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Mucous hypersecretion is a serious feature of chronic airway diseases such as asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis. Although mucins are produced via activation of an EGF receptor (EGFR) signaling cascade, the mechanisms leading to exaggerated mucin production in mucous hypersecretory diseases are unknown. Because expression of ICAM-1 and of the ICAM-1 ligand fibrinogen is increased in the airways of subjects with mucous hypersecretory diseases, we hypothesized that fibrinogen binding to ICAM-1 could increase EGFR-dependent mucin production in human airway (NCI-H292) epithelial cells. Consistent with this hypothesis, we found that an ICAM-1 neutralizing antibody and an ICAM-1(8-22) peptide that binds fibrinogen decreased mucin production induced by the EGFR ligand transforming growth factor (TGF)-alpha dose-dependently. Exogenous fibrinogen and a fibrinogen(117-133) peptide that binds ICAM-1 rescued mucin production in cells treated with the ICAM-1(8-22) peptide. Surprisingly, the ICAM-1(8-22) peptide increased EGFR phosphotyrosine and phospho-ERK1/2 in cells treated with TGF-alpha. The ICAM-1(8-22) peptide-induced increases in EGFR phosphotyrosine and phospho-ERK1/2 were prevented by exogenous fibrinogen, by the fibrinogen(117-133) peptide, and by selective inhibitors of phospholipase C (PLC), protein kinase C (PKC)-alpha/beta, and metalloproteases. These results suggest that fibrinogen binding to ICAM-1 promotes mucin production by decreasing TGF-alpha-induced EGFR and ERK1/2 activation and that the fibrinogen-ICAM-1-dependent decrease in EGFR and ERK1/2 activation occurs via inhibition of an early positive feedback pathway involving PLC- and PKC-alpha/beta-dependent metalloprotease activation and subsequent metalloprotease-dependent EGFR reactivation.

In previous work, we showed that epidermal growth factor receptor (EGFR) activation causes mucin expression in airway epithelium in vivo and in human NCI-H292 airway epithelial cells and normal human bronchial epithelial (NHBE) cells in vitro. Here we show that the cell surface adhesion molecule, E-cadherin, promotes EGFR-mediated mucin production in NCI-H292 cells in a cell density- and cell cycle-dependent fashion. The addition of the EGFR ligand, transforming growth factor (TGF)-α, increased MUC5AC protein expression markedly in dense, but not in sparse, cultures. MUC5AC-positive cells in dense cultures contained 2 N DNA content and did not incorporate bromodeoxyuridine, suggesting that they develop via cell differentiation and that a surface molecule involved in cell-cell contact is important for EGFR-mediated mucin production. In support of this hypothesis, in dense cultures of NCI-H292 cells and in NHBE cells at air-liquid interface, blockade of E-cadherin-mediated cell-cell contacts decreased EGFR-dependent mucin production. E-cadherin blockade also increased EGFR-dependent cell proliferation and TGF-α-induced EGFR tyrosine phosphorylation in dense cultures of NCI-H292 cells, suggesting that E-cadherin promotes EGFR-dependent mucin production and inhibits EGFR-dependent cell proliferation via modulation of EGFR phosphotyrosine levels. Furthermore, in dense cultures, E-cadherin blockade decreased the rate of EGFR tyrosine dephosphorylation, implicating an E-cadherin-dependent protein tyrosine phosphatase in EGFR dephosphorylation. Thus E-cadherin promotes EGFR-mediated cell differentiation and MUC5AC production, and our results suggest that this occurs via a pathway involving protein tyrosine phosphatase-dependent EGFR dephosphorylation.

