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Role of Neutrophil Elastase in Hypersecretion During COPD Exacerbations, and Proposed Therapies* FREE TO VIEW

Jay A. Nadel, MD
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*From the Cardiovascular Research Institute, Departments of Medicine and Physiology, University of California, San Francisco, CA.

Correspondence to: Jay A. Nadel, MD, University of California, San Francisco, CVRI, 505 Parnassus Ave, M-1325, San Francisco, CA 94143-0130; e-mail: janadel@itsa.ucsf.edu



Chest. 2000;117(5_suppl_2):386S-389S. doi:10.1378/chest.117.5_suppl_2.386S
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A common feature of COPD and other chronic lung diseases is hypersecretion of mucus into the airways, causing peripheral airway plugging and further airflow obstruction. The mucus is secreted by goblet cells, which are present in excessive numbers in COPD. This review describes how neutrophils in the airways of COPD patients stimulate the goblet cells to secrete their products. Recent findings on the mechanisms of neutrophil stimulation of goblet cell degranulation are discussed. These implicate the proteolytic enzyme elastase and cell surface adhesion molecules, and provide a basis for the investigation of potential novel therapies.

Figures in this Article

Airway hypersecretion is a characteristic feature in COPD and is a significant contributor to exacerbations. Excess mucus in the airways causes severe cough and discomfort, and can lead to further obstruction and inflammation. The mechanism of hypersecretion is, however, poorly understood. The airway epithelium undergoes metaplastic change in COPD, resulting in an excess of goblet cells; these cells, along with the submucosal glands, are responsible for the mucus production. In designing a therapeutic strategy to combat hypersecretion, two approaches might thus be considered: inhibiting the growth of the goblet cells, or preventing their degranulation, ie, the release of their secretory products. The mechanism of goblet cell degranulation in COPD is currently not known. This review highlights the possible role of neutrophils (which are present in inflammatory sites in the airways) in stimulating goblet cell degranulation and mucus hypersecretion in COPD and exacerbations.

Neutrophils are recruited into the airways in COPD and other chronic respiratory diseases12 by chemoattractants that are present in the airways35; in vitro and in vivo studies show that these neutrophils can stimulate goblet cell degranulation.6 The guinea pig provides a convenient model to study degranulation, since the airways normally contain goblet cells.7 Direct tracheal injection of neutrophil chemoattractants such as interleukin (IL)-8 or N-formyl-methyl-leucyl-phenylalanine (fMLP) results in migration of neutrophils into the lumen of the trachea. The tracheas of these animals can then be removed and examined microscopically, both for neutrophil infiltration (by myeloperoxidase staining) and for goblet cell degranulation (by Alcian blue [AB]/periodic acid-Schiff [PAS] staining of mucosubstances on the mucosal surface epithelium; Fig 1 ). Degranulation is quantified by volume density of staining using a semiautomatic imaging system (the public domain National Institutes of Health Image program, available at zippy.nimh.gov, or on disk from the National Technical Information Service, Springfield, VA; part number PB95–500195GEI).

Injection of IL-8 or fMLP was found, as expected, to result in neutrophil infiltration of the guinea pig trachea, maximal at 1 to 2 h.6 It was also demonstrated that this infiltration was followed by profound goblet cell degranulation (maximal at 4 h). Moreover, inhibition of neutrophil recruitment, using the leukocyte migration inhibitor NPC 15669, also inhibited goblet cell degranulation. These data, therefore, strongly suggest that neutrophils, which infiltrate the airways in COPD, promote in the degranulation of goblet cells and the resulting hypersecretion and concomitant problems.

