- Email: [email protected]
Morphometry Explains Variation in Airway Responsiveness in Transgenic Mice Overexpressing Interleukin-6 and Interleukin-11 in the Lung
76 77 78 79
80
81 82 83 84 85 86
antielastase hypothesis 30 years later. Proc Assoc Am Physicians 1995; 107:346 –352 Hautamaki RD, Kobayashi DK, Senior RM, et al. Requirement for macrophage elastase for cigarette smoke-induced emphysema in mice. Science 1997; 277:2002–2004 Finkelstein R, Fraser RS, Ghezzo H, et al. Alveolar inflammation and its relation to emphysema in smokers. Am J Respir Crit Care Med 1995; 152:1666 –1672 Kraft M, Djukanovic R, Wilson S, et al. Alveolar tissue inflammation in asthma. Am J Respir Crit Care Med 1996; 154:1505–1510 Fletcher CM. Chronic bronchitis and decline in pulmonary function with some suggestions on terminology. In: Cumming G, Bonsignore G, eds. Smoking and the lung (vol 17). New York, NY: Plenum Press, 1984; 397– 420 Peto R, Speitzer FE, Cochrane AL, et al. The relevance in adults of airflow obstruction, but not of mucus hypersecretion, to mortality from chronic lung disease. Am Rev Respir Dis 1983; 128:491–500 Prescott E, Lange P, Vestbo J. Chronic mucus hypersecretion in COPD and death from pulmonary infection. Eur Respir J 1995; 8:1333–1338 Vestbo J, Prescott E, Lange P. Association of chronic mucus hypersecretion with FEV1 decline and COPD morbidity. Am J Respir Crit Care Med 1996; 153:1530 –1535 Chanez P, Vignola AM, O’Shaughnessy T, et al. Corticosteroid reversibility in COPD is related to features of asthma. Am J Respir Crit Care Med 1997; 155:1529 –1534 Gibson PG, Dolivich J, Denburg J, et al. Chronic cough: eosinophilic bronchitis without asthma. Lancet 1989; 1:1346 – 1348 Hopkin JM, Tomlinson CS, Jenkins RM. Variations in response to cytotoxicity of cigarette smoke. BMJ 1981; 283: 1209 –1211 Amadori A, Zamarchi R, De Silvestro G, et al. Genetic control of the CD4/CD8 T-cell ratio in humans. Nat Med 1995; 1:1279 –1283
Expression of 15-Lipoxygenase and Evidence for Apoptosis in the Lungs From Patients With COPD* Yasunori Kasahara, MD; Rubin M. Tuder, MD; Carlyne D. Cool, MD; Norbert F. Voelkel, MD
(CHEST 2000; 117:260S) Abbreviations: KDR ⫽ kinase insert domain-containing receptor; VEGF ⫽ vascular endothelial growth factor
is a loss of alveolar structures and endothelial T here cells in emphysema. We hypothesize that the disap-
pearance of lung alveoli resulting in emphysema may occur by apoptosis following decreased expression or
*From the Pulmonary Hypertension Center, Department of Medicine (Drs. Kasahara and Voelkel) and Department of Pathology (Drs. Tuder and Cool), University of Colorado Health Sciences Center, Denver, CO. Correspondence to: Yasunori Kashahara, MD, Division of Pulmonary Science and Critical Care Medicine, University of Colorado Health Sciences Center, 4200 E. 9th Ave, Denver, CO 80262 260S
activity of lung vascular endothelial growth factor (VEGF) or its receptor, kinase insert domain-containing receptor(KDR). To examine this hypothesis, we carried out the TUNEL technique in lung samples obtained from 6 patients with emphysema and 11 normal control subjects (7 nonsmokers and 4 smokers). We found an increased number of TUNEL-positive cells/nucleic acids in alveolar septum in emphysema when compared with normal lungs. The cell death events were not different between healthy nonsmokers and nonemphysema smokers. Double staining with TUNEL and cytokeratin or factor VIII-related antigen confirmed endothelial cell and epithelial cell death in emphysema lungs. Western blot analysis for KDR of whole lung protein extracts showed that KDR protein expression was significantly reduced in emphysema lungs. In situ hybridization revealed a focally decreased expression of VEGF and KDR messenger RNA in emphysema lungs. Using immunohistochemistry, we localized and semiquantitatively estimated the abundance of 15-lipoxygenase in lungs obtained from 6 patients with emphysema, 6 normal control subjects, and 7 patients with severe pulmonary hypertension. Expression of 15-lipoxygenase in pulmonary arteries in emphysema was less intense than in severe pulmonary hypertension. We conclude that cell death events occur in emphysema, and that VEGF and its receptor KDR are decreased in emphysema lungs. We speculate that emphysema may occur by apoptosis following decreased expression or activity of lung VEGF or its receptor KDR.