Chronic obstructive pulmonary disease (COPD) is one of the leading causes of death in the U.S. Because cigarette smoking is so importantly implicated in the pathogenesis of COPD and because mucus hypersecretion plays such an important role in COPD, understanding of the mechanisms of smoking-induced mucus hypersecretion could lead to new therapies for COPD. Cigarette smoke causes mucin overproduction via EGF receptor (EGFR) in airway epithelial cells, but the cellular mechanism remains unknown. Airway epithelial cells contain EGFR proligands on their surfaces, which can be cleaved by metalloprotease and subsequently bind to EGFR resulting in mucin production. We hypothesize that TNF-alpha-converting enzyme (TACE) is activated by cigarette smoke, resulting in increased shedding of EGFR proligand, leading to EGFR phosphorylation and mucin induction in human airway epithelial (NCI-H292) cells. Here we show that cigarette smoke increases MUC5AC production in NCI-H292 cells, an effect that is prevented by an EGFR-neutralizing antibody and by specific knockdown of transforming growth factor-alpha (TGF-alpha) using small interfering RNA (siRNA) for TGF-alpha, implicating TGF-alpha-dependent EGFR activation in the responses. Cigarette smoke increases TGF-alpha shedding, EGFR phosphorylation, and mucin production, which are prevented by metalloprotease inhibitors (GM-6001 and TNF-alpha protease inhibitor-1) and by specific knockdown of TACE with TACE siRNA, implicating TACE in smoking-induced responses. Furthermore, pretreatment with antioxidants prevents smoking-induced TGF-alpha shedding and mucin production, suggesting that reactive oxygen species is involved in TACE activation. These results implicate TACE in smoking-induced mucin overproduction via the TACE-proligand-EGFR signal pathway in NCI-H292 cells.

Neutrophil products are implicated in hypersecretory airway diseases. To determine the mechanisms linking a proteolytic effect of human neutrophil elastase (HNE) and mucin overproduction, we examined the effects of HNE on MUC5AC mucin production in human airway epithelial (NCI-H292) cells. Stimulation with HNE for 5-30 min induced MUC5AC production 24 h later, which was prevented by HNE serine active site inhibitors, implicating a proteolytic effect of HNE. MUC5AC induction was preceded by epidermal growth factor receptor (EGFR) tyrosine phosphorylation and was prevented by selective EGFR tyrosine kinase inhibitors, implicating EGFR activation. HNE-induced MUC5AC production was inhibited by a neutralizing transforming growth factor-alpha (TGF-alpha, an EGFR ligand) antibody and by a neutralizing EGFR antibody but not by oxygen free radical scavengers, further implicating TGF-alpha and ligand-dependent EGFR activation in the response. HNE decreased pro-TGF-alpha in NCI-H292 cells and increased TGF-alpha in cell culture supernatant. From these results, we conclude that HNE-induced MUC5AC mucin production occurs via its proteolytic activation of an EGFR signaling cascade involving TGF-alpha.

Previous work showed that the Th2 cytokine interleukin (IL)-13 induces goblet cell metaplasia via an indirect mechanism involving the expression and subsequent activation of epidermal growth factor receptor (EGFR). Because Clara cell secretory protein (CCSP) expression has been reported in cells that express mucins, we examined the effect of IL-13 on CCSP gene and protein expression in pathogen-free rat airways and in pulmonary mucoepidermoid NCI-H292 cells. Intratracheal instillation of IL-13 induced CCSP mRNA in epithelial cells without cilia within 8–16 h, maximal between 24 and 48 h; CCSP immunostaining increased in a time-dependent fashion, maximal at 48 h. The CCSP immunostaining was localized in nongranulated secretory cells and goblet cells and in the lumen. Pretreatment with the selective EGFR tyrosine kinase inhibitor BIBX1522, cyclophosphamide (an inhibitor of bone marrow leukocyte mobilization), or a blocking antibody to IL-8 prevented CCSP staining. Treatment of NCI-H292 cells with the EGFR ligand transforming growth factor-α, but not with IL-13 alone, induced CCSP gene and protein expression. Selective EGFR tyrosine kinase inhibitors, BIBX1522 and AG1478, prevented CCSP expression in NCI-H292 cells, but the platelet-derived growth factor receptor tyrosine kinase inhibitor AG1295 had no effect. These findings indicate that IL-13 induces CCSP expression via an EGFR- and leukocyte-dependent pathway.

