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Background Idiopathic pulmonary fibrosis (IPF) is a disease with a poor prognosis. Although the median survival is 3 years, the clinical course varies to a large extent among IPF patients. To date, there has been no definitive prognostic marker. Extracellular vesicles (EVs) are known to hold nucleic acid, including microRNAs, and to regulate gene expression in the recipient cells. Moreover, EVs have been shown to express distinct surface proteins or enveloped microRNAs depending on the parent cell or pathological condition. We aimed to identify serum EV microRNAs that would be prognostic for IPF. Methods To determine target microRNAs in IPF, we measured serum EV microRNA expression profiles using microRNA PCR arrays in a bleomycin mouse model and validated the microRNAs in additional mice using RT-PCR. Secondly, we enrolled 41 IPF patients and conducted a 30-month prospective cohort study. Expression of serum EV miR-21-5p was normalized by dividing by the EV amount. The relative amount of EVs was measured using the ExoScreen method. We calculated the correlations between baseline serum EV miR-21-5p expression and other clinical variables. Furthermore, we determined if serum EV miR-21-5p can predict mortality during 30 months using the Cox hazard model. According to the median level, we divided the IPF patients into two groups. Then we compared the survival rate during 30 months between the two groups using the Kaplan-Meier method. Results Serum EV miR-21-5p was elevated in both the acute inflammatory phase (day 7) and the chronic fibrotic phase (day 28) in the mouse model. In the clinical setting, serum EV miR-21-5p was significantly higher in IPF patients than in healthy control subjects. The baseline serum EV miR-21-5p was correlated with the rate of decline in vital capacity over 6 months. Furthermore, serum EV miR-21-5p was independently associated with mortality during the following 30 months, even after adjustment for other variables. In the survival analysis, IPF patients whose baseline serum EV miR-21-5p was high had a significantly poorer prognosis over 30 months. Conclusions Our results suggest that serum EV miR-21-5p has potential as a prognostic biomarker for IPF. Electronic supplementary material The online version of this article (doi:10.1186/s12931-016-0427-3) contains supplementary material, which is available to authorized users.

Topical corticosteroid eye drops are commonly used for ocular sarcoidosis. That systemic absorption of corticosteroids by eye drops may influence the clinical course of sarcoidosis may be speculated because it has been reported that the serum concentration of corticosteroids after drop administration was dose-related. To evaluate the effects of corticosteroid eye drops on the clinical course of patients with stage I pulmonary sarcoidosis, we compared the serum levels of angiotensin converting enzyme (ACE) and bilateral hilar lymphadenopathy (BHL) on chest radiographs of group CS, which is consisted of patients who received topical therapy of betamethasone in the form of eye drops for anterior uveitis, and group CN, which is consisted of patients who did not receive any medications throughout the entire course of the disease. Although the serum ACE level was not significantly different between groups CS and CN at the time of the diagnosis of pulmonary sarcoidosis, the level of serum ACE in group CS was significantly higher than that in group CN 20 months after the topical corticosteroid treatment (24 IU/ml and 16 IU/ml, respectively). Further, the size of BHL on chest radiography in group CS was significantly larger than that in group CN 20 months after the topical treatment (82% and 37% of before control, respectively). These findings suggest the possibility that the topical corticosteroid therapy influenced the clinical course of pulmonary sarcoidosis, inducing some delay in the spontaneous remission in the long-term course.

Because nuclear factor (NF)-kappaB-regulated cytokines, including tumor necrosis factor-alpha (TNF-alpha), from monocytes and macrophages have been implicated in the pathogenesis and development of septic shock and acute respiratory distress syndrome (ARDS), the effect of the antisense oligonucleotide to the p65 subunit of NF-kappaB on the survival of lipopolysaccharide (LPS)-induced ARDS in BALB/c mice was examined. None and 70% of the animals died of diffuse hemorrhagic lung edema 1 to 2.5 days after intraperitoneal administration of 10 and 20 mg/kg LPS alone, respectively. Intravenously administered antisense oligonucleotide alone did not produce any significant changes in the behavior or lung histology. After intravenous administration of the anti-sense oligonucleotide, both peripheral blood monocytes and alveolar macrophages in bronchoalveolar lavage fluid were confirmed to contain sufficiently large amounts of intracellular antisense oligonucleotides for their function usingfluorescein isothiocyanate (FTCC)-labeled microscopy. The antisense oligonucleotide administered 6 hours before the intraperitoneal administration of LPS significantly decreased the survival rate with the progress of hemorrhagic edema in lung histology; 90% and 100% of animals treated with the antisense oligonuleotide died 0.5 to 1.5 days after the administration of 10 and 20 mg/kg LPS, respectively. These findings suggest that the suppression of cytokines and mediators in monocytes and alveolar macrophages by the antisense oligonucleotide to the p65 subunit of NF-kappaB worsens the survival of LPS-induced ARDS in mice with the progress of hemorrhagic lung edema.