The Role of Neutrophil Elastase

Purified neutrophil elastase has been shown previously to be a potent secretagogue for goblet cells in vitro.89 However, it is not clear whether this molecule is responsible for the effect of neutrophils on degranulation, since the cells themselves release little or no elastase in vitro, even after “activation” with a variety of molecules including IL-8, fMLP, leukotriene-B4, and tumor necrosis factor-α.10 Rather, activation of neutrophils causes a translocation of elastase from the azurophilic granules to the cell surface.11 In the guinea pig model, however, we have shown that an elastase inhibitor, ICI 200355, inhibited the neutrophil-dependent goblet cell degranulation seen after tracheal instillation of neutrophil chemoattractants.7 These results confirmed the role of neutrophil elastase in degranulation, but the question still remained as to how elastase exerts its effect, since neutrophil activators/chemoattractants do not appear to promote its release. To investigate this question, isolated human neutrophils were incubated with segments of guinea pig trachea, and the effects of a variety of agents on degranulation were assessed. It was confirmed, firstly, that activation of neutrophils with IL-8 and other chemoattractants resulted in expression of cell surface elastase. It was also shown that preactivated neutrophils, washed with sterile phosphate-buffered saline solution to avoid further contamination with IL-8, could still promote degranulation, while the cell-free supernatant from activated neutrophils had no effect. This demonstrated that it is cell-associated elastase, rather than the released enzyme, which is involved in degranulation.

The role of cell-associated elastase in goblet cell degranulation implies a direct interaction between the neutrophils and goblet cells. The possible role of adhesion molecules in this process has therefore been investigated. Human neutrophils and human tracheal segments, obtained from patients undergoing lung transplantation, were chosen for these experiments. These tracheae contained a high proportion of goblet cells (> 20%) due to the pathologic condition involved (pulmonary fibrosis, cystic fibrosis, COPD). The data from guinea pigs were confirmed in the human system; IL-8-activated neutrophils stimulated degranulation, and this was inhibited by elastase inhibitors. Furthermore, when neutrophils were pretreated with monoclonal antibody against cell surface adhesion molecules, antibodies to integrins of the β2 subfamily (CD11b [Mac-1] or CD18 [lymphocyte function-associated antigen-1β]) also inhibited IL-8 mediated degranulation (Fig 2 ). A similar inhibitory effect was obtained by pretreatment of the airway tissue with antibody to intercellular adhesion molecule-1. These results suggest that neutrophil-stimulated goblet cell degranulation is mediated by a direct interaction between the neutrophils and the goblet cells in the airway epithelium involving intercellular adhesion molecule-1 and integrins of the β2 subfamily.

Metaplasia of the airway epithelium in COPD and other airway diseases results in large numbers of goblet cells in the peripheral airways. When these goblet cells are stimulated to release their contents, the excess mucus in the airways can cause severe cough, airflow obstruction, peripheral airway plugging, and may lead to further inflammation and possibly infection. Neutrophils, which migrate into the airways in COPD, and particularly into inflammatory sites during exacerbations, stimulate goblet cell degranulation. These cells thus play a very important role in hypersecretion.

The experiments discussed here indicate the likely mechanism by which neutrophils promote hypersecretion, and thus offer a number of possible avenues for design of therapeutic strategies. Firstly, prevention of neutrophil migration into the airways is one obvious mechanism by which hypersecretion could be reduced. This might also reduce inflammation, but could also have unwanted side effects, including an impaired ability to combat infection. Selective inhibition of neutrophil elastase might, however, inhibit hypersecretion without other less desirable inhibitory effects on neutrophil activity. Similarly, interfering with the neutrophil/goblet cell interaction, either using integrin/adhesion molecule antagonists or regulating cell surface expression, offers another interesting possibility.

Neutrophils are undoubtedly crucial players in the pathogenesis of COPD and its exacerbations, and we are now beginning to understand the mechanisms by which these cells mediate hypersecretion. Armed with this knowledge, it is now possible to consider ways in which the debilitating consequences of hypersecretion can be alleviated.