Mucus hypersecretion from hyperplastic airway goblet cells is a hallmark of chronic obstructive pulmonary disease (COPD). Although cigarette smoking is thought to be involved in mucus hypersecretion in COPD, the mechanism by which cigarette smoke induces mucus overproduction is unknown. Here we show that activation of epidermal growth factor receptors (EGFR) is responsible for mucin production after inhalation of cigarette smoke in airways in vitro and in vivo. In the airway epithelial cell line NCI-H292, exposure to cigarette smoke upregulated the EGFR mRNA expression and induced activation of EGFR-specific tyrosine phosphorylation, resulting in upregulation of MUC5AC mRNA and protein production, effects that were inhibited completely by selective EGFR tyrosine kinase inhibitors (BIBX1522, AG-1478) and that were decreased by antioxidants. In vivo, cigarette smoke inhalation increased MUC5AC mRNA and goblet cell production in rat airways, effects that were prevented by pretreatment with BIBX1522. These effects may explain the goblet cell hyperplasia that occurs in COPD and may provide a novel strategy for therapy in airway hypersecretory diseases.

We hypothesized that foreign bodies in airways cause inflammation leading to goblet cell metaplasia. Instilled agarose plugs lodged in the bronchi of pathogen-free rats caused a time-dependent increase in Alcian blue-periodic acid-Schiff staining that was detected within 24 h and markedly increased at 72 h. Control bronchi contained no pregoblet or goblet cells, but plugged bronchi contained many pregoblet and goblet cells and a decrease in nongranulated secretory cells. In situ hybridization showed no expression of MUC5AC in control airways, but plugged airways showed a marked expression. Control bronchi showed sparse staining for epidermal growth factor receptor (EGFR) protein, but plugged bronchi showed intense EGFR staining in the epithelium. Pretreatment with an EGFR tyrosine kinase inhibitor (BIBX1522) prevented Alcian blue-periodic acid-Schiff staining and MUC5AC gene expression in plugged bronchi. Pretreatment with tumor necrosis factor-α neutralizing antibody or pretreatment with cyclophosphamide abolished plug-induced EGFR protein expression and goblet cell metaplasia. Thus instillation of agarose plugs induces profound goblet cell metaplasia by causing EGFR expression and activation.

Histamine can be recovered from the blood of ragweed-sensitized dogs after aerosol antigen challenge, although its source is unknown. Neutrophils and eosinophils have been recovered from bronchoalveolar lavage fluid (BALF) obtained under identical conditions. We investigated the time course of changes in histamine levels in plasma and BALF taken from ragweed-sensitized dogs after aerosol challenge. Changes in the numbers of circulating neutrophils, eosinophils, lymphocytes, monocytes, and platelets were also studied. After 3 min, total pulmonary resistance (RL) was maximally increased and systolic blood pressure was maximally decreased. Histamine levels in plasma and BALF were increased and circulating eosinophils and neutrophils were decreased. After 15 min, platelet numbers were reduced. By 90 min, changes in RL, blood pressure, plasma and BALF histamine concentrations, and circulating neutrophils and eosinophils had returned to base-line values, but platelet numbers remained significantly decreased. Sham challenge caused no significant changes in any of these variables. Intravenous administration of histamine in doses large enough to attain plasma levels comparable with those achieved after aerosol antigen challenge resulted in no concomitant rise in BALF histamine levels. We conclude that antigen challenge in sensitized dogs causes increases in BALF and plasma histamine levels and is associated with a reduction in circulating neutrophils, eosinophils, and platelets. It is likely that antigen causes airway mast cells to release mediators that move down a concentration gradient from the airways to the pulmonary circulation.