Morphometry Explains Variation in Airway Responsiveness in Transgenic Mice Overexpressing Interleukin-6 and Interleukin-11 in the Lung* Charles Kuhn, MD; Robert J. Homer, MD, PhD; Zhou Zhu, MD, PhD; Nicholas Ward, MD; and Jack A. Elias, MD
(CHEST 2000; 117:260S–262S) Abbreviations: AA ⫽ alveolar wall attached; AHR ⫽ airway hyperreactivity; CC10 ⫽ Clara cell 10-kd protein; IL ⫽ interleukin; S/V ⫽ surface/volume ratio
for airway hyperreactivity (AHR) accompanyT heingbasis COPD is poorly understood. Structural changes
may play a role. We have generated transgenic mice that overexpress related cytokines, interleukin (IL)-6 and IL11, in their airways, which were controlled by the Clara cell 10-kd protein (CC10) promoter. Despite some similar
*From the Department of Pathology (Dr. Kuhn), Brown University School of Medicine, Providence, RI; and the Departments of Pathology (Dr. Homer) and Pulmonary and Critical Care Medicine (Drs. Zhu, Ward, and Elias), Yale University, New Haven, CT. Correspondence to: Charles Kuhn, MD, Pathology Department, Memorial Hospital of Rhode Island, Pawtucket, RI 02860 Thomas L. Petty 42nd Annual Aspen Lung Conference: Mechanisms of COPD
Table 1—Morphometric Results on Lungs of Transgenic Mice and Littermate Controls* Mouse Strain Morphometric Variable
IL-6 (⫺)
IL-6 (⫹)
IL-11 (⫺)
IL-11 (⫹)
Cord length, m S/V, m⫺1 AAs, mm⫺1 Bronchiolar wall thickness, m External diameter, m Wall thickness, % external diameter Lumen diameter, m Thickness of mucosa and submucosa, m
29.5 ⫾ 5.5 0.142 ⫾ .009 34.2 ⫾ 1.3 2.77 ⫾ 0.34 127 ⫾ 10 2.2 ⫾ 0.2
65.3 ⫾ 10.5† 0.065 ⫾ .010† 16.4 ⫾ 1.8† 5.73 ⫾ 0.59† 188 ⫾ 26†‡ 3.1 ⫾ 0.3
33.4 ⫾ 4.3 0.134 ⫾ .017 30.6 ⫾ 3.5 2.43 ⫾ 0.49 138 ⫾ 15 1.9 ⫾ 0.4
65.6 ⫾ 9.4† 0.056 ⫾ .016† 15.6 ⫾ 1.6† 7.84 ⫾ 1.71† 148 ⫾ 21 5.6 ⫾ 1.3†
91.4 ⫾ 8.0 11.4 ⫾ 3.9
147.7 ⫾ 25.6† 12.5 ⫾ 1.8‡
96.8 ⫾ 8.1 11.5 ⫾ 1.9
90.5 ⫾ 20.9 15.8 ⫾ 2.5§
*Values given as the mean ⫾ SD of results for 6 to 12 mice in each group. (⫺) ⫽ littermate controls; (⫹) ⫽ mice expressing the transgene of the cytokine indicated. †Differs from littermate controls, p ⬍ 0.0001 by t test with Bonferroni’s correction for multiple comparisons. ‡Differs from IL-11, p ⬍ 0.01. §Differs from littermate controls, p ⬍ 0.01.
structural abnormalities, their airway physiology differed. CC10-IL-6 mice had normal expiratory flow and decreased reactivity to methacholine,1 while CC10-IL-11 mice had airflow obstruction and hyperreactivity.2 To clarify this difference, a morphometric study was undertaken of the lung parenchyma and bronchioles of the two transgenic strains and littermate controls.
caliber and external diameter of their airways. When the wall thickening was normalized to the diameter of the airway, it was proportional to the increased airway size. In contrast, the CC10-IL-11 strain showed submucosal thickening, increased muscle, and fibrosis in airways with normal external and luminal diameters. Their wall thickening was disproportionate to airway size.
