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(Chest. 2000;117:262S-266S.)
© 2000 American College of Chest Physicians

Mechanisms of Airway Hypersecretion and Novel Therapy*

Jay A. Nadel, MD

* 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: janadel{at}itsa.ucsf.edu

Key Words: airway hypersecretion • epidermal growth factor activation • goblet cell metaplasia


   Introduction
TOP
 Introduction
Materials and Methods
 Results
 Discussion
 References
 

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

Hypersecretion is an important feature in many chronic airway diseases, including COPD,1 cystic fibrosis,2 bronchiectasis,3 and acute asthma.4 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 6 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.

Goblet cells exist in variable numbers in the airway epithelium. In healthy humans and in pathogen-free animals, peripheral airways contain few goblet cells,8 but goblet cell metaplasia occurs in COPD,1 cystic fibrosis,2 and acute asthma.4 With the onset of lung transplantation and with extensive studies of COPD exacerbations, the importance of goblet cell metaplasia in peripheral airways is becoming recognized. Our hypothesis is that degranulation of these goblet cells leads to mucous plugging in peripheral airways. Further, we suggest that peripheral mucous plugging initially may cause few symptoms, but extensive plugging may lead to deterioration and death.

Goblet cell hypersecretion can be divided into two steps. First, airway epithelial cells are converted to mucus-containing goblet cells. Second, goblet cell degranulation of peripheral airways may be stimulated, leading to obstruction by plugging. We reasoned that if we could prevent goblet cells from being expressed, mucus secretion could not occur. Therefore, we have focused the present studies on the mechanism of goblet cell formation (rather than degranulation).

We hypothesized that a growth factor could be involved in goblet cell production, because hypersecretory diseases are associated with abnormal epithelial growth and proliferation. A possible candidate is epidermal growth factor (EGF) and its receptor EGF-R. EGF-R, a 70-kd membrane glycoprotein, is expressed in fetal airways, where it is important in cell proliferation, branching morphogenesis, and epithelial cell differentiation.9 In healthy adult human airways, expression of EGF-R is sparse, but EGF-R is expressed in malignant tumors and in asthma.10 In addition, EGF-R can be up-regulated by tumor necrosis factor (TNF)-{alpha} in lungs in hypersecretory diseases.11 Therefore, we hypothesized that the EGF-R system could play a role in goblet cell production in disease. We found that stimulation of airway epithelial cells with TNF-{alpha} induces EGF-R in epithelial cell cultures and in rats in vivo.8 Further, we showed that stimulation of EGF-R by its ligands results in mucus-producing goblet cells, and that ovalbumin (OVA) sensitization in rats causes induction of EGF-R and goblet cell production in rat airways. A key discovery is that selective EGF-R tyrosine kinase inhibitors prevent mucus production in each of these systems. We suggest that inhibitors of EGF-R could be useful in preventing goblet cell production and thus hypersecretion in disease. The studies are reported in detail elsewhere.8 Only the in vivo studies are reported here.


   Materials and Methods
TOP
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
In Vivo Studies
The experimental animal protocol was approved by the Committee on Animal Research, University of California San Francisco. Specific pathogen-free male F344 Fisher rats, weighing 230 to 250 g (Simonsen Laboratories; Gilroy, CA), were maintained in a temperature-controlled (21°C) room with standard laboratory food and water freely available.

Healthy Rats
Rats were anesthetized with methohexital sodium, 50 mg/kg intraperitoneal (IP), and allowed to breathe spontaneously. TNF-{alpha} (200 ng, 100 µL) was instilled into the trachea, and the animals were euthanized 24 h later. EGF (600 ng, 100 µL) or transforming growth factor (TGF)-{alpha} (rat synthetic TGF-{alpha}, 250 ng, 100 µL; Sigma; St. Louis, MO) was instilled into the trachea either alone or 24 h after the instillation of TNF-{alpha} (200 ng, 100 µL), and the animals were euthanized 48 h later. In each study, sterile phosphate-buffered saline solution (100 µL) was instilled into the trachea as control. In inhibition studies, rats were pretreated with BIBX1522 (3, 10, or 30 mg/kg IP, the dose estimated from studies using the inhibitor to prevent cancer growth), 1 h before and 24 h after instillation of TGF-{alpha}. The trachea and lungs were removed for examination 48 h after the instillation of TGF-{alpha}.