We exposed two awake dogs with a chronic tracheostomy and the cervical vagus nerves exteriorized in skin loops to 1.0 ppm of ozone (O3) for 2 h at intervals of 4 wk. We measured ventilatory variables before and after O3 exposure during rest and exercise before and after vagal block. We compared the effects of vagal blockade, exercise, and O3 on the primary determinants of breathing pattern (VT/TI, VT/TE, TI, and TE) in each of three conditions: base line (steady state), during hypercapnia, and after inhalation of 1% histamine. Under base-line conditions, O3 increased respiratory rate and decreased tidal volume (VT) by shortening time of expiration (TE) and time of inspiration (TI) without affecting VT/TI, an indicator of the neural drive to breathing. During progressive hypercapnia, O3 shortened TE and TI by effects both on tonic (nonvolume-related) and on phasic (volume-related) vagal inputs, and only the latter were prevented completely by cooling of the vagus nerves. Histamine-induced tachypnea was increased by O3 and was totally blocked by cooling the vagus nerves. We conclude that O3 shortens the timing of respiration without increasing ventilatory drive, shortens TI and TE through vagal and nonvagal pathways, increases tonic nonvagal and phasic vagal inputs, and stimulates more than one vagal fiber type.

Neuropeptides such as substance P are implicated in inflammation mediated by sensory nerves (neurogenic inflammation), but the roles in disease of these peptides and the peptidases that degrade them are not understood. It is well established that inflammation is a prominent feature of several airway diseases, including viral infections, asthma, bronchitis, and cystic fibrosis. These diseases are characterized by cough, airway edema, and abnormal secretory and bronchoconstrictor responses, all of which can be elicited by substance P. The effects of substance P and other peptides that may be involved in inflammation are decreased by endogenous neutral endopeptidase (NEP; also called enkephalinase, EC 3.4.24.11), which is a peptidase that degrades substance P and other peptides. In the present study, we report that rats with histories of infections caused by common respiratory tract pathogens (parainfluenza virus type 1, rat corona-virus, and Mycoplasma pulmonis) not only have greater susceptibility to neurogenic inflammatory responses than do pathogen-free rats but also have a lower activity of NEP in the trachea. This reduction in NEP activity may cause the increased susceptibility to neurogenic inflammation by allowing higher concentrations of substance P to reach tachykinin receptors in the trachea. Thus decreased NEP activity may exacerbate some of the pathological responses in animals with respiratory tract infections.

To determine which part of the parasympathetic bronchoconstrictor pathway is most sensitive to depression by general anesthetics, we stimulated different parts of the pathway in dogs after initial anesthesia with chloralose and urethan and then after additional anesthetic drugs. We stimulated the entire reflex pathway by producing apnea or hypoventilation, the sensory pathway by electrically stimulating the proximal end of cut superior laryngeal nerves, and the motor pathway by stimulating the distal end of a cut cervical vagus nerve. Bronchoconstrictor responses to all stimuli were assessed with a bypassed tracheal segment. When no additional anesthetic was administered, responses to all stimuli increased with time. Small additional doses of anesthetics (thiopental, 113;5 mg/kg; pentobarbital, 1–2 mg/kg; amobarbital, 1–2 mg/kg; or chloralose, 10 mg/kg) decreased responses to reflex and sensory stimulation markedly and reversibly, but they did not affect responses to motor stimulation. Increased doses decreased responses to motor stimulation as well. Our previous study (Skoogh et al., Am. Rev. Respir. Dis. 123: 202, 1981) showed that barbiturates depress parasympathetic ganglionic synapses; the present study suggests that central nervous system synapses may be even more sensitive to depression by general anesthetics.

We studied whether antigen-induced airway hyperresponsiveness was associated with pulmonary inflammation in 11 anesthetized ragweed-sensitized dogs. Airway responsiveness to acetylcholine aerosol was determined before and at 2, 6, and 24 h after ragweed or sham aerosol challenge. Pulmonary inflammation was assessed by bronchoalveolar lavage (BAL) performed at either 2 or 6 h. Total pulmonary resistance increased 11-fold at 5 min after ragweed. Airway responsiveness was unchanged at 2 h but was increased 6.6-fold at 6 h in 8 of 11 dogs (P less than 0.001); hyperresponsiveness persisted from 4 days to 4 mo. Airway responsiveness was unchanged by aerosols of diluent. Neutrophils in BAL fluid increased approximately sixfold at 2 h (P less than 0.02) and at 6 h (P less than 0.02) after antigen challenge. There were fewer eosinophils in fluid recovered at 6 h after antigen compared with 2 h lavages (P less than 0.05). In three nonresponders, BAL showed no significant changes in neutrophils and eosinophils after antigen. Thus antigen-induced hyperresponsiveness is associated with the presence of pulmonary inflammation, presumably arising from the airways and involving both neutrophils and eosinophils.