Both the superficial epithelium and submucosal glands play a role in airway secretion. Electrophysiological experiments showed that extracellular ATP induced an initial transient increase in Cl - secretion followed by a prolonged inhibition of Na + absorption in rabbit tracheal epithelium, which lacks submucosal glands. The response to ATP was mimicked by UTP or ATPyS in untreated normal epithelium, suggesting a P 2U -type receptor in the epithelium. Meanwhile, in tracheal epithelium from SO 2 -induced bronchitic rabbit ATP induced a prolonged increase in Cl - secretion without a decrease in Na + absorption, which was mimicked by adenosine or isoproterenol (ISP). The alteration in the bronchitic epithelium was shown to result from a newly expressed CFTR by both immunohistological and Northern blot analysis. Patch-clamp experiments showed that ATP induced an initial Cl - current followed by K + current in acinar cells of submucosal glands isolated from feline and human trachea. Although ISP alone or adenosine did not evoke any significant current responses, ISP augmented the ATP-induced Cl - and K + currents. A phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), mimicked the augmentation by ISP. ATP also induced an increase in [Ca 2+ ] i in acinar cells of submucosal glands, which was augmented by ISP. Mucus glycoprotein (MGP) secretion from isolated submucosal glands was also stimulated by ATP but not by adenosine. The ATP-induced MGP secretion was augmented by ISP, These findings suggest that P 2 -receptor stimulation and the resultant [Ca 2+ ] i -rise induce both electrolyte and MGP secretion, which is enhanced by [cAMP] i -rise in airway submucosal glands.

Submucosal glands were isolated within 4 h of death from tracheae and bronchi obtained from autopsied lungs, and the secretory response of secretory leukocyte protease inhibitor (SLPI) was examined with ELISA and a secretory index. Although human neutrophil elastase (HNE) at low concentrations increased SLPI secretion above the control level (i.e., 149% of control level at 10−11M), HNE at high concentrations significantly decreased it below the control level (i.e., 16% of control level at 10−7M). The decrease in SLPI concentration was shown to result from the degradation of SLPI by excessive HNE. Methacholine induced significant secretion (i.e., 363% of control level at 10−5M) that was abolished by both M1and M3receptor antagonists. A semiquantitative analysis of SLPI mRNA by RT-PCR and Southern blot showed that compared with the superficial epithelium, submucosal glands had a 30-fold or higher level of SLPI mRNA. Both HNE and methacholine significantly increased the level of SLPI mRNA in submucosal glands in a dose-dependent manner (i.e., 357% of control level at 10−7M and 175% of control level at 10−5M, respectively). These findings indicate that human airway submucosal glands can transcribe 30-fold or more SLPI mRNA than the superficial epithelium and that SLPI mRNA transcription and secretion are regulated by both HNE and muscarinic receptors.

Accumulating evidence suggests that thyrotropin (thyroid-stimulating hormone [TSH]) plays some roles in immunoregulation by an extrathyroidal action. Because airway submucosal glands are responsible for nonspecific and specific airway defense, we tested the effect of TSH on feline tracheal submucosal gland using a whole-cell patch-clamp technique, immunohistochemistry, and reverse transcription/polymerase chain reaction (RT-PCR). TSH potentiated neurotransmitter-induced ionic currents significantly in a dose-dependent manner. Acetylcholine (10(-)(8) M)- and norepinephrine (10(-)(7) M)-induced inward current (I(i)), which we previously showed to be a Cl(-) current, were increased to about 3-fold the pre-TSH control responses, respectively, by 2.0 ng/ml TSH; and to 6- and 23-fold the control values by 20.0 ng/ml TSH, respectively. TSH alone was without effect up to 20.0 ng/ml. Follicular stimulating hormone only slightly affected the I(i) (1. 5-fold the control). Analyses with immunohistochemistry and RT-PCR failed to identify TSH receptors on the glandular tissue. Maneuvers to raise the cellular adenosine 3',5'-cyclic monophosphate also failed to mimic the TSH-mediated potentiation. The TSH effect appeared to be mediated by a signaling pathway involving tyrosine kinase because its inhibitors (genistein and herbimycin A) abolished the augmentation completely, and interferon-gamma, a tyrosine kinase activator, imitated the TSH action on submucosal gland. Thus, TSH may be an important regulator of airway fluid secretion.

Background Activated eosinophils play an important role in the pathogenesis of bronchial asthma and other allergic diseases, and platelet-activating factor (PAF) is a potent activator of eosinophils. Objective To characterize the cytosolic Ca2+ ([Ca2+]i) mobilization in human eosinophils in response to PAF. Methods [Ca2+]i responses to PAF were examined in human eosinophils using a microscopic fura-2 fluorescence-ratio imaging system. Results PAF caused a significant and dose-dependent increase in (Ca2+)i, which consisted of an initial rapid rise followed by a sustained elevation. This PAF-induced (Ca2+)i rise was inhibited by WEB 2086, a specific PAF receptor antagonist. The addition of 5 m m EGTA or 1 m m Ni2+ to a nominally Ca2+-free solution did not appreciably reduce the initial rise but significantly inhibited the sustained rise. The application of a protein kinase C inhibitor, Ro31-8220, augmented the sustained increase by PAF. Thapsigargin, a microsomal Ca2+ ATPase inhibitor, induced no appreciable change in a nominally Ca2+-free solution but induced a marked increase in (Ca2+)i when changed to a Ca2+-containing solution. Conclusions The initial rapid rise and the following sustained rise in (Ca2+)i by PAF depends on Ca2+ release from the intracellular Ca2+ stores and Ca2+ influx, respectively, which are regulated by protein kinase C in human eosinophils. Furthermore, the so called Ca2+-capacitative entry is possibly involved in the Ca2+ influx from the extracellular solution in human eosinophils.