Sensitized Rats
Rats were sensitized on days 0 and 10 with IP injections of OVA (10 mg, grade V; Sigma), complexed with 100 mg of aluminum hydroxide in 0.5 mL of sterile saline solution. On days 20, 22, and 24, OVA (0.1%, 100 µL) was delivered by intratracheal (IT) instillation. Rats were euthanized either without IT instillation (day 20), or 48 h after the third IT instillation (day 26). To study the effect of BIBX1522 on goblet cell production in sensitized rats, BIBX1522 was given IP (10 mg/kg) 1 h before the IT instillation of OVA and instilled into the trachea (10-5mol/L, 100 µL) on days 20, 22 and 24. BIBX1522 was also injected IP (10 mg/kg) every 24 h until the day before the rats were euthanized. Forty-eight hours after the third IT instillation, the animals were euthanized and the trachea and lungs were removed.

Tissue Preparation
At preselected times during anesthesia, the systemic circulation was perfused with 1% paraformaldehyde in diethylenephosphoramide-treated phosphate-buffered saline solution at a pressure of 120 mm Hg. For frozen sections, tissues were removed and placed in 4% paraformaldehyde for 1 h and then replaced in 30% sucrose solution for cryoprotection overnight. The tissues were embedded in optimal cutting temperature compound. For plastic sections, the tissues were placed in 4% paraformaldehyde for 24 h, then dehydrated and embedded in JB-4 plus monomer solution A (Polysciences; Warrington, PA). The embedded tissues were cut as cross sections (4-µm thick) and placed on glass slides.

Cell Analysis
The total number of epithelial cells was determined by counting epithelial cell nuclei over 2 mm of the basal lamina with an oil immersion objective lens (x 1,000 magnification). The linear length of the basal lamina under each analyzed region of epithelium was determined by tracing the contour of the digitized image of the basal lamina. The epithelial cells were identified as described previously.12 13 In brief, goblet cells are goblet to low columnar in shape, with abundant alcian blue (AB)/periodic acid-Schiff (PAS)-stained granules filling most of the cytoplasm. Pregoblet cells contain smaller mucus-stained areas (< 1/3 height in epithelium from basement membrane to luminal surface) or with sparse granules stained with AB/PAS. Ciliated cells are recognized by their ciliated borders, lightly stained cytoplasm, and large round nuclei. Nongranulated secretory cells are columnar in shape and extend from the lumen to the basal lamina. The cytoplasm stains light pink, and a few small, PAS-positive and AB-negative granules are observed in the cytoplasm. Basal cells are small flattened cells with large nuclei located just above the basal lamina but not reaching the airway lumen.

Quantification of Goblet Cell Production
Goblet cell production was determined by the volume density of AB/PAS-stained mucous glycoconjugates on the epithelial mucosal surface, using a semi-automatic imaging system described elsewhere.14 We measured the AB/PAS-positively stained area and the total epithelial area and expressed the data as the percentage of the total area stained by AB-PAS. The analysis was performed with the public domain NIH Image program (developed at the US National Institutes of Health and available from the Internet by anonymous file transfer protocol from zippy.nimh.gov, or on floppy disk from the National Technical Information Service, Springfield, VA; part number PB95–500195GEI).

Immunohistochemical Localization of EGF-R in Rat Epithelium
The localization of EGF-R was examined using immunohistochemical staining with an antibody to EGF-R (Calbiochem; San Diego, CA) in frozen sections of rat trachea.

In Situ Hybridization
The complementary DNA for rat MUC5AC was generously provided by Dr. Carol Basbaum. A 320-base pair complementary DNA fragment of rat MUC5AC was subcloned into the Xba/hindIII site of the transcription vector, (pBluescript-SK(-); Stratagene; La Jolla, CA). The preparation of RNA probes and in situ hybridization were performed as described previously.12

Statistics
All data are expressed as mean ± SEM. One-way analysis of variance was used to determine statistically significant differences between groups. Scheffe’s F test was used to correct for multiple comparisons when statistical significances were identified in the analysis of variance. A probability < 0.05 for the null hypothesis was accepted as indicating a statistically significant difference.


   Results
TOP
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In the control state, the tracheal epithelium of pathogen-free rats contained few goblet and pregoblet cells. IT instillation of EGF-R ligands (EGF-R or TGF-{alpha}) alone had no effect on goblet cell production (Table 1 ). However, when TNF-{alpha} was first given, followed 24 h later by TGF-{alpha} or by EGF (data not shown), the numbers of goblet and pregoblet cells were increased markedly; the numbers of nongranulated secretory cells and basal cells decreased significantly, whereas there was no significant change in the total number of cells or in the number of ciliated cells. (Table 1) . In situ hybridization for MUC5AC gene showed no expression in control animals. When TNF-{alpha} was instilled intratracheally, followed by EGF or TGF-{alpha}, expression of MUC5AC was visible in the epithelium. Thus, induction of EGF-R alone or stimulation by EGF-R ligands alone was insufficient to induce goblet cell production. However, after the induction of EGF-R by TNF-{alpha}, instillation of EGF-R ligands stimulated goblet cell production markedly.


