cross-sections of the subsegmental bronchus of each patient were used. Immunostaining was performed using a Vectastain ABCPO mouse IgG kit (Vector Laboratories Inc., Burlin-
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game, USA) according to the avidin ^ biotin ^ peroxidase complex (ABC) method. After depara⁄nization, endogenous peroxidase deactivation was performed using 3% hydrogen peroxide solution for 3 min. Next, these sections were treated with 0.5% trypsin for 20 min to activate antigens. In order to block nonspeci¢c reactions, normal horse serum was allowed to react with sections for 20 min. As primary antibody, mouse anti-human laminin monoclonal antibody (Daiichi Fine Chemical Co., Ltd., Toyama, Japan) was diluted to 6.25 mg/mL and allowed to react with the sections at 301C overnight. After washing the sections using phosphate bu¡ered saline (PBS), biotin-labeled secondary antibody (horse antimouse IgG antibody) was allowed to react with the sections at 371C for 30 min.Then, the avidin ^ biotin ^peroxidase complex was allowed to react with the sections at 371C for 30 min., and the resulting sections were immersed in a DAB solution (0.02% 3, 3’-diaminobenzidine tetrahydrochloride, 5% H2O2 in 0.05 M Tris-HCl, pH 7.6) (Dojindo Laboratories Inc., Kumamoto, Japan). After staining nuclei using hematoxylin, ENTELLAN (Merck KgaA, Darmstadt, Germany) was used for mounting, and photographs were taken under a light microscope (PROVIS AX80, OLYMPUS, Tokyo, Japan). Furthermore, with some specimens, the relationship between immunoreactivity to laminin and such ¢ndings as, goblet cell hyperplasia, epithelial cell shedding, basal cell proliferation and basement membrane layer thickening, was investigated.
RESULTS Figure 1 shows laminin reactions in the subsegmental bronchus of a normal lung. The surface of the bronchus was covered with pseudostrati¢ed ciliated epithelia, and laminin reactions formed lines under the epithelial cells.
FIG. 1. Immunohistochemical staining with antibody anti-human laminin in normal bronchus. Antibody anti-laminin shows strong positivity along the epithelial cells.Thick laminin reaction lines under rows of pseudostrati¢ed ciliated epithelium (ABC method, original magni¢cation 700).
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Some laminin reactions were also seen in the lamina propria. Laminin reactions detected in the thickened basement membrane layer of the subsegmental bronchus of bronchial asthma patients were divided into four patterns: In Pattern A, laminin reactions branched out vertically in relation to the basement membrane layer; in Pattern B, laminin reactions formed lines along the lower margin of the basement membrane layer; in Pattern C, laminin reactions formed lines along the upper margin of the basement membrane layer; and in Pattern D, no laminin reactions were seen.When di¡erent patterns were seen, a predominating pattern was selected for classi¢cation purposes. In the lamina propria, ¢ndings such as, in£ammatory cell in¢ltration, edema, vasodilatation, smooth muscle hypertrophy and submucosal gland hypertrophy were seen, but there were no marked di¡erences among the four patterns. Nonetheless, as will be discussed later, there were slight di¡erences in the degree of capillary proliferation among the four patterns. In Pattern A, laminin reactions formed lines that ran vertically in relation to the basement membrane layer. Individual lines were narrow and thin. Also, some laminin reactions surrounded capillaries in the lamina propria and extended to the basement membrane layer. Although laminin reactions formed a single narrow thin line in the lamina propria, they branched out into several lines in the basement membrane layer. The tip of these branches extended mostly vertically or at an angle, but horizontally in some cases (Fig. 2).This pattern was seen around the entire circumference of the basement membrane layer in four patients and in one to two-thirds of the entire circumference in the remaining patients. In these patients with Patten A reactions that did not surround the entire circumference, Pattern B (laminin
FIG. 2. Pattern A immunoreactivity for laminin of the basement membrane layer for deceased bronchial asthma subject. Laminin reactions run perpendicular to the thickened basement membrane layer.Laminin reactions surrounding capillaries in the lamina propria extend into basement membrane layer in some areas (ABC method, (original magni¢cation 700).
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reactions along the lower margin of the basement membrane) or Patten D (no laminin reactions) was also seen. Capillary proliferation in the lamina propria, mostly ranging from moderate to severe in severity, was seen in almost all patients. Pattern B laminin reactions were seen along the lower margin of the basement membrane layer. Although most laminin reactions appeared to form lines, there were some notable spots, and where lines were terminated, some lines extended vertically in relation to the basement membrane layer. In all ¢ve patients, reactions were present partially, not all around the circumference of the basement membrane layer. Pattern A, C or D was observed where Pattern B laminin reactions were not detected. However, there was no connection between laminin reactions in the lamina propria and those in the basement membrane layer (Fig. 3). Also, when compared to the other patterns, thickening of the basement membrane layer was relatively milder for Pattern B. Furthermore, as shown in Fig. 4, Pattern C was characterized by strong thick laminin lines along the upper margin of the basement membrane layer. Pattern C reactions were seen around the entire circumference of the basement membrane layer in one patient, about1/ 3 the circumference in two patients, and 2/3 the circumference in three patients. When Pattern C reactions were partially seen around the circumference of the basement membrane layer, Pattern A-like reactions, where laminin reactions branched out in the basement membrane layer and extended narrowly and thinly, were seen in three of the six patients.These laminin reactions in the lamina propria were narrow but strong, extending toward the lower margin of the basement membrane layer in a reticular feature. Also, in Pattern D, although basement membrane layer thickening was most pronounced, no laminin
FIG. 3. Pattern B immunoreactivity for laminin of the basement membrane layer for the deceased bronchial asthma subject. Laminin reaction lines along lower margin of basement membrane layer. Although reactions appear to form lines, some pronounced dots are apparent. Among truncated lines, some reactions extend vertically (ABC method, /original magni¢cation 700).