We examined the effect of laminin (a major component of the basement membrane) on potential difference (PD) and short circuit current (SCC) in both posterior epithelial membranes (native tissue) and cultured epithelial cell layers from canine trachea using an Ussing chamber. Although laminin itself did not alter the baseline values of SCC or PD, it significantly inhibited in a dose-dependent manner the isoproterenol induced SCC and PD rises of the epithelial membrane. In the cultured epithelial layer treated with amiloride, laminin did not alter the baseline values of SCC, PD or resistance (R) but it significantly inhibited the isoproterenol induced SCC rises. However, laminin did not significantly inhibit the bradykinin induced SCC rise in the cultured epithelial layer treated with amiloride. These findings indicate that laminin is a determinant in ion transport (mainly Cl − secretion) across canine tracheal epithelium, inhibiting the β -agonist induced Cl − transport.

Background: IL-13 has been shown to induce IgE production in B cells by promoting class switching to IgE. Mast cells are known to play an important role in the pathogenesis of allergic diseases. We evaluated the ability of human mast cells to produce IL-13 using human mast cell line HMC-1 and freshly isolated lung mast cells and then examined the effect of dexamethasone on the gene expression and production of IL-13 by these cells. Methods: HMC-1 cells and lung mast cells were cultured with 10 ng/ml phorbol 12-myristate 13-acetate (PMA) and 1 μmol/L ionomycin and with 5 μg/ml phytohemagglutinin (PHA) and 10 ng/ml PMA, respectively, in the presence of dexamethasone. The gene expression of IL-13 at 3 hours (HMC-1 cells) or 12 hours (human lung mast cells) after stimulation was assessed semiquantitatively by sequential reverse transcription-polymerase chain reaction and Southern blot analysis. IL-13 production at 12 hours after stimulation was assayed by ELISA. Results: The gene expression of IL-13 by HMC-1 cells and human lung mast cells, which was detected at a low level in an unstimulated condition, was increased by PMA/ionomycin and suppressed by dexamethasone. The supernatant of HMC-1 cells and human lung mast cells showed a low level of IL-13, which was increased by the stimulation and suppressed by dexamethasone. Conclusion : These findings indicate that HMC-1 cells and human lung mast cells produce IL-13 and that dexamethasone suppresses the production of IL-13 by these cells through an inhibitory action on the gene expression. (J Allergy Clin Immunol 1998;102:134-42.)

Bronchial glands are abundant in human airways, playing a central role in airway secretion and the secretion of mucin, electrolytes and various defensive substances. Both mucin and electrolyte secretions from the bronchial glands maintain an effective mucociliary transport in the airways. Various defensive substances from the bronchial glands include lactoferrin, lysozyme, secretory IgA, secretory leukoprotease inhibitor (SLPI) and surfactant protein A (SP-A). Here, we focused on SLPI and SP-A secretion from human bronchial glands. First, we examined the secretory response and gene expression of SLPI in isolated human glands in response to human neutrophil elastase (HNE). The results indicate that, in human bronchial glands, HNE at low concentrations stimulates SLPI production and secretion, whereas HNE at high concentrations does not. Next, we examined SP-A secretion and gene expression in human bronchial glands. The findings indicate that human bronchial gland cells can transcribe the SP-A2 gene and produce SP-A in a manner different from alveoli, thus playing a role in airway defense mechanisms.

We examined the effect of dexamethasone on the gene expression and production of interleukin (IL)-13 by human peripheral blood mononuclear cells from healthy controls. The gene expression was assessed semiquantitatively by sequential transcription polymerase chain reaction and Southern blot analysis, and the production of this cytokine was assessed by enzyme-linked immunosorbent assay (ELISA). Dexamethasone suppressed IL-13 gene expression induced by stimulation with phytohemagglutinin and phorbol 12-myristate 13-acetate in a dose-dependent manner, with 96% suppression at 10(-6) M, and also suppressed the increased production of IL-13. This is suggested to be one of the mechanisms by which glucocorticoids suppress allergic inflammation.

There is growing evidence that extracellular ATP causes a dramatic change in the membrane conductance of a variety of inflammatory cells. In the present study, using the nystatin perforated patch recording configuration, we found that ATP (0.3-30 microM) induced a transient outward current in a concentration-dependent manner and that the reversal potential of the ATP-induced outward current was close to the K+ equilibrium potential, indicating that the membrane behaves like a K+ electrode in the presence of ATP. The first application of ATP to alveolar macrophages perfused with Ca2+-free external solution could induce the outward current, but the response to ATP was diminished with successive applications. Intracellular perfusion with a Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid, also diminished the response. When cyclic ADP-ribose (cADPR) was applied to the macrophage cytoplasm, a transient outward current was elicited. Thereafter, the successive outward current was inhibited, suggesting the involvement of cADPR in the response. Intracellular perfusion with inositol 1,4, 5-trisphosphate also induced a transient outward current, but the successive current was not inhibited. The ATP-induced outward current was abolished when 8-amino-cADPR (as a blocker of cADPR, 10(-6)-10(-5) M) was introduced into the cytoplasm. Homogenates of alveolar macrophages showed both ADP-ribosyl cyclase and cADPR hydrolase activities, and CD38 (ADP-ribosyl cyclase/cADPR hydrolase) expression was confirmed by reverse transcriptase-polymerase chain reaction and Western blot analyses. These results indicate that ATP activates K+ currents by releasing Ca2+ from cADPR-sensitive internal Ca2+ stores.