Materials and Methods
Discussion
The production of the transgenic mice has been described.1,2 Lungs from mice (age, 1 to 2 months) were fixed by intratracheal instillation of glutaraldehyde at a pressure of 25 cm of fixative. Two measures of airspace size, the mean cord length and surface/volume ratio (S/V) of the gas-exchanging parenchyma, were determined by a computerized method that has been described previously.3 The bronchiolar lumen, the external diameter of the bronchioles, and the total thickness of the bronchiolar wall and thickness of the wall internal to the smooth muscle were measured with a calibrated eyepiece reticle. To determine the density of alveolar walls attached (AAs) to the bronchioles (alveolar attachments per unit length of the bronchiolar perimeter), we measured both the long and short axis of elliptically shaped bronchiolar profiles and counted the number of AAs. The perimeter of the bronchiole, P, was calculated from the measured axes using the approximate formula for the perimeter of an ellipse, P ⫽ 2 公(a2 ⫹ b2)/2, where a and b are the semi-major and semi-minor axes, respectively. Knowing the calculated value for P, the value of the density of AA then is AA/P.
Results The morphometric results are summarized in Table 1. Both transgenic strains of mice had emphysema-like airspace enlargement that was of identical severity by three morphometric measures: cord length, S/V, and AA/P. This enlargement is thought to be the result of the failure of septation in both strains, although it has only been proven for the CC10-IL-11 mice.3 Both had lymphocytic nodules in airways and airway wall thickening with increased fibrosis and smooth muscle. Compared with controls, hyporeactive CC10-IL-6 mice had a 50% increase in the
The structural changes that lead to AHR have been modeled and studied in detail.4 – 6 Airway narrowing in response to agonists results from smooth muscle shortening working against the passive load of the recoil of the surrounding parenchyma. Emphysema, with its attendant loss of recoil and smooth muscle hypertrophy, which increases the force of contraction, will exaggerate the response to a given dose of agonist. Thickening of the bronchiolar wall internal to the smooth muscle also exaggerates airway narrowing,4 while thickening of the adventitial sheath may uncouple the interdependence of the airways and parenchyma, thereby decreasing the effect of parenchymal recoil. Hence, the airway remodeling and emphysema-like parenchymal changes both contribute to the airflow obstruction and AHR seen in the IL-11expressing mice. The IL-6-expressing mice had equally severe emphysema and also had airway wall thickening, but their bronchioles were 50% larger than those of either their littermates or the IL-11-expressing strain, and their airway wall thickening was proportional to airway size. Evidently, the increased lumen diameter overrode the effect of the emphysema and airway remodeling and resulted in relatively large airway diameter even after agonist inhalation. We conclude that airway remodeling and emphysema sometimes lead to AHR, but baseline airway size and caliber also determine airway responsiveness.
References 1 DiCosmo BF, Geba GP, Picarella D, et al. Airway epithelial cell expression of interleukin-6 in transgenic mice: uncouCHEST / 117 / 5 / MAY, 2000 SUPPLEMENT
261S
2
3
4 5 6
pling of airway inflammation and bronchial hyperreactivity. J Clin Invest 1994; 94:2028 –2035 Tang W, Geba GP, Zheng T, et al. Targeted expression of IL-11 in the murine airway causes lymphocytic inflammation, bronchial remodelling and airways obstruction. J Clin Invest 1996; 98:2845–2853 Ray R, Tang W, Wong P, et al. Regulated overexpression of interleukin-11 in the lung: Use to dissociate developmentdependent and -independent phenotypes. J Clin Invest 1997; 100:2501–2511 Wiggs, BR, Bosken C, Pare´ PD, et al. A model of airway narrowing in asthma and in chronic obstructive pulmonary disease. Am Rev Respir Dis 1992; 145:1251–1258 Lambert RK, Wiggs BR, Kuwano K, et al. Functional significance of increased airway smooth muscle in asthma and COPD. J Appl Physiol 1993; 74:2771–2781 Pare´ PD, Bai TR. Airway wall remodelling in chronic obstructive pulmonary disease. Eur Respir Rev 1996; 6:259 –263
Ongoing Airway Inflammation in Patients With COPD Who Do Not Currently Smoke* Steven R. Rutgers, MD; Dirkje S. Postma, MD; Nick H. ten Hacken, MD; Henk F. Kauffman, PhD; Thomas W. van der Mark, PhD; Gerard H. Koe¨ter, MD; and Wim Timens, MD
(CHEST 2000; 117:262S) Abbreviation: ECP ⫽ eosinophil cationic protein
I
Patients with COPD and high mucosal EG2⫹ cell numbers had high mucosal CD4⫹ cell numbers. Sputum eosinophilia was associated with a decrease in FEV1/vital capacity and BAL fluid eosinophilia with a decrease in mucosal NP57⫹ cells. We conclude that subjects with COPD who do not currently smoke have increased numbers of inflammatory cells. Eosinophils are increased in number in the airways in COPD but do not seem to be activated. The increased eosinophil numbers are probably due to recruitment as a result of ongoing inflammation. Macrophages and lymphocytes may play a role in this inflammation.