IMMUNOHISTOCHEMICAL CLASSIFICATION OF THE LOCALIZATION OF LAMININ
FIG. 4. Pattern C immunoreactivity for laminin of the basement membrane layer for deceased bronchial asthma subject. Thick laminin reaction lines along upper margin of basement membrane layer. Basal cells are seen above these reaction lines (ABC method, original magni¢cation 700).
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FIG. 5. Pattern D immunoreactivity for laminin of the basement membrane layer for deceased bronchial asthma subject. Absolutelynolamininreactionsinthe thickened basement membrane layer. Epithelia have been replaced with goblet cells (ABC method, original magni¢cation 700).
TABLE 1. Subject characteristics. Health statement Pattern* n A B C D
Sex
15 12 M/3 F 5 4 M/1F 6 3 M/3 F 17 11M/6 F
Mean age (y) Healthy Illness or nottreated Treated in the past Treated in the present Unknown 44 (22^71) 49 (25^68) 60 (24^74) 52 (27^74)
0 0 0 2
0 2 0 0
0 0 0 1
14 3 5 14
1 0 1 0
*Classi¢cation by di¡erence of laminin reaction in basement membrane layer of subsegmental bronchus.
reactions were seen. Capillary proliferation was mild in the lamina propria, and only narrow reticular laminin reactions were seen around capillaries (Fig. 5). In most Pattern D patients, no laminin reactions were seen around the entire circumference of the basement membrane layer, but some Pattern A-like reactions were seen in six patients. Table 1 shows the number, sex, age and health status for the patients with each pattern. There were no marked di¡erences in these parameters among the four patterns. Table 2 shows the laminin reactions and epithelial ¢ndings for the 43 patients classi¢ed into the four lamininreaction patterns. In the Pattern A patients, relatively severe epithelial cell shedding was seen, and thickening of the basement membrane layer was mostly moderate or severe. In the pattern B and C patients, there were no clear di¡erences in epithelial cell shedding and basement membrane layer thickening. In the Pattern D patients, epithelial cell shedding was hardly seen, but marked goblet cell hyperplasia was seen in many patients. Furthermore, basal cell proliferation and markedly thickened basement membrane layer were seen among the Pattern D patients.
DISCUSSION Bronchial asthma is characterized by reversible obstructive changes, reactive acceleration and chronic in£ammation of the airway (26 ^28). In persistent or severe bronchial asthma, various morphological changes, such as, epithelial cell shedding, goblet cell hyperplasia and basement membrane layer thickening, are seen (11,12, 29,30). These changes can serve as indicators of the severity of bronchial asthma in airway remodeling (11,18,21,29). Thickening of the bronchial epithelial basement membrane layer is considered one of the histological changes associated with airway remodeling (11,18,21,29). The results of several electron microscopic studies have shown that this thickening is caused by abnormal deposition of interstitial collagen types I, III and Vand extracellular matrix such as ¢bronectin and tenescin, in the lamina reticularis under the bronchial epithelia (12^21). This is referred to as subepithelial ¢brosis because it is caused by the proliferation of collagen ¢bers (12^22,26). Also, increases in collagen and collagen deposition are believed to be caused by the interaction between airway epithelial cells and ¢broblasts/myo¢broblasts (17), but the
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TABLE 2. Characteristics of study groups. Subject No.
Pattern A 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Pattern B 1 2 3 4 5 Pattern C 1 2 3 4 5 6 Pattern D 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Sex*
Age (y)
Shedding of epithelial cells
Hyperplasia of goblet cells
Proliferation of globels cells
Thickness of basement membrane layer
M M M M M F F M M M F M M M M
43 71 70 24 23 68 22 25 22 29 70 62 54 42 28
w +++ +++ ++ +++ +++ ++ +++
+++ + +++
++ + ++ ++ + + +++ ++
++ +++ ++ ++ ++ ++ ++ +++ + +++ ++ + ++ +++ ++
M M M M F
25 26 67 57 68
++ +++
+++
++ + +++
++ + + ++ +
M M F F F M
24 69 74 73 68 50
+++ +++
+ ++
+ ++ ++ +++
++ ++ + +++ ++ ++
F F F M M F M M M M M F M M M F M
69 53 40 49 58 58 41 45 30 64 74 33 67 55 60 59 27
+++ +++
+++ +++ +++ + +++ ++ +++ + +++ +++ +++ +++
++ ++ +++ ++ + +++ ++ + + ++ ++ + +++ ++ +++
+ +++ +++ +++ ++ + ++ +++ ++ ++ +++ +++ + ++ +++ +++ +++
*Abbreviations: M, male; F, female. wSymbols: , negative; +, weak, ++, moderate; +++, strong.