The aim of this study was to elucidate the mechanism of the formation of the widespread mucous-plugging observed in autopsied lungs from patients with bronchial asthma. We performed morphometric analysis of airways of autopsied lungs from eight patients with bronchial asthma (Group BA), and compared it with those of six chronic bronchitics (Group CB) and four control patients (Control). The following parameters were measured in paraffin sections: volume proportion of bronchial glands to bronchial wall (Gland%); goblet cell granules to total epithelial layer (Goblet %); intraluminal mucus expressed as the mucus occupying ratio (MOR); volume ratio of intraluminal mucus continuous with goblet cells to total intraluminal mucus (Vc/Vtol %); and surface ratio of the contact surface of intraluminal mucus continuous with goblet cells to the total luminal surface (Sc/Stot %). Gland%, Goblet %, and MOR or inflammatory cell numbers in the airway walls both from Group BA and CB were larger than those from the Control group. However, no significant differences were observed between Group BA and CB in Gland%, Goblet %, MOR or inflammatory cell numbers, except for the eosinophil number: i.e. 23 +/- 3, 22 +/- 3 and 6 +/- 2% in Gland%; 22 +/- 9, 5 +/- 4 and 2 +/- 2% in Goblet%; 10 +/- 3, 18 +/- 3 and 0.3 +/- 0.5% in MOR; 199 +/- 68, 10 +/- 3 and 2 +/- 2 cells. mm-2 in eosinophil number of the peripheral airways from Groups BA, CB and Control, respectively. In contrast, marked and significant increases were observed both in Vc/Vtot% and Sc/Stot% in Group BA compared to Groups CB and Control both in central and peripheral airways: i.e. Vc/Vtot% in the peripheral airways was 53 +/- 5, 4 +/- 3 and 0.8 +/- 0.8% from Groups BA, CB and Control, respectively (BA vs CB or BA vs Control, p < 0.01 each). These findings suggest that the continuity of goblet cells and intraluminal mucus or lack of full release of mucus, from goblet cells, is peculiar to asthmatic airways, and may contribute to the formation of mucous-plugs.

To clarify the role of bradykinin receptor subtypes, we examined the effect of bradykinin on feline tracheal and human airway submucosal gland secretion using an isolated gland preparation. Bradykinin induced a significant increase in [3H]glycoconjugate secretion in a dose-dependent manner from isolated glands, which was significantly inhibited by D-Arg-(Hyp3, Thi5,8, D-Phe7)-bradykinin (the B2-receptor antagonist), whereas Des-Arg9-(Leu8)-bradykinin (B1-receptor antagonist) or indomethacin did not significantly alter it. Nitric oxide synthase inhibitor (nitro-L-arginine methyl ester) caused a significant inhibition of bradykinin-induced glycoconjugate secretion, which was reversed by the addition of L-arginine. Bradykinin evoked bidirectional current responses, and an initial inward current (Cl- current) was followed by an outward current (K+ current) of the acinar cells in a whole cell configuration by patch-clamp technique. Bradykinin induced an immediate increase in intracellular calcium concentration ([Ca2+]i) of the acinar cells followed by a prolonged plateau, and Ca2+ removal resulted in an initial increase alone. [Ca2+]i rise was significantly inhibited by the B2-receptor antagonist, whereas the B1-receptor antagonist did not significantly alter it. These findings suggest that B2-receptor stimulation and the resultant [Ca2+]i rise induced both mucus glycoprotein and electrolyte secretions, involving NO formation in airway submucosal gland cells.

Morphometric analysis of airways was performed in autopsied lungs from four patients with pulmonary emphysema (PE) followed by bronchial-asthma (BA)-like attacks (Group PE+BA) (four males, 72 +/- 9 yr). The results were compared with those from five pulmonary emphysema patients (Group PE) (five males, age 71 +/- 4 hr), three patients with bronchial asthma (Group BA) (one female and two males, age 65 +/- 7 yr), and four control subjects with no pulmonary diseases (Group Cont) (one female, three males, age 64 +/- 4 yr). The proportion of gland area to bronchial wall (gland%), ratio of goblet-cell occupancy to the total epithelial layer (goblet%), thickness of the basement membrane, amount of intraluminal mucus (mucus occupying ratio; MOR%), and number of various cell types per square millimeter in airway walls in a section 4 microns thick were measured in central (3 to 8 mm diameter) and peripheral airways (2 mm or less diameter). Gland% for the PE+BA group was significantly greater than that for the Cont group, whereas it did not differ significantly from that of the PE or BA groups. Goblet% and thickness of the basement membrane in central and/or peripheral airways in Group PE+BA were significantly greater than those in Group Cont, whereas those in Group PE were similar to those in Group Cont. Although not statistically significant, MOR% in central and peripheral airways from Group PE+BA showed a similar value to that in Group BA, whereas MOR% in Group PE was the same as that in Group Cont. The eosinophil number in peripheral airways walls in Group PE+BA showed a similar value to that in Group BA, which was significantly greater than in Group Cont. Other cells (macrophages, lymphocytes, and neutrophils) showed similar values among Groups PE+BA, PE, and BA. The number of eosinophils in central and/or peripheral airways correlated significantly with both goblet% and BMT, whereas other cells did not. These findings indicate that the airways of Group PE+BA are morphologically similar to those of Group BA, suggesting a combination of pulmonary emphysema with bronchial asthma.

