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Inflammatory Processes in Bronchial Asthma l R. Beasley, W. Roche and

s.T.

Holgate

Medicine 1 and Pathology, Southampton General Hospital, Southhampton, United Kingdom

Summary

Bronchial asthma is an inflammatory disease. The characteristic pathological features of epithelial cell loss. goblet cell hyperplasia. increased deposition of collagen beneath the basement membrane. mast cell degranulation. and inflammatory cell infiltration of the mucosa are not limited to fatal asthma. Similar inflammatory events have been observed in subjects who would be considered to have clinically stable asthma. These observations would suggest that pharmacological treatment directed against the underlying inflammatory processes in asthma should not be limited to those patients with severe forms of the disease.

1. Fatal Asthma There is now convincing evidence to suggest that an intense inflammatory process occurs within the airways of asthmatic subjects dying from severe asthma (Dunnill 1960; Houston et al. 1953; Huber & Koessler 1922). The airway lumen is filled with tenacious, viscid plugs formed from an exudate containing plasma proteins, inflammatory cells, epitMlium and mucus. These occluding plugs may be so extensive and dense that numerous areas of focal atelectasis may occur and the lungs may not collapse when the pleural cavities are opened at post mortem. The bronchial wall is thickened, congested and oedematous, and there is extensive damage to the ciliated respiratory epithelium. De1 This presentation is based on a paper by Beasley R, et al. American Review of Respiratory Disease 139: 806-817, 1989. Publication of this article is by kind permission of the editor of the American Review of Respiratory Disease and the authors.

tachment of these epithelial cells may be marked, leaving a layer of basal cells, or leading to complete replacement by goblet cells, especially in the smaller bronchi. Increased deposition of collagen occurs beneath the basement membrane, leading to an apparent 3-fold increase in thickness of the basement membrane at light microscopy (Nowak 1969). The vessels beneath the basement membrane are dilated and the mucosa is oedematous and infiltrated with eosinophils, lymphocytes and plasma cells. This cellular infiltration is predominantly composed of eosinophils which, although found at all sites throughout the airway wall, are specifically localised to areas of damage to the epithelium (Gleich et al. 1987). There is increased thickness of the bronchial muscle, and it has been demonstrated that this increase is largely due to hyperplasia and not hypertrophy (Heard & Hossain 1973). There is considerable enlargement of the mucous glands in the segmental bronchi, which is comparable to that present in chronic bronchitis (Dunnill et al. 1969).

Inflammatory Processes in Bronchial Asthma

2. Non-Fatal Asthma 2.1 Sputum There is evidence to suggest that this marked inflammatory reaction present in fatal asthma may also occur in less severe forms of the disease which do not lead to death. Sputum produced by asthmatics is usually thick and tenacious, and when recovered may take the shape ofa cast of the bronchus, as observed by Curschmann more than 100 years ago (1883). The presence of large clusters of epithelial cells in the sputum reflects the degree of damage to the respiratory epithelium. This detachment is seen almost exclusively in asthmatics and increases considerably during asthmatic attacks (Naylor 1962). In addition to epithelial elements, the sputum may also contain eosinophils and Charcot-Leyden crystals, reflecting the eosinophil infiltration and activation within the bronchial mucosa. More recently, eosinophil activation has been confirmed by the finding of eosinophilderived major basic protein (MBP) in the sputum of subjects with stable and acute asthma (Dor et al. 1984). It has been demonstrated that the concentration of MBP in sputum during an attack of asthma is increased to levels that have been associated with toxicity to the respiratory epithelium in vitro. The non-cellular elements of asthmatic sputum are composed of mucus and plasma proteins, due to both increased vascular permeability and increased goblet cell and mucous gland secretion. 2.2 Biopsy Studies Information on the inflammatory processes in non-fatal asthma has also been obtained from pathological studies in which bronchial biopsies have been performed on asthmatic patients. In the initial study of Glynn and Michaels (1960), the predominant histological characteristic of the asthmatic subjects was eosinophil infiltration of the lamina propria. Eosinophils were commonly observed throughout the bronchial wall and were present within the lumen of capillaries, the submucosal tissue, basement membrane and epithelium.

