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 Brian L. Bradley, MD,* May Azzawi, MSc,** Mikila Jacobson, PhD,* Basil Assoufi, MD, PhD,* John V. Collins, MD,*** Anne-Marie A. Irani, MD,**** Lawrence B. Schwartz, MD, PhD,**** Stephen R. Durham, MD,* Peter K. Jeffery, PhD,** and A. Barry Kay, MD, PhD* London, England, and Richmond, Vu. Bronchial biopsy specimens were obtained by fiberoptic bronchoscopy from 21 atopic subjects with asthma, 10 atopic subjects without asthma, and 12 normal healthy control subjects. With immunohistochemical techniques and a panel of monoclonal antibodies, inflammatory cells were identiJied and counted in the bronchial mucosa. The mean number of leukocytes (CD45’) and T-lymphocytes (CD3‘, CD4+, and CDS+) at two airway levels in the subjects with asthma tended to be higher than in the other groups, but this difference did not achieve statistical signijcance. Similarly, there were no sign&ant differences in the numbers of mucosal-type or connective tissue-type mast cells, elastase-positive neutrophils, or Leu-M3 + cells in the airway mucosa of subjects with asthma compared with atopic subjects without asthma and healthy control subjects. In contrast, significantly more interleukin-2 receptor-positive (CD2.5’) cells and “activated” (EG2’) eosinophils (EOSs) were present in the airways of subjects with asthma at both proximal and subsegmental biopsy sites. When the relationships between numbers of T-lymphocytes, activated (CD25’) cells, and EOSs were analyzed, there were positive correlations between CD3 and EG2, between CD3 and CD25, and between CD25 and EG2 positive cells in the airways of subjects with asthma. Furthermore, the ratio of EG2 + to CD45 + cells correlated with the provocative concentration of methacholine that caused a 20% decrease of FEV, in hyperresponsive subjects. Although these associations do not prove a causal relationship, the results support the hypothesis that activated (CD25) T-lymphocytes release products which regulate recruitment of EOSs into the airway wall. In addition, our Jindings suggest that, in the large airways at least, asthma is not associated with hyperplasia of either mucosal-type or connective tissue-type mast cell. (.I ALLERGY CUN IMMUNOL 1991;88:661-74.) Key words: Eosinophils, biopsies, asthma
T-lymphocytes, mast cells, neutrophils,
From the Departments of *Allergy and Clinical Immunology and **Lung Pathology, National Heart & Lung Institute, and ***Royal Brompton and National Heart Hospital, London, and ****Medical College of Virginia, Virginia Commonwealth University. Richmond, Va. Supported by grants from the Chest, Heart, and Stroke Association, and the National Asthma Campaign, U. K.
macrophages,
bronchial
Received for publication Nov. 20, 1990. Revised April 29, 1991. Accepted for publication April 30, 1991. Reprint requests: A. B. Kay, MD, Professor and Director, Dept. of Allergy and Clinical Immunology, National Heart & Lung Institute, Dovehouse St., London, SW3 6LY, England, l/1/30591
661
662
Bradley
et al.
It is now clear from a number of studies that bronchial asthma, even in its mild form, is characterized by local infiltration of inflammatory cells. I-4 The technique of BAL has identified and quantified cells and mediators associated with the asthmatic process.5-‘3 However, BAL reveals only an indirect profile of events occurring within the bronchial mucosa, since cells and their products are retrieved from the peripheral airways and alveoli of the respiratory tract as well as the bronchi. Alternatively, endobronchial mucosal biopsy specimens examined by immunohistology allow precise identification of the various cell types infiltrating the bronchial mucosa. We have recently used immunohistology to quantify EOSs, T-lymphocytes, and T cell subsets at two airway levels in 11 atopic subjects with asthma.4 Our findings were compared with biopsy specimens from nine atopic subjects without asthma and 10 normal control subjects. Although we observed significant elevations in IL-2 receptor (CD25)-positive cells (as an index of T cell activation), this finding was significant at a proximal airway level only. Increased numbers of subepithelial “activated” (EG2+) EOSs were also observed at proximal and distal airway sites, but their numbers did not correlate significantly with the severity of disease as indicated by the methacholine PC,, (an index of airway hyperresponsiveness). The lack of statistical significance may have been attributable to the relatively small numbers studied. One of the objectives of the present study was to reevaluate the relationship between mucosal EOSs, T-lymphocytes (CD3), CD4 and CD8 positive cells, and activated (CD25 ‘) T cells in a considerably larger asthma study group (21 subjects) . In