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  		Table 1. Cell Analysis in Tracheal Epithelium*

 
Next, we examined the effect of OVA sensitization on EGF-R and goblet cell production. IP injections of OVA did not alter the total number of epithelial cells and did not increase the number of goblet cells or pregoblet cells (Table 1) . However, when this was followed by three IT instillations of OVA, the total number of epithelial cells increased significantly compared with the control state. The numbers of goblet and pregoblet cells were increased markedly, but the numbers of ciliated and basal cells were unchanged (Table 1) . Thus, OVA IP followed by OVA IT caused goblet cell hyperplasia.

Because BIBX1522 prevented mucin production in cultured cells, the effect of this inhibitor was examined in pathogen-free rats. AB/PAS staining, which was increased by tracheal instillation of TNF-{alpha} followed by EGF-R ligand TGF-{alpha}, was inhibited dose-dependently by pretreatment with BIBX1522 (Fig 1 , top, A). Similarly, three intratracheal instillations of OVA caused a significant increase in goblet cell production, which was inhibited by pretreatment with BIBX1522 (Fig 1 , bottom, B).



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  		Figure 1. Effect of EGF-R tyrosine kinase inhibitor (BIBX1522) on production of goblet cells (expressed as percent of stained area of airway epithelium occupied by AB/PAS-positive stained cells). Top, A: Stimulation with TNF-{alpha} (200 ng, 100 µL). Tracheal instillation of TNF-{alpha} followed by the EGF-R ligand TGF-{alpha} increased goblet cell production significantly (n = 5; *p < 0.0001), an effect that was inhibited by pretreatment with BIBX1522 (3 to 30 mg/kg, IP) dose-dependently (n = 5; p* compared to TNF-{alpha} followed by TGF-{alpha}, {dagger} p = 0.0034, {dagger}{dagger} p < 0.0001). Bottom, B: OVA sensitization. Animals given OVA IP only showed little AB/PAS-positive staining in bronchial epithelium. Animals first sensitized with OVA IP, followed by three IT instillations of OVA, showed a marked increase in AB/PAS-positive staining (n = 5 *p < 0.0001). Pretreatment with BIBX1522 (10 mg/kg, IP) inhibited OVA-induced production of goblet cells (n = 5, {dagger}{dagger} p < 0.0001).8

 

   Discussion
TOP
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this investigation, we examined the role of EGF-R activation in the production of mucins in airways. The hypothesis was that activation of EGF-R tyrosine kinase results in goblet cell production of mucins in airway epithelium. Because the use of these inhibitors cannot yet be used in such studies in man, we examined a permanent line of cultured human airway epithelial (NCI-H292) cells known to produce mucins.8 We found that TNF-{alpha} induced the expression of EGF-R in the cells. Subsequent stimulation with EGF-R ligands (EGF, TGF-{alpha}) resulted in the MUC5AC production at both gene expression and protein levels. Selective inhibitors of EGF-R tyrosine kinase blocked this expression, implicating EGF-R signaling in MUC5AC production.

In pathogen-free rats (as in healthy humans), the airway epithelium contains few EGF-R constitutively. However, when TNF-{alpha} was instilled IT, EGF-R were expressed in the airway epithelium. Subsequent stimulation with an EGF-R ligand resulted in mucin gene and protein expression in the epithelium. Similarly, active sensitization by instillation of OVA into the airways resulted in the expression of EGF-R in the epithelium and the conversion of epithelial cells to the goblet cell phenotype. Most interestingly, a selective inhibitor of EGF-R tyrosine kinase completely inhibited goblet cell production in rats stimulated with TNF-{alpha} plus an EGF-R ligand or sensitized with OVA. These results incriminate EGF-R activation in goblet cell metaplasia.

Previous studies showed that various stimuli such as ozone,15 sulfur dioxide,16 viruses,16 lipopolysaccharide15 17 and platelet-activating factor12 up-regulate mucin expression. Chronic cigarette smoking is incriminated in the production of chronic bronchitis. Additional studies will be required to evaluate the relationship of these and other stimuli to the production of mucin-producing cells by the EGF-R system.