details of this interaction are not known (20,22). In bronchial asthma patients, it has been known that laminin reactions localize notably along the upper margin of the
basement membrane layer (31,32), and that collagen type IV also localizes in the same area (33). In this study, we focused our attention on the thickened basement
IMMUNOHISTOCHEMICAL CLASSIFICATION OF THE LOCALIZATION OF LAMININ
membrane layer, and investigated histomorphological changes in the lamina lucida immunohistochemically using anti-laminin antibodies. The results showed that the localization of immunoreactivity in severe bronchial asthma is not as simple as previously thought and that laminin reactions can roughly be classi¢ed into four patterns. As shown in Table 2, there was a relationship with epithelial cell shedding. In Pattern A, epithelial cell shedding was seen in 7 of the 15 patients, while goblet cell hyperplasia was only seen in 3 of the 15 patients. In Pattern A, lines either extended vertically in relation to the basement membrane layer or they originated from the lamina propria and branched out in the basement membrane layer. Also, capillary proliferation in the lamina propria was observed in almost every patient. Pattern A laminin reactions could form as follows: the basement membrane under epithelial cells is made of epithelial cells and ¢broblasts (5,7), and since epithelial cells are generally detached and eliminated in Pattern A patients, it is di⁄cult to form a basement membrane. Nonetheless, as shown in Fig. 2, in some patients, laminin reactions in the lamina propria, in particular around capillaries, were marked and extended into the thickened basement membrane layer. We believe this is one of the reasons for seeing characteristic immunoreactivity to laminin in deceased bronchial asthma patients. In Patterns B and C, laminin reactions formed lines extending under epithelial cells: in Pattern B, lines formed along the lower margin of the basement membrane layer, whereas in Pattern C, lines formed along the upper margin of the basement membrane layer. Although laminin reactions were totally di¡erent between these two patterns, there were no clear di¡erences in epithelial cell shedding and basement membrane layer thickening. We could not, however, gather enough data to identify the cause of the di¡erence between Patterns B and C. Furthermore, in Pattern D, although immunoreactivity to laminin in the basement membrane layer was not seen at all, the proliferation of basal and goblet cells was clearly elevated in many patients when compared to the other three patterns. One of the causes for the absence of immunoreactivity to laminin in the thickened basement membrane layer could be that since capillary proliferation was hardly seen in the lamina propria in Pattern D patients, the level of laminin did not increase in the lamina propria. Also, this has not been documented but it is possible that goblet cells, which form epithelia, are not capable of producing laminin. The degree of basement membrane layer thickening for Pattern D was severer than that for the other patterns, but the reason for this was not clear. Between Patterns A and D, there were clear di¡erences in the four items shown inTable 2. In other words, in Pattern A, epithelial cell shedding was seen, but clear signs of goblet cell hyperplasia and basal cell proliferation
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were not seen. On the other hand, in Pattern D, epithelial cell shedding was hardly seen, but goblet cell hyperplasia and basal cell proliferation were marked. As a result, Patterns A and D may be on the opposite ends of the morphological changes associated with severe bronchial asthma. The above ¢ndings clarify that immunoreactivity to laminin in the basement membrane layer of patients with severe bronchial asthma shows a great degree of variation. The prerequisite for having basement membrane layer thickening, one component of airway remodeling, is the presence of the basal lamina, but some patients showed no immunoreactivity to laminin (Pattern D) or did not have a normal laminin structure (Pattern A). As a result, we concluded that conditions were too severe for remodeling to occur in patients with severe bronchial asthma. In other words, the histological characteristics of laminin are very unique in patients with severe bronchial asthma.