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There was also evidence of eosinophil activation, since in some areas eosinophils appeared to have disintegrated, with numerous free granules present. Goblet cell hyperplasia was also frequently observed in this study. Salvato (1968) subsequently confirmed the intense tissue eosinophilia and goblet cell hyperplasia in subjects with asthma and also reported that mast cell degranulation may occur. In a study of asthmatic children, Cutz et al. (1978) noted that the overall histopathological changes in 2 children with severe asthma were similar to those in children who died in status asthmaticus. The only differences between the 2 groups were the presence of larger numbers of peribronchial eosinophils and focal denudation of mucosa in the children who died in an asthmatic attack, presumably reflecting the more acute and active disease process. Bronchial changes such as goblet cell hyperplasia, mucous plugging and increased collagen under the basement membrane were comparable in both groups. In a recent study of the bronchial epithelium, Laitinen et al. (1985) reported that asthma patients can have profound epithelial destruction at all levels of the airways. Epithelial damage was observed in subjects with asthma of varying degrees of severity, some with only mild bronchial hyperreactivity. The ciliated cells appeared to be the cells in the respiratory epithelium that were destroyed most often. In another bronchoscopic study of the respiratory epithelium of asthmatic subjects, Lundgren (1977) also identified large areas of epithelium with either abnormal cilia, or even complete ciliary loss.

3. Mild Allergic Asthma In most of these histological studies of non-fatal asthma, the asthmatic subjects represented a clinically heterogeneous group, particularly with respect to the severity of, previous treatment for, or the allergic basis of, this disorder. As a result, there has been uncertainty as to the nature and degree of the inflammatory processes that may occur in clinically mild asthma. We specifically investigated this

Inflammatory Processes in Bronchial Asthma

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Fig. 1. Marked eosinophil granule heterogeneity in asthmatic subject (uranyl acetate-lead citrate. transmission electron microscopy. magnification x 20.000).

problem in a recent study in which we compared the histopathological features of bronchial biopsy specimens from mild allergic asthmatics with those from non-asthmatic controls (Beasley et al. 1989). All the asthmatic subjects had clinically mild allergic asthma, with normal spirometry, moderately increased nonspecific bronchial responsiveness and positive skin tests to common allergens. All asthmatic subjects were clinically stable before the bronchoscopy and required only intermittent inhaled bronchodilat~r treatment; certainly none were taking anti-inflammatory drugs that could affect the underlying biopsy appearances. 3. 1 Eosinophils Cellular and ultra-structural examination of the bronchial biopsy specimens revealed inflammatory

changes in the bronchi of the asthmatic subjects similar to those observed in more severe forms of the disease. The most distinctive feature was the inflammatory cell infiltration of the mucosa of all the asthmatic subjects studied. In particular, eosinophils were present at all sites throughout the airway wall. In the lamina propria there was evidence of active recruitment of eosinophils, with adherence to the vascular endothelium and emigration to the interstitial tissue. Eosinophils had migrated to the bronchial epithelium and were present in both the subepithelial collagen layer and among the bronchial epithelial cells. In addition to this active migration, there was morphological evidence of activation, with marked heterogeneity of the granular structure (fig. I). Eosinophils were partly degranulated, some containing granules with electron lucent matrix only and others MBP dense core ma-

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Inflammatory Processes in Bronchi al Asthma

terial alone, while at some sites extracellular granular material was present. 3.2 Mast Cells Mast cells present in the bronchial mucosa of the asthmatic subjects also appeared activated, showing various stages of degranulation (fig. 2). Their appearance could not be attributed to the biopsy procedure, since the mast cells of the control subjects were always normal, with intact secretory granules. This would suggest that even in clinically mild allergic asthma, the airways may be under some degree of histaminic tone, and this would certainly be consistent with the previous observation that histamine H I-receptor blockade results in significant bronchodilatation in such subjects (Rafferty & Holgate 1987).

3.3 Vascular Events The inflammatory events within the bronchial mucosa of the asthmatic subjects were not limited to eosinophil infiltration and activation or mast cell degranulation. In addition to that of eosinophils, there was active recruitment of monocytes, neutrophils, platelets and lymphocytes from the vessels within the lamina propria. These inflammatory cells adhered to the endothelial cells of the capillaries and post-capillary venules. These intravascular events may be crucial in the initial development and subsequent potentiation of the inflammatory reaction within the bronchial wall in asthma. These cells are likely to be involved in the IgE-mediated inflammatory response, since they have low affinity IgE receptors which, after stimulation, release inflammatory mediators which have

Fig. 2. Mast cell in asthmatic subject showing extensive degranulation (uranyl acetate-lead Citrate. transmission electron microscopy. magnification x 20,000).

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mens, because of artefactual epithelial loss occurring in both the asthmatic and control subjects, analysis of the bronchial lavage specimens suggested that considerable epithelial cell loss may occur in mild asthma. The asthmatic subjects had a 5-fold increase in epithelial cells in the lavage fluid compared with the normal controls (fig. 3). These epithelial cells appeared viable and often occurred in clumps, suggesting that the primary defect was one of adherence to the basement membrane and not widespread epithelial cell destruction. The observation that there was a significant inverse correlation between the epithelial cell count in the lavage fluid and the airway responsiveness supports the hypothesis that epithelial cell loss may be important in the pathogenesis of bronchial hyper-reactivity in asthma. This would also be consistent with the previous reports of Naylor (1962) that increased shedding of epithelial cells into the sputum occurs with worsening asthma. Fig. 3. Shed epithelial cells in SAL fluid from asthmatic subject (May-Grunwald-Giemsa. light microscopy. magnification x 638).

been implicated in the pathogenesis of inflammation in asthma. 3.4 Collagen Deposition Another characteristic feature of the asthmatic subjects was the presence of marked collagen deposition beneath the basement membrane, which was not observed in the non-asthmatic subjects. Although the causative mechanism is uncertain, it is likely to relate to the chronic inflammatory process occurring within the mucosa. The asthmatic subjects in this study had mild asthma, and in some the disease had been present for only a short duration, suggesting that this increased deposition of collagen may occur early in the disease process.