A case of diffuse alveolar hemorrhage due to lung metastasis of ovarian angiosarcoma is described. A 33-year-old woman developed persistent cough and recurrent hemoptyses with resultant anemia. Chest radiograph and computerized tomography (CT) showed diffuse and patchy shadows in both lungs with nodules in the central area of the shadows. In spite of intensive care and administration of large amounts of glucocorticoids, she died of respiratory failure 5 months after the onset of her symptoms. Histological examination by both thoracoscopic biopsy and autopsy revealed diffuse tumor emboli of disseminated angiosarcoma which originated from the right ovary and diffuse alveolar hemorrhage which appeared to be caused by the tumor emboli with resultant invasion and destruction to vascular walls. This is the first description of diffuse alveolar hemorrhage probably caused by tumor emboli of metastatic angiosarcoma.

We investigated the mechanisms of eosinophil penetration and mannitol permeability through a multilayer of cultured human tracheal epithelial cells. Wells of tissue culture plates were separated into the upper and the lower chambers by the cultured epithelial cell layer. 51Cr-labeled eosinophils or 3H-mannitol were put into the lower chamber. To stimulate the epithelial cells, platelet-activating factor (PAF) and/or phorbol myristate acetate (PMA) were added to the upper chamber. After 4 h of incubation, the eosinophil penetration rate was determined as a percentage of the total count added to the lower chamber. PMA significantly increased the eosinophil penetration rate in a dose-dependent manner (4.0% at 10(-5) M), compared with control (0.67%), whereas PAF itself did not. Activation of eosinophils by the addition of PAF to the lower chamber produced a significant increase in the eosinophil penetration (6.5% at 10(-6) M), which was inhibited by staurosporine. For determining the mannitol permeability, PMA, PAF, and/or supernatant from eosinophils were added to both upper and lower chambers and incubated for 30 min. PMA induced a significant increase in the mannitol permeability (175% of controls at 10(-5) M), whereas PAF itself did not alter it. Supernatant from eosinophils activated by PAF (10(-6) M) significantly increased the permeability (451% of controls), which was blocked by staurosporine. Supernatants from AA861 (a 5-lipoxygenase inhibitor)-treated or phenidon (a phospholipase A2 inhibitor)-treated eosinophils activated by PAF failed to alter the supernatant-induced increases in mannitol permeability.(ABSTRACT TRUNCATED AT 250 WORDS)

To investigate cellular differentials in natural airway narrowing of steroid-dependent asthmatic individuals, we performed bronchoalveolar lavage (BAL) on 10 inpatients with asthma treated only with bronchodilators during episodes of natural airway narrowing evaluated by serial monitoring of peak expiratory flow (PEF), and on nine normal volunteers. We confirmed that the airway narrowing was not completely reversed by salbutamol aerosol just before the BAL study, but was completely reversed by administration of systemic steroid after the BAL study. Thus, the natural airway narrowing investigated in this study consisted not only of the constriction of airway smooth muscle, but also of edema of the airway mucosa and/or secretion in airways. Both the numbers and percentages of eosinophils and alcian blue-positive cells in BAL fluids from the asthmatic subjects were significantly higher than those of normals, but the numbers and percentages of neutrophils, lymphocytes, and macrophages were not. Thus, eosinophils and alcian blue-positive cells selectively increased in the airways during the natural airway narrowing. Because we found that the metachromatic cells consisted of two types, with a single nucleus and with segmented nuclei, we further examined basophil chemotactic activity (BCA) in BAL fluids. We showed that BCA was significantly higher in the asthmatic (79.3 +/- 17.2 cells/5 hpf) than in the normal subjects (6.2 +/- 1.6 cells/5 hpf), and also that the activity was more strongly correlated with the cells having segmented nuclei (p = 0.95) than with all of the cells (p = 0.73).(ABSTRACT TRUNCATED AT 250 WORDS)

The intracellular Ca2+ concentration ([Ca2+]i) of acinar cells of isolated submucosal glands from trachea was measured using a fluorescent dye, Fura-2. Neurokinin A (NK-A) produced a sustained rise in [Ca2+]i in a dose-dependent manner, reaching a response of 500 to 600% of the prior baseline value at 10(-6) or 10(-5) M, and the NK-A evoked [Ca2+]i was significantly higher than that by substance P (SP) at similar concentrations. NK-B did not induce significant increases in [Ca2+]i. In a Ca(2+)-free solution, NK-A produced a transient rise in [Ca2+]i, which returned to the baseline within 3 min. Mucus glycoprotein (MGP) secretion, estimated by measuring trichloroacetic-acid (TCA) precipitable glycoconjugates, was stimulated by NK-A or SP. These findings indicate that tachykinins produce a rise in [Ca2+]i by both entry from the extracellular solution and release from intracellular storage, probably by NK-2 receptor stimulation, and stimulate MGP secretion from airway submucosal glands.