REFERENCES 1. Yaar M, Foidart JM, Brown KS, Rennard SI, Martin GR, Liotta L. The Goodpasture-like syndrome in mice induced by intravenous injections of anti-typeIV collagen and anti-laminin antibody. Am J Pathol 1982; 107: 79–91. 2. Raghu G, Striker LJ, Hudson LD, Striker GE. Extracellular matrix in normal and fibrotic human lungs. Am Rev Respir Dis 1985; 131: 281–289. 3. Gil J, Martinez-Hernandez A. The connective tissue of the rat lung: electron immunohistochemical studies. JHistochem Cytochem 1984; 32: 230–238. 4. Timpl R. Structure and biological activity of basement membrane proteins. Eur J Biochem 1989; 180: 487–502. 5. Martinez-Hernandez A, Amenta PS. The basement membrane in pathology. Lab Invest 1983; 48: 656–677. 6. Gartner LP, Hiatt JL. Extracellular Matrix. In: Schmitt B, Thorp D, Robins CJ, Ware N, editors. Color textbookof histology. Philadelphia: W.B. Saunders Company, 2001. 7. Pierce GB, Nakane PK. Basement membranes. Synthesis and deposition in response to cellular injury. Lab Invest 1969; 21: 27–41. 8. Yurchenco PD, Cheng YS, Colognato H. Laminin forms an independent network in basement membranes. J Cell Biol 1992; 117: 1119–1133. 9. Foidart JM, Bere Jr EW , Yaar M, et al. Distribution and immunoelectron microscopic localization of laminin, a noncollagenous basement membrane glycoprotein. Lab Invest 1980; 42: 336–342. 10. Matsubara O, Tamura A, Ohdama S, Mark EJ. Alveolar basement membrane breaks down in diffuse alveolar damage: an immunohistochemical study. Pathol Int 1995; 45: 473–482. 11. Jeffery PK, Laitinen A, Venge P. Biopsy markers of airway inflammation and remodelling. Respir Med 2000; 94: Suppl F S9–15. 12. Vignola AM, Kips J, Bousquet J. Tissue remodeling as a feature of persistent asthma. J Allergy Clin Immunol 2000; 105: 1041–1053. 13. Roche WR, Beasley R, Williams JH, Holgate ST. Subepithelial fibrosis in the bronchi of asthmatics. Lancet 1989; 1: 520–524. 14. Laitinen LA, Altraja A, Karjalainen EM, Laitinen A. Early interventions in asthma with inhaled corticosteroids. J Allergy Clin Immunol 2000; 105: S582–585.
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15. Boulet LP, Laviolette M, Turcotte H, et al. Bronchial subepithelial fibrosis correlates with airway responsiveness to methacoline. Chest 1997; 112: 45–52. 16. Chakir J, Laviolette M, Boutet M, Laliberte R, Dube J, Boulet LP. Lower airways remodeling in nonasthmatic subjects with allergic rhinitis. Lab Invest 1996; 75: 735–744. 17. Brewster CE, Howarth PH, Djukanovic R, Wilson J, Holgate ST, Roche WR. Myofibroblasts and subepithelial fibrosis in bronchial asthma. Am J Respir Cell Mol Biol 1990; 3: 507–511. 18. Hoshino M, Nakamura Y, Sim J, Shimojo J, Isogai S. Bronchial subepithelial fibrosis and expression of matrix metalloproteinase-9 in asthmatic airway inflammation. J Allergy Clin Immunol 1998; 102: 783–788. 19. Bousquet J, Vignola AM, Chanez P, Champbell AM, Bonsignore G, Michel FB. Airways remodelling in asthma: no doubt, no more? Int Arch Allergy Immunol 1995; 107: 211–214. 20. Chetta A, Foresi A, Del Donno M, Bertorelli G, Pesci A, Olivieri D. Airways remodeling is a distinctive feature of asthma and is related to severity of disease. Chest 1997; 111: 852–857. 21. Redington AE. Fibrosis and airway remodeling. Clin Exp Allergy 2000; 30: Suppl 1 42–45. 22. Busse W, Elias J, Sheppard D, Banks-Schlegel S. Airway remodeling and repair. Am J Respir Crit Care Med 1999; 160: 1035–1042. 23. Bradley BL, Azzawi M, Jacobson M, et al. Eosinophils, T-lymphocytes, mast cells, neutrophils, and macrophages in bronchial biopsy specimens from atopic subjects with asthma: comparison with biopsy specimens from atopic subjects without asthma and normal control subjects and relationship to bronchial hyperresponsiveness. J Allergy Clin Immunol 1991; 88: 661–674.
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24. Azzawi M, Bradley B, Jeffery PK, et al. Identification of activated T lymphocytes and eosinophils in bronchial biopsies in stable atopic asthma. Am Rev Respir Dis 1990; 142: 1407–1413. 25. Metzger WJ, Zavala D, Richerson HB, et al. Local allergen challenge and bronchoalveolar lavage of allergic asthmatic lungs. Description of the model and local airway inflammation. Am Rev Respir Dis 1987; 135: 433–440. 26. Laitinen A, Laitinen LA. Pathology of asthma. Allergy Proc 1994; 15: 323–328. 27. O’Byrne PM, Inman MD. The clinical expression of allergy in the lungs. Allergy 1999; 54: 7–14. 28. Kay AB. Asthma and inflammation. J Allergy Clin Immunol 1991; 87: 893–910. 29. Stirling RG, Chung KF. Severe asthma: definition and mechanisms. Allergy 2001; 56: 825–840. 30. Bai TR, Cooper J, Koelmeyer T, Pare PD, Weir TD. The effect of age and duration of disease on airway structure in fatal asthma. Am J Respir Crit Care Med 2000; 162: 663–669. 31. Altraja A, Laitinen A, Virtanen I, et al. Expression of laminins in the airways in various types of asthmatic patients: a morphometric study. Am J Respir Cell Mol Biol 1996; 15: 482–488. 32. Hashimoto H, Hoshino K. Immunohistochemical localization of laminin in fetal mouse lungs. Acta Histochem Cytochem 1988; 21: 125–136. 33. Chu HW, Halliday JL, Martin RJ, Leung DY, Szefler SJ, Wenzel SE. Collagen deposition in large airways may not differentiate severe asthma from milder forms of the disease. Am J Respir Crit Care Med 1998; 158: 1936–1944.