Mechanisms of Airway Hypersecretion and Novel Therapy* Jay A. Nadel, MD
(CHEST 2000; 117:262S–266S) Key words: airway hypersecretion; epidermal growth factor activation; goblet cell metaplasia Abbreviations: AB ⫽ alcian blue; EGF ⫽ epidermal growth factor; IP ⫽ intraperitoneal; IT ⫽ intratracheal; OVA ⫽ ovalbumin; PAS ⫽ periodic acid-Schiff; TGF ⫽ transforming growth factor; TNF ⫽ tumor necrosis factor
is an important feature in many chronic H ypersecretion airway diseases, including COPD, cystic fibrosis,
nflammation in the airways in COPD is largely attributed to smoking, yet this may be present even in ex-smokers. We studied changes in inflammatory cells and factors contributing to these changes in the airways of patients with COPD who do not currently smoke. We studied 18 nonatopic subjects with COPD (14 men and 4 women; mean ⫾ SD age, 62 ⫾ 8 years; FEV1, 59 ⫾ 13% predicted) and 11 nonatopic healthy subjects (8 men and 3 women; age, 58 ⫾ 8 years; FEV1, 104 ⫾ 11% predicted). Sputum induction and bronchoscopy with BAL and biopsies were performed. Subjects with COPD had more mucosal CD68⫹ cells (median, 1,115 vs 590 cells/mm, p ⫽ 0.03) and EG2⫹ cells (40 vs. 5 cells/mm, p ⫽ 0.049) and a tendency towards more CD4⫹ but not CD8⫹ cells than healthy control subjects. Furthermore, subjects with COPD had higher percentages of sputum neutrophils (77% vs 36%, p ⫽ 0.001) and eosinophils (1.2% vs 0.2%, p ⫽ 0.008) and BAL fluid eosinophils (0.4% vs 0.2%, p ⫽ 0.03) and higher concentrations of sputum, eosinophil cationic protein (ECP) (838 vs 121 ng/mL, p ⬍ 0.001). Concentrations of ECP per eosinophil were not higher.
bronchiectasis, and acute asthma. Mucus secretion is derived from airway submucosal glands and from goblet cells lining the airway epithelium. Submucosal glands are located in large conducting airways, where their ducts empty onto the airway luminal surface, preferentially at airway bifurcations adjacent to cough receptor endings. Therefore, it is not surprising that gland hypersecretion is associated with cough. Early investigators showed that airway obstruction in COPD originates in the periphery, and they showed that this obstruction is related to mortality. In 1989, Speizer et al5 concluded that a simple measure of lung function (FEV1) is an important predictor of COPD mortality. Cough and sputum production showed no (or only a weak) correlation with mortality. Subsequently, many investigators provided evidence that phlegm was of no predictive value when controlling for level of ventilatory impairment and smoking.5–7 From these findings, one may conclude that airway hypersecretion from hyperplastic glands may cause distressing symptoms, but is not likely to be a major cause of death COPD.