The standard patch-clamp technique was employed on enzymatically digested acinar cells of submucosal glands isolated from feline trachea. ATP (-10(-3) M) evoked bidirectional current responses and an initial inward current at -80 mV (Cl- current) was followed by an outward current at 0 mV of membrane potential (K+ current). Isoproterenol (ISO) alone did not evoke any significant current responses. However, ISO augmented the ATP-induced inward and outward currents. A phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, mimicked the augmentation by ISO. [Ca2+]i of acinar cells in isolated gland was measured using a fluorescent dye, fura 2. ATP (-10(-3) M) induced an immediate increase in [Ca2+]i followed by a prolonged plateau, and Ca2+ removal resulted in an initial increase alone without the prolonged phase. ISO also augmented the ATP-evoked increases in [Ca2+]i mainly in the plateau phases. Mucus glycoprotein (MGP) secretion was estimated by measuring trichloroacetic acid-precipitable [3H]glycoconjugates from isolated glands. ATP (-10(-3) M) evoked significant MGP secretion and ISO enhanced the ATP-induced MGP secretion. In contrast, adenosine (-10(-3) M) produced no significant responses in current, MGP secretion, or [Ca2+]i. These findings suggest that 1) P2-receptor stimulation and the resultant [Ca2+]i rise induced both electrolyte and MGP secretions and 2) ATP-induced secretion is enhanced by an adenosine 3',5'-cyclic monophosphate intracellular concentration [cAMP]i-rise after beta-receptor stimulation in airway submucosal glands.

We examined the role of inositol 1,4,5-trisphosphate (IP3) receptors in acetylcholine (ACh)-induced chloride (Cl-) current in acinar cells of human and feline airway submucosal glands, using whole cell patch-clamp analysis. ACh (10 nM-1 microM) induced an initial Cl- current followed by a K+ current, and lower doses of ACh (1-10 nM) often induced oscillations of both currents, which were mimicked by the application of intracellular IP3. Neither isoproterenol (-10 microM) nor raising intracellular adenosine 3,5-cyclic monophosphate induced any current. Caffeine (20-50 mM) and intracellular ryanodine (1-100 microM) induced a K+ current alone without Cl- current. Monoclonal antibodies to the IP3 receptor abolished both ACh-induced K+ and Cl- currents. Immunohistochemical analysis revealed the localization of IP3 receptors on both the cytosol and some regions of the endoplasmic reticulum beneath the apical membrane of acinar cells. These results indicate that apically localized IP3 receptors control Cl- secretion from airway submucosal gland cells.

We examined the effect of Mg 2+ on potential difference (PD) and short circuit current (SCC) of the posterior epithelial membrane of canine trachea using an Ussing chamber. After the exchange to a Mg 2+ -free solution, PD and SCC rapidly increased, reaching maximal values within 3 min, followed by a gradual return towards the baseline over 60 min. In a Ca 2+ -free solution, Mg 2+ removal did not alter PD and SCC values. Increased Mg 2+ in the solution produced significant gradual decreases in PD and SCC. The decreases in PD and SCC were reversed by the addition of excessive Ca 2+ to the solution. Mg 2+ removal did not alter significantly isoproterenol-induced increases in PD and SCC values, while increased Mg 2+ significantly reduced the increases. These findings indicate that extracellular Mg 2+ is an important determinant in ion transport across the airway epithelium, probably through antagonistic actions of Mg 2+ and Ca 2+ .

Stepronin (SPN) is clinically used as an expectorant, and thenoic acid (TA) is its metabolite. We examined the effects of these drugs on the bioelectric parameters [potential difference (PD), short circuit current (SCC), conductance (G)] of the posterior epithelial membrane of canine trachea and on those of the mucus glycoprotein secretion from feline tracheal isolated glands. PD and SCC were obtained using an Ussing chamber and G was calculated as the ratio SCC/PD. Neither SPN nor TA significantly altered the baseline values of PD and SCC. However, in the mucosal solution, both SPN and TA significantly inhibited PD and SCC evoked by isoproterenol (ISOP), whereas G remained unchanged. Amirolide did not alter the inhibitory action of SPN and TA. Mucus glycoprotein secretion from isolated glands was estimated by measuring trichloride acetic acid-precipitable [3H]-glycoconjugates. SPN and TA significantly reduced mucus glycoprotein secretion. Further, when stimulated by methacholine, these agents significantly inhibited mucus glycoprotein secretion from isolated glands. These findings suggest that SPN inhibits airway secretion in vitro by both decreasing Cl- secretion from epithelial cells and mucus glycoprotein secretion from submucosal glands.