addition, we have taken the opportunity to quantify the numbers of subepithelial MC, (tryptase positive, chymase negative, or MC,) and MC, (tryptase/chymase positive or MC,) in subjects with asthma and healthy subjects. The possible role of the mast cell in bronchial asthma is suggested by a number of observations. For example, histamine and mast cell tryptase have been recovered from BAL after inhalation of antigen in atopic allergic subjects with asthma.6 Furthermore, mast cell numbers are reported to be increased in BAL from subjects with asthma.7. lo* ‘I, I3 For these reasons, it appeared important to determine whether hyperplasia of either the MC, or MC, was a feature of the bronchial mucosa in asthma. Finally, we have enumerated monocytel macrophages and neutrophils with Leu-M3 and a polyclonal neutrophil elastase, respectively. Both of these cell types”* l3 have also been implicated in asthma, and functional changes in those cells ob-
J. ALLERGY
CLIN. IMMUNOL. OCTOBER 1991
Abbreviations used APAAP: 2 Alkaline phosphatase-antialkaline phosphatase BAL: Bronchoalveolar lavage IL: Interleukin MC,: Mucosal-type mast cell MC,: Connective tissue-type mast cell PEFR: Peak expiratory flow rate EOS: Eosinophil SPT: Skin prick test PC,: Provocative concentration causing a 20% decrease in FEV, Ab: Antibody NHS: Normal human serum MAb: Monoclonal antibody BH: Bronchial hyperresponsiveness AR: Allergic rhinitis TBS: Tris-buffered saline
tained from BAL from subjects with asthma have been recognized. However, changes in BAL may not be representative of changes in the airway ~all.‘~* l5 It is unclear whether neutrophils or macrophage numbers are higher in the bronchial mucosa of subjects with stable, mild, ongoing asthma rather than in normal control subjects. Thus, the overall hypothesis is that general EOS infiltration in atopic asthma and EOS-mediated damage results in increased bronchial hyperresponsiveness. It is further proposed that EOS accumulation is under the control of T-lymphocyte or mast cell products, or both, and that neutrophils and macrophages further amplify mucosal inflammation through release of their own distinct granule and lipid mediators. By comparing results obtained in subjects with asthma with results from atopic subjects without asthma and from normal control subjects, the specificity of the observed changes for asthma, as opposed to atopy, can also be evaluated. MATERIAL AND METHODS Patients and control subjects Forty-three subjects participated in the study (21 atopic subjects with asthma, 10 atopic subjects without asthma, and 12 normal control subjects). The patients with asthma and AR were volunteers from the Allergy Clinic, Royal Brompton National Heart and Lung Hospital, London. Normal healthy nonatopic control subjects were recruited from laboratory staff and students. Subjects aged 18 to 65 years were eligible for the study. All subjects with asthma and atopic subjects without asthma (either AR sufferers or symptomless) were SPT positive to one or more extracts of eight common aeroallergens. Healthy control subjects were SPT
VOLUME NUMBER
TABLE
Bronchial
88 4
I. Clinical
details
of the subjects
Group
No. of subjects Sex (M/F) Age (yr) Mean Range FEV, (% of predicted) Mean Range MethacholinePC,, (mg/ml) Geometricmean Range Blood EOS counts Mean Range
biopsies
in asthma
663
studied
With atopic asthma
Atopic
subjects asthma
21
10
12/9
911
without
Nonatopic normal control subjects
12 616
25.4 19-58
22.9 19-35
23.7 19-30
95.7 69.4-l 14.3
105 90-126
107 87.2-132
1.04 0.16-15.0
12.3 3.2->32.0
27.1 5.4->32.0
155.6 57-310
123.5 o-459
348.7 46-1392
negative. The subjectswith asthmahad clinically stable disease with an FEV, of >70% of predictednormalvalues. All 43 subjectswere nonsmokers,andnonehad received inhaledor oral corticosteroidsfor at least3 monthsbefore the study. The atopic subjectswithout asthmaand control subjectsal;1hadFEV, >80% of predictednormal.Theyhad no history suggestiveof asthma,andtheir increasein FEV, in response to inhaledP,-agonistswas
T-lymphocytes, mast cells, neutrophils,
From the Departments of *Allergy and Clinical Immunology and **Lung Pathology, National Heart & Lung Institute, and ***Royal Brompton and National Heart Hospital, London, and ****Medical College of Virginia, Virginia Commonwealth University. Richmond, Va. Supported by grants from the Chest, Heart, and Stroke Association, and the National Asthma Campaign, U. K.
macrophages,
bronchial
Received for publication Nov. 20, 1990. Revised April 29, 1991. Accepted for publication April 30, 1991. Reprint requests: A. B. Kay, MD, Professor and Director, Dept. of Allergy and Clinical Immunology, National Heart & Lung Institute, Dovehouse St., London, SW3 6LY, England, l/1/30591
661
662
Bradley
et al.