*From the Departments of Pulmonology (Drs. Rutgers, Postma, ten Hacken, van der Mark, and Koe¨ter), Allergology (Dr. Kauffman), and Pathology (Dr. Timens), University Hospital Groningen, The Netherlands. Correspondence to: W. Timens, MD, Professor of Pathology, University Hospital Groningen, PO Box 30.001, NL-9700 RB Groningen, Netherlands
*From the Cardiovascular Research Institute, and Departments of Medicine and Physiology, University of California San Francisco, San Francisco, CA. Correspondence to: Jay A. Nadel, MD, Division of Pulmonary and Critical Care Medicine, University of California San Francisco, CVRI, 505 Parnassus Ave, M-1325, San Francisco, CA 94143-0130; e-mail: [email protected]
262S
1
3
2
4
Thomas L. Petty 42nd Annual Aspen Lung Conference: Mechanisms of COPD
80
81 82 83 84 85 86
antielastase hypothesis 30 years later. Proc Assoc Am Physicians 1995; 107:346 –352 Hautamaki RD, Kobayashi DK, Senior RM, et al. Requirement for macrophage elastase for cigarette smoke-induced emphysema in mice. Science 1997; 277:2002–2004 Finkelstein R, Fraser RS, Ghezzo H, et al. Alveolar inflammation and its relation to emphysema in smokers. Am J Respir Crit Care Med 1995; 152:1666 –1672 Kraft M, Djukanovic R, Wilson S, et al. Alveolar tissue inflammation in asthma. Am J Respir Crit Care Med 1996; 154:1505–1510 Fletcher CM. Chronic bronchitis and decline in pulmonary function with some suggestions on terminology. In: Cumming G, Bonsignore G, eds. Smoking and the lung (vol 17). New York, NY: Plenum Press, 1984; 397– 420 Peto R, Speitzer FE, Cochrane AL, et al. The relevance in adults of airflow obstruction, but not of mucus hypersecretion, to mortality from chronic lung disease. Am Rev Respir Dis 1983; 128:491–500 Prescott E, Lange P, Vestbo J. Chronic mucus hypersecretion in COPD and death from pulmonary infection. Eur Respir J 1995; 8:1333–1338 Vestbo J, Prescott E, Lange P. Association of chronic mucus hypersecretion with FEV1 decline and COPD morbidity. Am J Respir Crit Care Med 1996; 153:1530 –1535 Chanez P, Vignola AM, O’Shaughnessy T, et al. Corticosteroid reversibility in COPD is related to features of asthma. Am J Respir Crit Care Med 1997; 155:1529 –1534 Gibson PG, Dolivich J, Denburg J, et al. Chronic cough: eosinophilic bronchitis without asthma. Lancet 1989; 1:1346 – 1348 Hopkin JM, Tomlinson CS, Jenkins RM. Variations in response to cytotoxicity of cigarette smoke. BMJ 1981; 283: 1209 –1211 Amadori A, Zamarchi R, De Silvestro G, et al. Genetic control of the CD4/CD8 T-cell ratio in humans. Nat Med 1995; 1:1279 –1283
Expression of 15-Lipoxygenase and Evidence for Apoptosis in the Lungs From Patients With COPD* Yasunori Kasahara, MD; Rubin M. Tuder, MD; Carlyne D. Cool, MD; Norbert F. Voelkel, MD
(CHEST 2000; 117:260S) Abbreviations: KDR ⫽ kinase insert domain-containing receptor; VEGF ⫽ vascular endothelial growth factor
is a loss of alveolar structures and endothelial T here cells in emphysema. We hypothesize that the disap-
pearance of lung alveoli resulting in emphysema may occur by apoptosis following decreased expression or
*From the Pulmonary Hypertension Center, Department of Medicine (Drs. Kasahara and Voelkel) and Department of Pathology (Drs. Tuder and Cool), University of Colorado Health Sciences Center, Denver, CO. Correspondence to: Yasunori Kashahara, MD, Division of Pulmonary Science and Critical Care Medicine, University of Colorado Health Sciences Center, 4200 E. 9th Ave, Denver, CO 80262 260S
activity of lung vascular endothelial growth factor (VEGF) or its receptor, kinase insert domain-containing receptor(KDR). To examine this hypothesis, we carried out the TUNEL technique in lung samples obtained from 6 patients with emphysema and 11 normal control subjects (7 nonsmokers and 4 smokers). We found an increased number of TUNEL-positive cells/nucleic acids in alveolar septum in emphysema when compared with normal lungs. The cell death events were not different between healthy nonsmokers and nonemphysema smokers. Double staining with TUNEL and cytokeratin or factor VIII-related antigen confirmed endothelial cell and epithelial cell death in emphysema lungs. Western blot analysis for KDR of whole lung protein extracts showed that KDR protein expression was significantly reduced in emphysema lungs. In situ hybridization revealed a focally decreased expression of VEGF and KDR messenger RNA in emphysema lungs. Using immunohistochemistry, we localized and semiquantitatively estimated the abundance of 15-lipoxygenase in lungs obtained from 6 patients with emphysema, 6 normal control subjects, and 7 patients with severe pulmonary hypertension. Expression of 15-lipoxygenase in pulmonary arteries in emphysema was less intense than in severe pulmonary hypertension. We conclude that cell death events occur in emphysema, and that VEGF and its receptor KDR are decreased in emphysema lungs. We speculate that emphysema may occur by apoptosis following decreased expression or activity of lung VEGF or its receptor KDR.