To determine the role of protein kinase C (PKC) in airway submucosal gland secretion, we examined the effect of a selective PKC stimulant, phorbol 12-myristate 13-acetate (PMA), on mucus glycoprotein (MGP) secretion, fluid secretion and intracellular Ca 2+ concentration ([Ca 2+ ]i) in isolated feline submucosal glands. MGP and fluid secretions were estimated by measuring trichloroacetic acid (TCA)-precipitable glycoconjugates and 22 Na-efflux, respectively, from isolated glands. [Ca 2+ ]i was measured using a Ca 2+ -sensitive fluorescent dye, Fura 2. PMA itself produced a significant increase in MGP secretion in a dose-dependent fashion (173% of control at 10 −5 M). PMA also produced a significant increase in 22 Na-efflux (151% of baseline rate constant at 10 −5 M). Indomethacin failed to alter the increase in MGP secretion or in 22 Na-efflux in response to PMA. Two PKC inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) and sphingosine, inhibited both MGP secretion and 22 Na-efflux stimulated by PMA; there was only a partial inhibition after stimulation by methacholine (MCh). PMA did not significantly alter [Ca 2+ ]i and H-7 did not alter the MCh-induced [Ca 2+ ]i rise. These findings indicate that PKC has a direct stimulatory role in stimulus-secretion coupling of airway submucosal gland secretion.

Electrolyte or fluid is secreted across airway mucosa by both superficial epithelium and submucosal glands. To understand the effect of glucocorticoid on fluid movement across airway mucosa, we examined the effects of dexamethasone (Dex) on bioelectric properties of canine and feline tracheal epithelium and on 22Na efflux from isolated feline tracheal submucosal glands. Potential difference (PD) and short-circuit current (SCC) across tracheal epithelium were measured using an Ussing chamber, and conductance (G) was calculated as the ratio SCC/PD. Isolated glands were loaded with 22Na, and the rate constant (RC) of Na2+ efflux was calculated by measuring the radioactivity of each effluent sample. After treatment with 10(-9) to 10(-5) M Dex for up to 6 h, the epithelium and isolated glands were stimulated with isoproterenol (ISP) and methacholine (MCh), respectively. Dex treatment did not alter significantly baseline values of PD, SCC, or RC. However, Dex treatment produced a dose-dependent attenuation of ISP-evoked epithelial PD and SCC and of MCh-evoked glandular RC. In canine epithelium, pretreatment with 10(-5) M Dex for 6 h reduced by 40% the ISP (10(-6) M)-evoked rise in PD and SCC, whereas G remained unchanged. After 10(-5) M Dex treatment for 6 h, MCh (10(-5) M)-evoked RC in isolated glands was significantly less than in control glands (MCh alone) by 23%. These findings suggest that glucocorticoid decreases the fluid secretion across the airway mucosa, especially when the mucosa are stimulated.

Idiopathic pulmonary fibrosis (IPF) and pulmonary emphysema (PE) have distinct clinical and pathological characteristics, and have been considered to be separate disorders. However, recent animal experiments have suggested that, with regard to their pathogenesis, the diseases have some features in common. However, there are no clinical data supporting this hypothesis. We report here 9 patients (all male, 67 +/- 2 years, mean +/- SE) who had PE followed by IPF. They were found among 152 PE patients who came to Tohoku University Hospital during the past 15 years (1976-1991). All patients were male and heavy smokers and 2 patients also had prostate cancer and gastric cancer, respectively. Three patients were alive during this study and had been diagnosed as having IPF and PE by the combination of transbronchial biopsy, selective alveolobronchogram, CT examination and lung function tests. The diagnosis of IPF and PE in the other patients was based on the pathological findings of autopsied lungs in addition to clinical findings. All patients showed PE mainly in the upper lobes and IPF in the lower lobes. In all patients, in addition to all known causes of pulmonary fibrosis, the possibilities that chronic or recurrent infections in PE induced pulmonary fibrosis and that IPF produced emphysematous changes were carefully excluded by medical records and pathological findings. It is not clear whether the occurrence of emphysema and pulmonary fibrosis in these cases is coincidental, or whether the two diseases are linked by a common pathogenetic pathway.

We performed a morphometric analysis of peribronchiolar and perivascular fibrosis in lungs obtained at autopsy from six patients with chronic bronchitis, six with pulmonary emphysema, and four normal control subjects. The areas of fibrosis outside the smooth muscle layer of bronchioles and outside the external elastic lamina of muscular pulmonary arteries were measured and their thickness was then calculated by assuming a round airway or artery. Patients with chronic bronchitis had significantly thicker peribronchiolar fibrosis in bronchioles of 1 mm or less in diameter and also thicker perivascular fibrosis of the adjacent muscular pulmonary arteries than the other two groups. The extent of perivascular fibrosis was significantly correlated with peribronchiolar fibrosis only in the muscular pulmonary arteries adjacent to the bronchioles but not in those away from the bronchioles. These findings suggest direct extension of chronic inflammation from bronchioles to the adjacent muscular pulmonary arteries in chronic bronchitis but not in pulmonary emphysema. Such perivascular fibrosis might lead to sustained pulmonary hypertension.