Abbreviations: AB = Alcian blue; fMLP = N-formyl-methionyl-leucyl-phenylalanine; IL = interleukin; PAS = periodic acid-Schiff

Figure Jump LinkFigure 1. Photomicrographs of human bronchial epithelium removed from patient with idiopathic pulmonary fibrosis at the time of lung transplantation and stained with AB/PAS. In the unstimulated condition (top left, a), AB-positive cells are conspicuous in the bronchial epithelium. Bronchial segments that are incubated with neutrophils alone (top right, b) or with IL-8 alone (bottom left, c) do not show degranulation. However, segments incubated with IL-8 plus neutrophils show profound goblet cell degranulation (arrows; bottom right, d). Reprinted with permission from Takeyama et al.6Grahic Jump Location
Figure Jump LinkFigure 2. Effects of neutrophils (106 cells/mL) alone, IL-8 (10-7 M) alone, neutrophils plus IL-8, or neutrophils “activated” by IL-8 on goblet cell degranulation (expressed as percent area of epithelium stained with AB/PAS in human bronchial segments in vitro) and effects of anti-CD18 antibody on goblet cell degranulation caused by “activated” neutrophils. For explanation, see text. Responses are expressed as mean ± SEM; n = 4 for each group. *p < 0.01, significantly different from control values. †p < 0.05, significantly different from the response to “activated” neutrophils. M = molar.Grahic Jump Location
Snider, GL, Faling, LJ, Rennard, SI (1994) Chronic bronchitis and emphysema. Murray, JF Nadel, JA eds.Textbook of respiratory medicine 2nd ed. ,1331-1397 Saunders. New York, NY:
 
Richman-Eisenstat, JB, Jorens, PG, Hebert, CA, et al Interleukin-8: an important chemoattractant in sputum of patients with chronic inflammatory airway diseases.Am J Physiol1993;264 (Lung Cell Mol Physiol 8),L413-L418
 
Keatings, VM, Collins, PD, Scott, DM, et al Differences in interleukin-8 and tumor necrosis factor-alpha in induced sputum from patients with chronic obstructive pulmonary disease or asthma.Am J Respir Crit Care Med1996;153,530-534
 
Nocker, RE, Shcoonbrood, DF, van de Graaf, EA, et al Interleukin-8 in airway inflammation in patients with asthma and chronic obstructive pulmonary disease.Int Arch Allergy Immunol1996;109,183-191
 
Ozaki, T, Hayashi, H, Tani, K, et al Neutrophil chemotactic factors in the respiratory tract of patients with chronic airway diseases or idiopathic pulmonary fibrosis.Am Rev Respir Dis1992;145,85-91
 
Takeyama, K, Agusti, C, Ueki, I, et al Neutrophil-dependent goblet cell degranulation: role of membrane-bound elastase and adhesion molecules.Am J Physiol1998;275 (Lung Cell Mol Physiol 19),L294-L302
 
Tokuyama, K, Kuo, HP, Rohde, JA, et al Neural control of goblet cell secretion in guinea pig airways.Am J Physiol1990;259 (Lung Cell Mol Physiol 3),L108-L115
 
Breuer, R, Christensen, TG, Lucey, EC, et al An ultrastructural morphometric analysis of elastase-treated hamster bronchi shows discharge followed by progressive accumulation of secretory granules.Am Rev Respir Dis1987;136,698-703
 
Sommerhoff, CP, Nadel, JA, Basbaum, CB, et al Neutrophil elastase and cathepsin G stimulate secretion from cultured bovine airway gland serous cells.J Clin Invest1990;85,682-689
 
Jorens, PG, Richman-Eisenstat, JB, Housset, BP, et al Interleukin-8 induces neutrophil accumulation but not protease secretion in the canine trachea.Am J Physiol1992;263 (Lung Cell Mol Physiol 7),L708-L713
 
Owen, CA, Campbell, MA, Sannes, PL, et al Cell surface-bound elastase and cathepsin G on human neutrophils: a novel, non-oxidative mechanism by which neutrophils focus and preserve catalytic activity of serine proteinases.J Cell Biol1995;131,775-789
 