Morphometry Explains Variation in Airway Responsiveness in Transgenic Mice Overexpressing Interleukin-6 and Interleukin-11 in the Lung* Charles Kuhn, MD; Robert J. Homer, MD, PhD; Zhou Zhu, MD, PhD; Nicholas Ward, MD; and Jack A. Elias, MD
(CHEST 2000; 117:260S–262S) Abbreviations: AA ⫽ alveolar wall attached; AHR ⫽ airway hyperreactivity; CC10 ⫽ Clara cell 10-kd protein; IL ⫽ interleukin; S/V ⫽ surface/volume ratio
for airway hyperreactivity (AHR) accompanyT heingbasis COPD is poorly understood. Structural changes
may play a role. We have generated transgenic mice that overexpress related cytokines, interleukin (IL)-6 and IL11, in their airways, which were controlled by the Clara cell 10-kd protein (CC10) promoter. Despite some similar
*From the Department of Pathology (Dr. Kuhn), Brown University School of Medicine, Providence, RI; and the Departments of Pathology (Dr. Homer) and Pulmonary and Critical Care Medicine (Drs. Zhu, Ward, and Elias), Yale University, New Haven, CT. Correspondence to: Charles Kuhn, MD, Pathology Department, Memorial Hospital of Rhode Island, Pawtucket, RI 02860 Thomas L. Petty 42nd Annual Aspen Lung Conference: Mechanisms of COPD
Table 1—Morphometric Results on Lungs of Transgenic Mice and Littermate Controls* Mouse Strain Morphometric Variable
IL-6 (⫺)
IL-6 (⫹)
IL-11 (⫺)
IL-11 (⫹)
Cord length, m S/V, m⫺1 AAs, mm⫺1 Bronchiolar wall thickness, m External diameter, m Wall thickness, % external diameter Lumen diameter, m Thickness of mucosa and submucosa, m
29.5 ⫾ 5.5 0.142 ⫾ .009 34.2 ⫾ 1.3 2.77 ⫾ 0.34 127 ⫾ 10 2.2 ⫾ 0.2
65.3 ⫾ 10.5† 0.065 ⫾ .010† 16.4 ⫾ 1.8† 5.73 ⫾ 0.59† 188 ⫾ 26†‡ 3.1 ⫾ 0.3
33.4 ⫾ 4.3 0.134 ⫾ .017 30.6 ⫾ 3.5 2.43 ⫾ 0.49 138 ⫾ 15 1.9 ⫾ 0.4
65.6 ⫾ 9.4† 0.056 ⫾ .016† 15.6 ⫾ 1.6† 7.84 ⫾ 1.71† 148 ⫾ 21 5.6 ⫾ 1.3†
91.4 ⫾ 8.0 11.4 ⫾ 3.9
147.7 ⫾ 25.6† 12.5 ⫾ 1.8‡
96.8 ⫾ 8.1 11.5 ⫾ 1.9
90.5 ⫾ 20.9 15.8 ⫾ 2.5§
*Values given as the mean ⫾ SD of results for 6 to 12 mice in each group. (⫺) ⫽ littermate controls; (⫹) ⫽ mice expressing the transgene of the cytokine indicated. †Differs from littermate controls, p ⬍ 0.0001 by t test with Bonferroni’s correction for multiple comparisons. ‡Differs from IL-11, p ⬍ 0.01. §Differs from littermate controls, p ⬍ 0.01.
structural abnormalities, their airway physiology differed. CC10-IL-6 mice had normal expiratory flow and decreased reactivity to methacholine,1 while CC10-IL-11 mice had airflow obstruction and hyperreactivity.2 To clarify this difference, a morphometric study was undertaken of the lung parenchyma and bronchioles of the two transgenic strains and littermate controls.
caliber and external diameter of their airways. When the wall thickening was normalized to the diameter of the airway, it was proportional to the increased airway size. In contrast, the CC10-IL-11 strain showed submucosal thickening, increased muscle, and fibrosis in airways with normal external and luminal diameters. Their wall thickening was disproportionate to airway size.