Inhalation of SO2 (4–6 ppm) for 10 min decreased airway conductance (increased airway resistance) in seven healthy subjects. Subcutaneous injection of atropine prevented this effect. In anesthetized, paralyzed, artifically ventilated cats, SO2 increased pulmonary resistance, whether delivered to the upper or to the lower airways. This was prevented by complete cold block of the cervical vagosympathetic nerves or by injecting atropine intravenously before the SO2 was inhaled. These results establish the reflex nature of bronchoconstriction during inhalation of SO2. Note: With the Technical Assistance of Earl H. White reflex bronchoconstriction; airway resistance Submitted on February 18, 1964

Equations for optimal frequencies of breathing at which mechanical work and mean inspiratory force are minimal and which assume dead space and resistance are constant ignore possible changes in airway caliber. We assumed an inverse relationship between resistance and dead space and plotted a series of curves relating mechanical work and mean inspiratory force to frequency of breathing for various dead spaces and corresponding resistances. As dead space increases, the levels of work and inspiratory force fall to minima, then rise; the frequencies at which work and force are minimal increase, then decrease. Adding a series resistance or dead space, changing the resistance/dead space relationship, decreasing lung compliance, or increasing alveolar ventilation do not alter the general shapes of the curves, but change the values of optimal dead space. The optimal dead spaces for minimal work (0.125 liter) and for minimal inspiratory force (0.14 liter) lie within the normal range. Nervously mediated airway tone in healthy subjects may represent optimal adjustment of airway caliber. dead space, optimal; bronchial caliber Submitted on October 26, 1962

Our aim was to determine whether flow from the submucosal glands of the trachea is reflexly regulated by sensory stimuli from the stomach in the cat, and, if such a gastropulmonary reflex exists, what sensory and motor pathways are important. We found that mechanical stimulation of the gastric mucosa increased submucosal gland secretions from 7.9 +/- 0.7 to 17.4 +/- 1.7 nl/min (mean +/- SE, P less than 0.001). This effect was prevented reversibly by cooling both abdominal vagus nerves to -3 degrees C before stimulation and was restored by rewarming the nerves. The effect was prevented irreversibly by cutting both abdominal vagus nerves and was then mimicked by electrically stimulating the central cut end of one of the nerves. This increase in secretions caused by electrical stimulation of the nerve was prevented by administration of atropine sulfate before stimulation. We conclude that stimuli from the stomach reflexly affect the rate of submucosal gland secretion. The sensory limb of this reflex lies in the abdominal vagus nerves, and the motor pathways are mediated by cholinergic muscarinic receptors.

We made concurrent measurements of the functional residual capacity (FRC) with the body plethysmograph (thoracic gas volume) and by 7-min and prolonged open-circuit nitrogen dilution methods (communicating gas volume). The mean difference between the 7-min communicating gas volume and the thoracic gas volume in 13 healthy subjects was only 0.13 liters. The thoracic gas volume averaged 0.99 liters larger than the communicating gas volume after 7 min of O2 breathing in 13 patients with emphysema. The communicating gas volume at 12–18 min was the same as the thoracic gas volume in 11 of 13 patients but was smaller in the other 2. When the thoracic gas volume was used to measure FRC, the total lung capacity averaged 142% of predicted normal in 13 patients with emphysema. Submitted on January 4, 1962

We measured airway resistance and thoracic gas volume by the body plethysmograph technique, and transpulmonary pressure in seven healthy, adult subjects, before and after induction of bronchoconstriction. A deep inspiration never altered airway resistance, measured at functional residual capacity in the control state, but always reduced it for 1—2 min when bronchoconstriction was present. Discrepancies in data published on airway resistance may be due to use of methods which require a deep inspiration, or to occurrence of a spontaneous deep inspiration shortly before the test. Submitted on January 13, 1961