Immunohistochemical classi¢cation of the localization of laminin in the thickened bronchial epithelial basement membrane of deceased bronchial asthma patients A. SAOTOME*, N. KANAIw,T. NAGAI*,T.YASHIROz, AND S.TOKUDOME* *Department of Legal Medicine, Dokkyo University School of Medicine, 880, Kitakobayashi, Mibu, Tochigi 321- 0293, Japan; wDepartment of Pathology, Jichi Medical School, Tochigi, Japan; zDepartment of Anatomy, Jichi Medical School, Tochigi, Japan Abstract To ascertain histological changes in the basal lamina of the bronchial epithelial basement membrane in patients with severe bronchial asthma, an immunohistochemical study was conducted in 43 patients who died of bronchial asthma. Antibodies againstlaminin, a component ofthe lamina lucida, were utilized.The results revealed various patterns for immunoreactivity to laminin in the thickened basement membrane layer.We were able to classify these reactivities into four patterns. In Pattern A, laminin reactions branched vertically in relation to the thickened basement membrane layer.In Pattern B, laminin reactions formed lines along the lower margin of the thickened basement membrane layer. In Pattern C, laminin reactions formed lines along the upper margin of the thickened basement membrane layer. Finally, in Pattern D, no laminin reactions were observed.In addition, relationships between immunohistological characteristics of laminin and findings such as epithelial cell shedding, basal cell proliferation and basement membrane layer thickening were investigated.In many Pattern A patients, epithelial cell shedding was observed, but goblet cell hyperplasia and basal cell proliferation were barely detectable.Conversely, in numerous Pattern D patients, epithelial cell shedding was barely seen, butgobletcellhyperplasia and basal cellproliferation were marked.Hence,Patterns A and D were on opposite ends of the spectrum of morphological characteristics associated with severe bronchial asthma. In Patterns B and C, laminin reactions formedlines along the lower and upper margin ofthe thickened basement membranelayer, respectively. However, no marked differences existed in epithelial cell shedding and basement membrane layer thickening. The present study is thus the firstto clarify that laminin reactions in the thickened basement membrane layer vary, and this feature is unique to the bronchi of patients with severe bronchial asthma. r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1053/rmed.2003.1502, available online at http://www.sciencedirect.com
Keywords immunohistochemistry; laminin; severe bronchial asthma; epithelial basement membrane.
INTRODUCTION The principal components of the basement membrane are glycoproteins such as collagen type IV, glycosaminoglycan, laminin, ¢bronectin and entactin (1^5). Under electron microscopy, the basement membrane has three layers: lamina lucida, lamina densa and lamina reticularis, in order from the cell side (5^7). Laminin, the principal component of the lamina lucida, is secreted from endothelial and epithelial cells, and binds strongly with collagen type IV (8 ^10) to play an important role in the Received 28 May 2002; accepted in revised form 4 November 2002 Corresponding author should be addressed to author. Dr. Shogo Tokudome, Department of Legal Medicine, Dokkyo University School of Medicine, 880, Kitakobayashi, Mibu, Tochigi 321- 0293, Japan. Fax: +81-282- 86 -7678
adhesion, di¡erentiation and migration of these cells (4,9,10).When injury causes epithelial cells to detach, cells are regenerated along the existing basement membrane. Since a lack of the basement membrane hinders cell regeneration, this membrane is considered to act as an anchor for cell proliferation (8). For a long time, basement membrane layer thickening has been considered one of the typical morphological changes in the bronchi of patients with bronchial asthma (11). This thickening is seen as a uniform amorphic thick layer under light microscopy. Nonetheless, electron microscopy reveals thickening of the lamina reticularis, while the basal lamina, consisting of the lamina lucida and lamina densa, does not thicken (12,13). Many studies have been conducted to investigate histological changes in the lamina reticularis (12^22), but
IMMUNOHISTOCHEMICAL CLASSIFICATION OF THE LOCALIZATION OF LAMININ
few studies have investigated the basal lamina (12,13),and virtually no studies have examined the basal lamina of patients with severe bronchial asthma. The present study immunohistochemically investigated laminin, the principal component of the lamina lucida, to ascertain histological changes in the thickened basement membrane layer in the lungs of individuals who died of severe bronchial asthma. The results demonstrated that immunoreactivity to laminin in the basement membrane layer was not uniform in all patients, showing a great degree of variation.