It is now clear from a number of studies that bronchial asthma, even in its mild form, is characterized by local infiltration of inflammatory cells. I-4 The technique of BAL has identified and quantified cells and mediators associated with the asthmatic process.5-‘3 However, BAL reveals only an indirect profile of events occurring within the bronchial mucosa, since cells and their products are retrieved from the peripheral airways and alveoli of the respiratory tract as well as the bronchi. Alternatively, endobronchial mucosal biopsy specimens examined by immunohistology allow precise identification of the various cell types infiltrating the bronchial mucosa. We have recently used immunohistology to quantify EOSs, T-lymphocytes, and T cell subsets at two airway levels in 11 atopic subjects with asthma.4 Our findings were compared with biopsy specimens from nine atopic subjects without asthma and 10 normal control subjects. Although we observed significant elevations in IL-2 receptor (CD25)-positive cells (as an index of T cell activation), this finding was significant at a proximal airway level only. Increased numbers of subepithelial “activated” (EG2+) EOSs were also observed at proximal and distal airway sites, but their numbers did not correlate significantly with the severity of disease as indicated by the methacholine PC,, (an index of airway hyperresponsiveness). The lack of statistical significance may have been attributable to the relatively small numbers studied. One of the objectives of the present study was to reevaluate the relationship between mucosal EOSs, T-lymphocytes (CD3), CD4 and CD8 positive cells, and activated (CD25 ‘) T cells in a considerably larger asthma study group (21 subjects) . In addition, we have taken the opportunity to quantify the numbers of subepithelial MC, (tryptase positive, chymase negative, or MC,) and MC, (tryptase/chymase positive or MC,) in subjects with asthma and healthy subjects. The possible role of the mast cell in bronchial asthma is suggested by a number of observations. For example, histamine and mast cell tryptase have been recovered from BAL after inhalation of antigen in atopic allergic subjects with asthma.6 Furthermore, mast cell numbers are reported to be increased in BAL from subjects with asthma.7. lo* ‘I, I3 For these reasons, it appeared important to determine whether hyperplasia of either the MC, or MC, was a feature of the bronchial mucosa in asthma. Finally, we have enumerated monocytel macrophages and neutrophils with Leu-M3 and a polyclonal neutrophil elastase, respectively. Both of these cell types”* l3 have also been implicated in asthma, and functional changes in those cells ob-
J. ALLERGY
CLIN. IMMUNOL. OCTOBER 1991
Abbreviations used APAAP: 2 Alkaline phosphatase-antialkaline phosphatase BAL: Bronchoalveolar lavage IL: Interleukin MC,: Mucosal-type mast cell MC,: Connective tissue-type mast cell PEFR: Peak expiratory flow rate EOS: Eosinophil SPT: Skin prick test PC,: Provocative concentration causing a 20% decrease in FEV, Ab: Antibody NHS: Normal human serum MAb: Monoclonal antibody BH: Bronchial hyperresponsiveness AR: Allergic rhinitis TBS: Tris-buffered saline
tained from BAL from subjects with asthma have been recognized. However, changes in BAL may not be representative of changes in the airway ~all.‘~* l5 It is unclear whether neutrophils or macrophage numbers are higher in the bronchial mucosa of subjects with stable, mild, ongoing asthma rather than in normal control subjects. Thus, the overall hypothesis is that general EOS infiltration in atopic asthma and EOS-mediated damage results in increased bronchial hyperresponsiveness. It is further proposed that EOS accumulation is under the control of T-lymphocyte or mast cell products, or both, and that neutrophils and macrophages further amplify mucosal inflammation through release of their own distinct granule and lipid mediators. By comparing results obtained in subjects with asthma with results from atopic subjects without asthma and from normal control subjects, the specificity of the observed changes for asthma, as opposed to atopy, can also be evaluated. MATERIAL AND METHODS Patients and control subjects Forty-three subjects participated in the study (21 atopic subjects with asthma, 10 atopic subjects without asthma, and 12 normal control subjects). The patients with asthma and AR were volunteers from the Allergy Clinic, Royal Brompton National Heart and Lung Hospital, London. Normal healthy nonatopic control subjects were recruited from laboratory staff and students. Subjects aged 18 to 65 years were eligible for the study. All subjects with asthma and atopic subjects without asthma (either AR sufferers or symptomless) were SPT positive to one or more extracts of eight common aeroallergens. Healthy control subjects were SPT
VOLUME NUMBER
TABLE
Bronchial
88 4
I. Clinical
details
of the subjects
Group
No. of subjects Sex (M/F) Age (yr) Mean Range FEV, (% of predicted) Mean Range MethacholinePC,, (mg/ml) Geometricmean Range Blood EOS counts Mean Range
biopsies
in asthma
663
studied
With atopic asthma
Atopic
subjects asthma
21
10
12/9
911
without
Nonatopic normal control subjects
12 616
25.4 19-58
22.9 19-35
23.7 19-30
95.7 69.4-l 14.3
105 90-126
107 87.2-132
1.04 0.16-15.0
12.3 3.2->32.0
27.1 5.4->32.0
155.6 57-310
123.5 o-459
348.7 46-1392
negative. The subjectswith asthmahad clinically stable disease with an FEV, of >70% of predictednormalvalues. All 43 subjectswere nonsmokers,andnonehad received inhaledor oral corticosteroidsfor at least3 monthsbefore the study. The atopic subjectswithout asthmaand control subjectsal;1hadFEV, >80% of predictednormal.Theyhad no history suggestiveof asthma,andtheir increasein FEV, in response to inhaledP,-agonistswas