Figures

Figure Jump LinkFigure 1. Photomicrographs of human bronchial epithelium removed from patient with idiopathic pulmonary fibrosis at the time of lung transplantation and stained with AB/PAS. In the unstimulated condition (top left, a), AB-positive cells are conspicuous in the bronchial epithelium. Bronchial segments that are incubated with neutrophils alone (top right, b) or with IL-8 alone (bottom left, c) do not show degranulation. However, segments incubated with IL-8 plus neutrophils show profound goblet cell degranulation (arrows; bottom right, d). Reprinted with permission from Takeyama et al.6Grahic Jump Location
Figure Jump LinkFigure 2. Effects of neutrophils (106 cells/mL) alone, IL-8 (10-7 M) alone, neutrophils plus IL-8, or neutrophils “activated” by IL-8 on goblet cell degranulation (expressed as percent area of epithelium stained with AB/PAS in human bronchial segments in vitro) and effects of anti-CD18 antibody on goblet cell degranulation caused by “activated” neutrophils. For explanation, see text. Responses are expressed as mean ± SEM; n = 4 for each group. *p < 0.01, significantly different from control values. †p < 0.05, significantly different from the response to “activated” neutrophils. M = molar.Grahic Jump Location

Tables

References

Snider, GL, Faling, LJ, Rennard, SI (1994) Chronic bronchitis and emphysema. Murray, JF Nadel, JA eds.Textbook of respiratory medicine 2nd ed. ,1331-1397 Saunders. New York, NY:
 
Richman-Eisenstat, JB, Jorens, PG, Hebert, CA, et al Interleukin-8: an important chemoattractant in sputum of patients with chronic inflammatory airway diseases.Am J Physiol1993;264 (Lung Cell Mol Physiol 8),L413-L418
 
Keatings, VM, Collins, PD, Scott, DM, et al Differences in interleukin-8 and tumor necrosis factor-alpha in induced sputum from patients with chronic obstructive pulmonary disease or asthma.Am J Respir Crit Care Med1996;153,530-534
 
Nocker, RE, Shcoonbrood, DF, van de Graaf, EA, et al Interleukin-8 in airway inflammation in patients with asthma and chronic obstructive pulmonary disease.Int Arch Allergy Immunol1996;109,183-191
 
Ozaki, T, Hayashi, H, Tani, K, et al Neutrophil chemotactic factors in the respiratory tract of patients with chronic airway diseases or idiopathic pulmonary fibrosis.Am Rev Respir Dis1992;145,85-91
 
Takeyama, K, Agusti, C, Ueki, I, et al Neutrophil-dependent goblet cell degranulation: role of membrane-bound elastase and adhesion molecules.Am J Physiol1998;275 (Lung Cell Mol Physiol 19),L294-L302
 
Tokuyama, K, Kuo, HP, Rohde, JA, et al Neural control of goblet cell secretion in guinea pig airways.Am J Physiol1990;259 (Lung Cell Mol Physiol 3),L108-L115
 
Breuer, R, Christensen, TG, Lucey, EC, et al An ultrastructural morphometric analysis of elastase-treated hamster bronchi shows discharge followed by progressive accumulation of secretory granules.Am Rev Respir Dis1987;136,698-703
 
Sommerhoff, CP, Nadel, JA, Basbaum, CB, et al Neutrophil elastase and cathepsin G stimulate secretion from cultured bovine airway gland serous cells.J Clin Invest1990;85,682-689
 
Jorens, PG, Richman-Eisenstat, JB, Housset, BP, et al Interleukin-8 induces neutrophil accumulation but not protease secretion in the canine trachea.Am J Physiol1992;263 (Lung Cell Mol Physiol 7),L708-L713
 
Owen, CA, Campbell, MA, Sannes, PL, et al Cell surface-bound elastase and cathepsin G on human neutrophils: a novel, non-oxidative mechanism by which neutrophils focus and preserve catalytic activity of serine proteinases.J Cell Biol1995;131,775-789
 
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