MATERIALS AND METHODS Subjects were 43 patients who died of bronchial asthma and were autopsied at theTokyo Medical Examiner’s Of¢ce during about 10 years since 1989. The subject group comprised 30 men and 13 women, ranging in age from 22 to 74 years (mean: 50 years) at time of death. In every patient, autopsy revealed lung hyperin£ation and mucus plug in the bronchial lumen, and pathological ¢ndings revealed severe bronchial lesions. No other lethal abnormalities were observed in other organs. Although the duration of bronchial asthma could not be ascertained, inspection reports yielded information about the health status of most patients at the time of death. The general health of two patients was ¢ne; 2 patients were sick but not receiving medical care; 1 patient had received medical care in the past, but not at the time of death; and 36 patients were receiving treatments. Health status at time of death was unknown for 2 patients. For controls, 20 patients younger than 65 years, who were autopsied within 24 h of death at our department, were subjected to the same set of tests. These control patients had not died of respiratory organ injuries such as carbon monoxide poisoning, burns, asphyxiation caused by neck compression, or drowning, and they did not display pulmonary abnormalities. As a general rule, a 10% formaldehyde solution was injected via the bronchi into the lungs at a low pressure for ¢xation. However, this could not be performed in some patients. After ¢xation for 2^ 4 days, a total of 15 specimens were collected from three locations in each left and right lung, and created an incision perpendicular to the longitudinal axis of the airway. Each formalin-¢xed specimens were embedded in para⁄n and stored at room temperature. Then, 3^ 4 mm sections were prepared from each specimens, depara⁄nized, and subjected to immunohistochemical staining using antilaminin antibodies. Since changes associated with bronchial asthma have been reported in the segmental and subsegmental bronchi (23^25), the cross-sections of the subsegmental bronchus of each patient were used. Immunostaining was performed using a Vectastain ABCPO mouse IgG kit (Vector Laboratories Inc., Burlin-
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game, USA) according to the avidin ^ biotin ^ peroxidase complex (ABC) method. After depara⁄nization, endogenous peroxidase deactivation was performed using 3% hydrogen peroxide solution for 3 min. Next, these sections were treated with 0.5% trypsin for 20 min to activate antigens. In order to block nonspeci¢c reactions, normal horse serum was allowed to react with sections for 20 min. As primary antibody, mouse anti-human laminin monoclonal antibody (Daiichi Fine Chemical Co., Ltd., Toyama, Japan) was diluted to 6.25 mg/mL and allowed to react with the sections at 301C overnight. After washing the sections using phosphate bu¡ered saline (PBS), biotin-labeled secondary antibody (horse antimouse IgG antibody) was allowed to react with the sections at 371C for 30 min.Then, the avidin ^ biotin ^peroxidase complex was allowed to react with the sections at 371C for 30 min., and the resulting sections were immersed in a DAB solution (0.02% 3, 3’-diaminobenzidine tetrahydrochloride, 5% H2O2 in 0.05 M Tris-HCl, pH 7.6) (Dojindo Laboratories Inc., Kumamoto, Japan). After staining nuclei using hematoxylin, ENTELLAN (Merck KgaA, Darmstadt, Germany) was used for mounting, and photographs were taken under a light microscope (PROVIS AX80, OLYMPUS, Tokyo, Japan). Furthermore, with some specimens, the relationship between immunoreactivity to laminin and such ¢ndings as, goblet cell hyperplasia, epithelial cell shedding, basal cell proliferation and basement membrane layer thickening, was investigated.
RESULTS Figure 1 shows laminin reactions in the subsegmental bronchus of a normal lung. The surface of the bronchus was covered with pseudostrati¢ed ciliated epithelia, and laminin reactions formed lines under the epithelial cells.
FIG. 1. Immunohistochemical staining with antibody anti-human laminin in normal bronchus. Antibody anti-laminin shows strong positivity along the epithelial cells.Thick laminin reaction lines under rows of pseudostrati¢ed ciliated epithelium (ABC method, original magni¢cation 700).
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Some laminin reactions were also seen in the lamina propria. Laminin reactions detected in the thickened basement membrane layer of the subsegmental bronchus of bronchial asthma patients were divided into four patterns: In Pattern A, laminin reactions branched out vertically in relation to the basement membrane layer; in Pattern B, laminin reactions formed lines along the lower margin of the basement membrane layer; in Pattern C, laminin reactions formed lines along the upper margin of the basement membrane layer; and in Pattern D, no laminin reactions were seen.When di¡erent patterns were seen, a predominating pattern was selected for classi¢cation purposes. In the lamina propria, ¢ndings such as, in£ammatory cell in¢ltration, edema, vasodilatation, smooth muscle hypertrophy and submucosal gland hypertrophy were seen, but there were no marked di¡erences among the four patterns. Nonetheless, as will be discussed later, there were slight di¡erences in the degree of capillary proliferation among the four patterns. In Pattern A, laminin reactions formed lines that ran vertically in relation to the basement membrane layer. Individual lines were narrow and thin. Also, some laminin reactions surrounded capillaries in the lamina propria and extended to the basement membrane layer. Although laminin reactions formed a single narrow thin line in the lamina propria, they branched out into several lines in the basement membrane layer. The tip of these branches extended mostly vertically or at an angle, but horizontally in some cases (Fig. 2).This pattern was seen around the entire circumference of the basement membrane layer in four patients and in one to two-thirds of the entire circumference in the remaining patients. In these patients with Patten A reactions that did not surround the entire circumference, Pattern B (laminin
FIG. 2. Pattern A immunoreactivity for laminin of the basement membrane layer for deceased bronchial asthma subject. Laminin reactions run perpendicular to the thickened basement membrane layer.Laminin reactions surrounding capillaries in the lamina propria extend into basement membrane layer in some areas (ABC method, (original magni¢cation 700).