Materials and Methods
Discussion
The production of the transgenic mice has been described.1,2 Lungs from mice (age, 1 to 2 months) were fixed by intratracheal instillation of glutaraldehyde at a pressure of 25 cm of fixative. Two measures of airspace size, the mean cord length and surface/volume ratio (S/V) of the gas-exchanging parenchyma, were determined by a computerized method that has been described previously.3 The bronchiolar lumen, the external diameter of the bronchioles, and the total thickness of the bronchiolar wall and thickness of the wall internal to the smooth muscle were measured with a calibrated eyepiece reticle. To determine the density of alveolar walls attached (AAs) to the bronchioles (alveolar attachments per unit length of the bronchiolar perimeter), we measured both the long and short axis of elliptically shaped bronchiolar profiles and counted the number of AAs. The perimeter of the bronchiole, P, was calculated from the measured axes using the approximate formula for the perimeter of an ellipse, P ⫽ 2 公(a2 ⫹ b2)/2, where a and b are the semi-major and semi-minor axes, respectively. Knowing the calculated value for P, the value of the density of AA then is AA/P.
Results The morphometric results are summarized in Table 1. Both transgenic strains of mice had emphysema-like airspace enlargement that was of identical severity by three morphometric measures: cord length, S/V, and AA/P. This enlargement is thought to be the result of the failure of septation in both strains, although it has only been proven for the CC10-IL-11 mice.3 Both had lymphocytic nodules in airways and airway wall thickening with increased fibrosis and smooth muscle. Compared with controls, hyporeactive CC10-IL-6 mice had a 50% increase in the
The structural changes that lead to AHR have been modeled and studied in detail.4 – 6 Airway narrowing in response to agonists results from smooth muscle shortening working against the passive load of the recoil of the surrounding parenchyma. Emphysema, with its attendant loss of recoil and smooth muscle hypertrophy, which increases the force of contraction, will exaggerate the response to a given dose of agonist. Thickening of the bronchiolar wall internal to the smooth muscle also exaggerates airway narrowing,4 while thickening of the adventitial sheath may uncouple the interdependence of the airways and parenchyma, thereby decreasing the effect of parenchymal recoil. Hence, the airway remodeling and emphysema-like parenchymal changes both contribute to the airflow obstruction and AHR seen in the IL-11expressing mice. The IL-6-expressing mice had equally severe emphysema and also had airway wall thickening, but their bronchioles were 50% larger than those of either their littermates or the IL-11-expressing strain, and their airway wall thickening was proportional to airway size. Evidently, the increased lumen diameter overrode the effect of the emphysema and airway remodeling and resulted in relatively large airway diameter even after agonist inhalation. We conclude that airway remodeling and emphysema sometimes lead to AHR, but baseline airway size and caliber also determine airway responsiveness.
References 1 DiCosmo BF, Geba GP, Picarella D, et al. Airway epithelial cell expression of interleukin-6 in transgenic mice: uncouCHEST / 117 / 5 / MAY, 2000 SUPPLEMENT
261S
2
3
4 5 6
pling of airway inflammation and bronchial hyperreactivity. J Clin Invest 1994; 94:2028 –2035 Tang W, Geba GP, Zheng T, et al. Targeted expression of IL-11 in the murine airway causes lymphocytic inflammation, bronchial remodelling and airways obstruction. J Clin Invest 1996; 98:2845–2853 Ray R, Tang W, Wong P, et al. Regulated overexpression of interleukin-11 in the lung: Use to dissociate developmentdependent and -independent phenotypes. J Clin Invest 1997; 100:2501–2511 Wiggs, BR, Bosken C, Pare´ PD, et al. A model of airway narrowing in asthma and in chronic obstructive pulmonary disease. Am Rev Respir Dis 1992; 145:1251–1258 Lambert RK, Wiggs BR, Kuwano K, et al. Functional significance of increased airway smooth muscle in asthma and COPD. J Appl Physiol 1993; 74:2771–2781 Pare´ PD, Bai TR. Airway wall remodelling in chronic obstructive pulmonary disease. Eur Respir Rev 1996; 6:259 –263
Ongoing Airway Inflammation in Patients With COPD Who Do Not Currently Smoke* Steven R. Rutgers, MD; Dirkje S. Postma, MD; Nick H. ten Hacken, MD; Henk F. Kauffman, PhD; Thomas W. van der Mark, PhD; Gerard H. Koe¨ter, MD; and Wim Timens, MD
(CHEST 2000; 117:262S) Abbreviation: ECP ⫽ eosinophil cationic protein
I
Patients with COPD and high mucosal EG2⫹ cell numbers had high mucosal CD4⫹ cell numbers. Sputum eosinophilia was associated with a decrease in FEV1/vital capacity and BAL fluid eosinophilia with a decrease in mucosal NP57⫹ cells. We conclude that subjects with COPD who do not currently smoke have increased numbers of inflammatory cells. Eosinophils are increased in number in the airways in COPD but do not seem to be activated. The increased eosinophil numbers are probably due to recruitment as a result of ongoing inflammation. Macrophages and lymphocytes may play a role in this inflammation.