4. Conclusion Although it may not be recognised clinically, bronchial asthma is an inflammatory disease with distinctive pathological features. Our recent study has demonstrated that this characteristic inflammatory process is not limited to severe or fatal forms of the disease. Inflammatory changes similar to those seen in subjects who had died from a severe attack of asthma were also observed in subjects who would clinically be considered to have mild stable asthma. These observations would suggest that pharmacological treatment directed against the underlying inflammatory processes in asthma should not be limited to those patients with severe forms of the disease.

Acknowledgement 3.5 Epithelial Cell Loss Although it was difficult to determine the epithelial appearance of the bronchial biopsy speci-

We gratefully acknowledge the assistance of Dr G.P. Twentyman, Mr c.B. Inman, Mr R. Barnett, Miss S. Cox, Mrs J. Williams, Dr D.B. Jones and Miss M.D. Harris.

Inflammatory Processes in Bronchial Asthma

References Beasley R, Roche WR, Roberts JA, Holgate ST. Cellular events in the bronchi in mild asthma and after bronchial provocation. American Review of Respiratory Disease 139: 806-817, 1989 Curschmann H. Uber Bronchiolitis exsudativa und ihr Verhaltnis zum Asthma nervosum. Deutsches Archives fUr Klinische Medizinische (Leipzig) 32: 1-34, 1883 Cutz E, Levison H, Cooper DM. Ultrastructure of airways in children with asthma. Histopathology 2: 407-421, 1978 Dor PJ, Ackerman SJ, Gleich GJ. Charcot-Leyden crystal protein and eosinophil granule major basic protein in sputum of patients with respiratory disease. American Review of Respiratory Disease 130: 1072-1077 1984 Dunnill MS. The pathology of asthma with special reference to changes in the bronchial mucosa. Journal of Clinical Pathology 13: 27-33, 1960 Dunnill MS, Massarella GR, Anderson JA. A comparison of the quantitative anatomy of the bronchi in normal subjects. in status asthmaticus, in chronic bronchitis and in emphysema. Thorax 24: 176- 179, 1969 Gleich GJ, Motojima S, Frigas E, Kephart GM, Fujisawa T. et al. The eosinophilic leukocyte and the pathology of fatal bronchial asthma: evidence for pathologic heterogeneity. Journal of Allergy and Clinical Immunology 80: 412-414, 1987 Glynn AA, Michaels L. Bronchial biopsy in chronic bronchitis and asthma. Thorax 15: 142-153, 1960 Heard BE, Hossain S. Hyperplasia of bronchial muscle in asthma. Journal of Pathology 110: 319-331, 1973

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Houston Jc. de Navasquez S, Trounce JR. A clinical and pathological study of fatal cases of status asthmaticus. Thorax 8: 207-213, 1953 Huber HL, Koessler KK. The pathology of bronchial asthma. Archives of Internal Medicine 30: 689-760. 1922 Laitinen LA, Heino M, Laitinen A, Kava T. Haahtela T. Damage of the airway epithelium and bronchial reactivity in patients with asthma. American Review of Respiratory Disease 131: 599-606, 1985 Lundgren R. Scanning electron microscopic studies of bronchial mucosa before and during treatment with beclomethasone dlproprionate inhalations. Scandinavian Journal of Respiratory Diseases (Suppl. 101): 179-187. 1977 Naylor B. The shedding of the mucosa of the bronchial tree in asthma. Thorax 17: 69-72, 1962 Nowak J. Anatomopathologic changes in the bronchial walls in chronic inflammation, with special reference to the basement membrane, in the course of bronchial asthma. Acta Medica Polona 2: 153-172, 1969 Rafferty P, Holgate ST. Terfenadine (Seldane) is a pote~t a~d selective histamine H I receptor antagonist in asthmatic airways. American Review of Respiratory Disease 135: 181-184, 1987 Salvato G. Some histological changes in chronic bronchitis and asthma. Thorax 23: 168-172. 1968 Author's address: Dr R. BC'as/C'y, Department of Medicine. Wellington School of Medicine. P.O. Box 7343, Wellington South, Wellington (New Zealand).