In the past, the role of peripheral airways in mucus hypersecretory diseases has been relatively neglected for several reasons. First, the symptoms of hypersecretion deriving from the large conducting airways has drawn major attention because of the associated symptom of cough. Narrowing of the major conducting airways results in significant changes in the mechanical properties of the lungs and consequent symptoms associated with the increased resistive work of breathing. On the other hand, because the resistive work performed by individual peripheral airways is small, even complete obstruction of multiple peripheral airways may not cause significant symptoms. Complete obstruction of peripheral airways could cause significant hypoxemia, but decreased oxygen saturation is not a prominent cause of dyspnea. Furthermore, tests presently available (eg, pulmonary function, radiographic techniques) are not diagnostic of obstruction in peripheral airways. New methodologies need to be developed to improve the ability to diagnose these lesions.

When metaplasia of airway epithelium to produce goblet cells occurs, little or no change in airway geometry may occur as long as the mucins remain tightly packed inside goblet cells. However, when goblet cells degranulate, the released mucins become hydrated and expand in volume, so airway obstruction occurs.

Therapy of hypersecretion can be divided into two strategies: (1) prevention of excytosis of goblet cell mucins; and (2) prevention of the development of goblet cells. This study focuses on prevention of goblet cell development. A discussion of some recent studies on a potent mechanism of goblet cell degranulation is described elsewhere.14

In summary, the EGF-R cascade is shown by the present studies to be important in stimulating the growth of airway goblet cells, which are implicated in mucus hypersecretion, especially in peripheral airways where lesions are difficult to detect and potentially lethal. Treatment with selective inhibitors of EGF-R tyrosine kinase may provide effective therapy in hypersecretory diseases of airways.


   References
TOP
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. 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.
  2. Boat, TF, Boucher, RC (1994) Cystic fibrosis. Murray, JF Nadel, JA eds. Textbook of respiratory medicine 2nd ed. ,1418-1450 Saunders New York, NY.
  3. Fahy, JV, Schuster, A, Ueki, I, et al (1992) Mucus hypersecretion in bronchiectasis: the role of neutrophil proteases. Am Rev Respir Dis 146,1430-1433[ISI][Medline]
  4. Shimura, S, Andoh, Y, Haraguchi, M, et al (1996) Continuity of airway goblet cells and intraluminal mucus in the airways of patients with bronchial asthma. Eur Respir J 9,1395-1401[Abstract]
  5. Speizer, FE, Fay, ME, Dockery, DW, et al (1989) Chronic obstructive pulmonary disease mortality in six U.S. cities Am Rev Respir Dis 140,S49-S55[ISI][Medline]
  6. Lange P, Nyboe J, Appleyard M, et al. Relation of ventilatory impairment and of chronic mucus hypersecretion to mortality from obstructive lung disease and from all causes. Thorax 1990; 45:8:579–585
  7. Annesi, I, Kauffmann, F (1986) Is respiratory mucus hypersecretion really an innocent disorder? Am Rev Respir Dis 134,688-693[ISI][Medline]
  8. Takeyama, K, Dabbagh, K, Lee, H-M, et al (1999) Epidermal growth factor system regulates mucin production in airways. Proc Natl Acad Sci USA 96,3081-3086[Abstract/Free Full Text]
  9. Ruocco, S, Lallemand, A, Tournier, JM, et al (1996) Expression and localization of epidermal growth factor, transforming growth factor-alpha, and localization of their common receptor in fetal human lung development. Pediatr Res 39,448-455[ISI][Medline]
  10. Amishima, M, Munakata, M, Nasuhara, Y, et al (1998) Expression of epidermal growth factor and epidermal growth factor receptor immunoreactivity in the asthmatic human airway. Am J Crit Care Med 157,1907-1912[Abstract/Free Full Text]
  11. Ulich TR. In: Kelley J, ed. Cytokines of the lung. New York, NY: Dekker, 1993; 307–332
  12. Lou, Y-P, Takeyama, K, Grattan, K, et al (1998) Platelet-activating factor induces goblet cell hyperplasia and mucin gene expression in airways. Am J Respir Crit Care Med 157,1927-1934[Abstract/Free Full Text]
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  15. Harkema, JR, Hotchkiss, JA (1993) Ozone- and endotoxin-induced mucous cell metaplasias in rat airway epithelium: novel animal models to study toxicant-induced epithelial transformation in airways. Toxicol Lett 68,251-263[CrossRef][ISI][Medline]
  16. Jany, B, Gallup, M, Tsuda, T, et al (1991) Mucin gene expression in rat airways following infection and irritation. Biochem Biophys Res Commun 181,1-8[CrossRef][ISI][Medline]
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