Mechanisms of Airway Hypersecretion and Novel Therapy* Jay A. Nadel, MD
(CHEST 2000; 117:262S–266S) Key words: airway hypersecretion; epidermal growth factor activation; goblet cell metaplasia Abbreviations: AB ⫽ alcian blue; EGF ⫽ epidermal growth factor; IP ⫽ intraperitoneal; IT ⫽ intratracheal; OVA ⫽ ovalbumin; PAS ⫽ periodic acid-Schiff; TGF ⫽ transforming growth factor; TNF ⫽ tumor necrosis factor
is an important feature in many chronic H ypersecretion airway diseases, including COPD, cystic fibrosis,
nflammation in the airways in COPD is largely attributed to smoking, yet this may be present even in ex-smokers. We studied changes in inflammatory cells and factors contributing to these changes in the airways of patients with COPD who do not currently smoke. We studied 18 nonatopic subjects with COPD (14 men and 4 women; mean ⫾ SD age, 62 ⫾ 8 years; FEV1, 59 ⫾ 13% predicted) and 11 nonatopic healthy subjects (8 men and 3 women; age, 58 ⫾ 8 years; FEV1, 104 ⫾ 11% predicted). Sputum induction and bronchoscopy with BAL and biopsies were performed. Subjects with COPD had more mucosal CD68⫹ cells (median, 1,115 vs 590 cells/mm, p ⫽ 0.03) and EG2⫹ cells (40 vs. 5 cells/mm, p ⫽ 0.049) and a tendency towards more CD4⫹ but not CD8⫹ cells than healthy control subjects. Furthermore, subjects with COPD had higher percentages of sputum neutrophils (77% vs 36%, p ⫽ 0.001) and eosinophils (1.2% vs 0.2%, p ⫽ 0.008) and BAL fluid eosinophils (0.4% vs 0.2%, p ⫽ 0.03) and higher concentrations of sputum, eosinophil cationic protein (ECP) (838 vs 121 ng/mL, p ⬍ 0.001). Concentrations of ECP per eosinophil were not higher.
bronchiectasis, and acute asthma. Mucus secretion is derived from airway submucosal glands and from goblet cells lining the airway epithelium. Submucosal glands are located in large conducting airways, where their ducts empty onto the airway luminal surface, preferentially at airway bifurcations adjacent to cough receptor endings. Therefore, it is not surprising that gland hypersecretion is associated with cough. Early investigators showed that airway obstruction in COPD originates in the periphery, and they showed that this obstruction is related to mortality. In 1989, Speizer et al5 concluded that a simple measure of lung function (FEV1) is an important predictor of COPD mortality. Cough and sputum production showed no (or only a weak) correlation with mortality. Subsequently, many investigators provided evidence that phlegm was of no predictive value when controlling for level of ventilatory impairment and smoking.5–7 From these findings, one may conclude that airway hypersecretion from hyperplastic glands may cause distressing symptoms, but is not likely to be a major cause of death COPD.
*From the Departments of Pulmonology (Drs. Rutgers, Postma, ten Hacken, van der Mark, and Koe¨ter), Allergology (Dr. Kauffman), and Pathology (Dr. Timens), University Hospital Groningen, The Netherlands. Correspondence to: W. Timens, MD, Professor of Pathology, University Hospital Groningen, PO Box 30.001, NL-9700 RB Groningen, Netherlands
*From the Cardiovascular Research Institute, and Departments of Medicine and Physiology, University of California San Francisco, San Francisco, CA. Correspondence to: Jay A. Nadel, MD, Division of Pulmonary and Critical Care Medicine, University of California San Francisco, CVRI, 505 Parnassus Ave, M-1325, San Francisco, CA 94143-0130; e-mail: [email protected]
262S
1
3
2
4
Thomas L. Petty 42nd Annual Aspen Lung Conference: Mechanisms of COPD