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reactions along the lower margin of the basement membrane) or Patten D (no laminin reactions) was also seen. Capillary proliferation in the lamina propria, mostly ranging from moderate to severe in severity, was seen in almost all patients. Pattern B laminin reactions were seen along the lower margin of the basement membrane layer. Although most laminin reactions appeared to form lines, there were some notable spots, and where lines were terminated, some lines extended vertically in relation to the basement membrane layer. In all ¢ve patients, reactions were present partially, not all around the circumference of the basement membrane layer. Pattern A, C or D was observed where Pattern B laminin reactions were not detected. However, there was no connection between laminin reactions in the lamina propria and those in the basement membrane layer (Fig. 3). Also, when compared to the other patterns, thickening of the basement membrane layer was relatively milder for Pattern B. Furthermore, as shown in Fig. 4, Pattern C was characterized by strong thick laminin lines along the upper margin of the basement membrane layer. Pattern C reactions were seen around the entire circumference of the basement membrane layer in one patient, about1/ 3 the circumference in two patients, and 2/3 the circumference in three patients. When Pattern C reactions were partially seen around the circumference of the basement membrane layer, Pattern A-like reactions, where laminin reactions branched out in the basement membrane layer and extended narrowly and thinly, were seen in three of the six patients.These laminin reactions in the lamina propria were narrow but strong, extending toward the lower margin of the basement membrane layer in a reticular feature. Also, in Pattern D, although basement membrane layer thickening was most pronounced, no laminin
FIG. 3. Pattern B immunoreactivity for laminin of the basement membrane layer for the deceased bronchial asthma subject. Laminin reaction lines along lower margin of basement membrane layer. Although reactions appear to form lines, some pronounced dots are apparent. Among truncated lines, some reactions extend vertically (ABC method, /original magni¢cation 700).
IMMUNOHISTOCHEMICAL CLASSIFICATION OF THE LOCALIZATION OF LAMININ
FIG. 4. Pattern C immunoreactivity for laminin of the basement membrane layer for deceased bronchial asthma subject. Thick laminin reaction lines along upper margin of basement membrane layer. Basal cells are seen above these reaction lines (ABC method, original magni¢cation 700).
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FIG. 5. Pattern D immunoreactivity for laminin of the basement membrane layer for deceased bronchial asthma subject. Absolutelynolamininreactionsinthe thickened basement membrane layer. Epithelia have been replaced with goblet cells (ABC method, original magni¢cation 700).
TABLE 1. Subject characteristics. Health statement Pattern* n A B C D
Sex
15 12 M/3 F 5 4 M/1F 6 3 M/3 F 17 11M/6 F
Mean age (y) Healthy Illness or nottreated Treated in the past Treated in the present Unknown 44 (22^71) 49 (25^68) 60 (24^74) 52 (27^74)
0 0 0 2
0 2 0 0
0 0 0 1
14 3 5 14
1 0 1 0
*Classi¢cation by di¡erence of laminin reaction in basement membrane layer of subsegmental bronchus.
reactions were seen. Capillary proliferation was mild in the lamina propria, and only narrow reticular laminin reactions were seen around capillaries (Fig. 5). In most Pattern D patients, no laminin reactions were seen around the entire circumference of the basement membrane layer, but some Pattern A-like reactions were seen in six patients. Table 1 shows the number, sex, age and health status for the patients with each pattern. There were no marked di¡erences in these parameters among the four patterns. Table 2 shows the laminin reactions and epithelial ¢ndings for the 43 patients classi¢ed into the four lamininreaction patterns. In the Pattern A patients, relatively severe epithelial cell shedding was seen, and thickening of the basement membrane layer was mostly moderate or severe. In the pattern B and C patients, there were no clear di¡erences in epithelial cell shedding and basement membrane layer thickening. In the Pattern D patients, epithelial cell shedding was hardly seen, but marked goblet cell hyperplasia was seen in many patients. Furthermore, basal cell proliferation and markedly thickened basement membrane layer were seen among the Pattern D patients.
DISCUSSION Bronchial asthma is characterized by reversible obstructive changes, reactive acceleration and chronic in£ammation of the airway (26 ^28). In persistent or severe bronchial asthma, various morphological changes, such as, epithelial cell shedding, goblet cell hyperplasia and basement membrane layer thickening, are seen (11,12, 29,30). These changes can serve as indicators of the severity of bronchial asthma in airway remodeling (11,18,21,29). Thickening of the bronchial epithelial basement membrane layer is considered one of the histological changes associated with airway remodeling (11,18,21,29). The results of several electron microscopic studies have shown that this thickening is caused by abnormal deposition of interstitial collagen types I, III and Vand extracellular matrix such as ¢bronectin and tenescin, in the lamina reticularis under the bronchial epithelia (12^21). This is referred to as subepithelial ¢brosis because it is caused by the proliferation of collagen ¢bers (12^22,26). Also, increases in collagen and collagen deposition are believed to be caused by the interaction between airway epithelial cells and ¢broblasts/myo¢broblasts (17), but the
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TABLE 2. Characteristics of study groups. Subject No.
Pattern A 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Pattern B 1 2 3 4 5 Pattern C 1 2 3 4 5 6 Pattern D 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Sex*
Age (y)
Shedding of epithelial cells
Hyperplasia of goblet cells
Proliferation of globels cells
Thickness of basement membrane layer
M M M M M F F M M M F M M M M
43 71 70 24 23 68 22 25 22 29 70 62 54 42 28
w +++ +++ ++ +++ +++ ++ +++
+++ + +++
++ + ++ ++ + + +++ ++
++ +++ ++ ++ ++ ++ ++ +++ + +++ ++ + ++ +++ ++
M M M M F
25 26 67 57 68
++ +++
+++
++ + +++
++ + + ++ +
M M F F F M
24 69 74 73 68 50
+++ +++
+ ++
+ ++ ++ +++
++ ++ + +++ ++ ++
F F F M M F M M M M M F M M M F M
69 53 40 49 58 58 41 45 30 64 74 33 67 55 60 59 27
+++ +++
+++ +++ +++ + +++ ++ +++ + +++ +++ +++ +++
++ ++ +++ ++ + +++ ++ + + ++ ++ + +++ ++ +++
+ +++ +++ +++ ++ + ++ +++ ++ ++ +++ +++ + ++ +++ +++ +++
*Abbreviations: M, male; F, female. wSymbols: , negative; +, weak, ++, moderate; +++, strong.
details of this interaction are not known (20,22). In bronchial asthma patients, it has been known that laminin reactions localize notably along the upper margin of the
basement membrane layer (31,32), and that collagen type IV also localizes in the same area (33). In this study, we focused our attention on the thickened basement
IMMUNOHISTOCHEMICAL CLASSIFICATION OF THE LOCALIZATION OF LAMININ
membrane layer, and investigated histomorphological changes in the lamina lucida immunohistochemically using anti-laminin antibodies. The results showed that the localization of immunoreactivity in severe bronchial asthma is not as simple as previously thought and that laminin reactions can roughly be classi¢ed into four patterns. As shown in Table 2, there was a relationship with epithelial cell shedding. In Pattern A, epithelial cell shedding was seen in 7 of the 15 patients, while goblet cell hyperplasia was only seen in 3 of the 15 patients. In Pattern A, lines either extended vertically in relation to the basement membrane layer or they originated from the lamina propria and branched out in the basement membrane layer. Also, capillary proliferation in the lamina propria was observed in almost every patient. Pattern A laminin reactions could form as follows: the basement membrane under epithelial cells is made of epithelial cells and ¢broblasts (5,7), and since epithelial cells are generally detached and eliminated in Pattern A patients, it is di⁄cult to form a basement membrane. Nonetheless, as shown in Fig. 2, in some patients, laminin reactions in the lamina propria, in particular around capillaries, were marked and extended into the thickened basement membrane layer. We believe this is one of the reasons for seeing characteristic immunoreactivity to laminin in deceased bronchial asthma patients. In Patterns B and C, laminin reactions formed lines extending under epithelial cells: in Pattern B, lines formed along the lower margin of the basement membrane layer, whereas in Pattern C, lines formed along the upper margin of the basement membrane layer. Although laminin reactions were totally di¡erent between these two patterns, there were no clear di¡erences in epithelial cell shedding and basement membrane layer thickening. We could not, however, gather enough data to identify the cause of the di¡erence between Patterns B and C. Furthermore, in Pattern D, although immunoreactivity to laminin in the basement membrane layer was not seen at all, the proliferation of basal and goblet cells was clearly elevated in many patients when compared to the other three patterns. One of the causes for the absence of immunoreactivity to laminin in the thickened basement membrane layer could be that since capillary proliferation was hardly seen in the lamina propria in Pattern D patients, the level of laminin did not increase in the lamina propria. Also, this has not been documented but it is possible that goblet cells, which form epithelia, are not capable of producing laminin. The degree of basement membrane layer thickening for Pattern D was severer than that for the other patterns, but the reason for this was not clear. Between Patterns A and D, there were clear di¡erences in the four items shown inTable 2. In other words, in Pattern A, epithelial cell shedding was seen, but clear signs of goblet cell hyperplasia and basal cell proliferation
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were not seen. On the other hand, in Pattern D, epithelial cell shedding was hardly seen, but goblet cell hyperplasia and basal cell proliferation were marked. As a result, Patterns A and D may be on the opposite ends of the morphological changes associated with severe bronchial asthma. The above ¢ndings clarify that immunoreactivity to laminin in the basement membrane layer of patients with severe bronchial asthma shows a great degree of variation. The prerequisite for having basement membrane layer thickening, one component of airway remodeling, is the presence of the basal lamina, but some patients showed no immunoreactivity to laminin (Pattern D) or did not have a normal laminin structure (Pattern A). As a result, we concluded that conditions were too severe for remodeling to occur in patients with severe bronchial asthma. In other words, the histological characteristics of laminin are very unique in patients with severe bronchial asthma.
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