We examined the effects of endothelin on both the trichloroacetic acid precipitable 3H-labeled glycoconjugate release and intracellular Ca2+ concentration ([Ca2+]i]) measured by the usage of fura-2 in submucosal glands isolated from feline trachea. Endothelin-1 produced a significant increase in glycoconjugate release from the isolated glands in a dose-dependent fashion, reaching a response of 161% of the control at 10(-6) M. Atropine, propranolol, phentolamine, or indomethacin did not produce any significant alterations in the ET-1-evoked glycoconjugate secretion from the isolated glands. In contrast, in tracheal explants which contained epithelium, ET-1 produced a significant reduction in the glycoconjugate secretion in a dose-dependent fashion, reaching a response of 59% of the control at 10(-6) M. In the presence of cultured epithelial cells, ET-1 also produced a significant reduction in the glycoconjugate secretion from isolated glands. In isolated glands, ET-1 produced a sustained increase in the [Ca2+]i which was abolished by the removal of Ca2+ from the medium or by the presence of cultured epithelial cells. Pretreatment with indomethacin failed to alter the epithelial inhibitory action evoked by ET-1 in both the glycoconjugate secretion and the [Ca2+]i in isolated glands. ET-2 and ET-3 failed to produce significant alterations in the glycoconjugate secretion or [Ca2+]i. These findings indicate 1) that ET-1 induces mucus glycoprotein secretion via a Ca2+ influx and 2) that it possibly augments the an epithelial action inhibitory to the mucus glycoprotein secretion from airway submucosal glands.

To determine what muscarinic receptor subtype regulates [Ca2+]i mediating airway submucosal gland secretion, we examined the effects of atropine (Atr), pirenzepine (PZ), 11([2-(diethylamino)methyl-1-piperidinyl] acetyl)-5,11-dihydro-6H-pyrido (2,3-b)(1,4)-benzo-diazepin-6-one (AF-DX116) and 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP) on methacholine (MCh)-evoked [Ca2+]i rise in acinar cells, and compared this with mucus glycoprotein (MGP) and electrolyte secretion evoked by MCh from submucosal glands isolated from feline trachea. [Ca2+]i was measured with the Ca(2+)-sensitive fluorescent dye, fura 2. We determined MGP secretion by measuring TCA-precipitable 3H-labeled glycoconjugates and electrolyte secretion by the change in the rate constant of 22Na-efflux from isolated glands. Half-maximal inhibitory concentrations (IC50) of PZ, AF-DX116, 4-DAMP, and Atr against MCh-evoked [Ca2+]i rise were 10(-7) M, 6 x 10(-6) M, 8 x 10(-9) M, and 6 x 10(-9) M, respectively. IC50 of PZ, AF-DX116, 4-DAMP, and Atr against MCh-evoked MGP secretion were 10(-6) M, 2 x 10(-5) M, 8 x 10(-9) M, and 6 x 10(-9) M, respectively. MCh (10(-5) M)-evoked 22Na efflux was significantly inhibited by 10(-7) M 4-DAMP and 10(-7) M Atr (P less than 0.01, each) but not by 10(-7) M PZ. Receptor binding assays with [3H]quinuclidinyl benzilate showed that the Ki values for PZ, AF-D x 116, 4-DAMP and Atr were 2.2 x 10(-8) M, 6.6 x 10(-7) M, 6.2 x 10(-10) M, and 2.9 x 10(-10) M, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Five idiopathic pulmonary fibrosis (IPF) patients with sputum since the initial period of the disease (IPF SP+, more than 15 ml/day) were compared with five IPF patients without sputum throughout the course of the disease (IPF SP-) and four control subjects without pulmonary disease matched for age and sex. No significant differences in the duration of symptoms, pulmonary functions, or glucocorticoid therapy were observed between the two IPF groups. Autopsied lungs fixed by immersion into formaldehyde were used for morphometry by digitizing computer. The volume proportion of glands to bronchial wall thickness (gland%), volume proportion of goblet cells to total epithelial layer (goblet%), and luminal mucous volume were measured in central and peripheral airways. The gland percentage in the central airways of the IPF SP+ group was 18 +/- 1% (mean +/- SE), which was significantly greater than 7 +/- 0.6% of the IPF SP- group (p less than 0.001), similar to the 6 +/- 1% of control subjects. Luminal mucous volume in the peripheral airways of the IPF SP+ group was 11 +/- 2%, which was significantly greater than 3 +/- 1% of the IPF SP- group (p less than 0.05) or 0.6 +/- 0.3% of the control subjects (p less than 0.01). Furthermore, luminal mucous volume in both the central and peripheral airways significantly correlated with gland% (p less than 0.01, each). No significant difference in other parameters such as goblet% and cell infiltration between the IPF SP+ group and IPF SP- group was observed. These findings suggest that IPF with hypersecretion is associated with mucous glandular hypertrophy and the accumulation of mucus in the airways.

Guinea pig and human eosinophils were co-cultured with or without epithelial cells (controls). Eosinophil survival was enhanced in the presence of the cultured epithelial cells in a number-dependent fashion. Further, supernatants from cultured epithelial cells also enhanced eosinophil survival. Both a monoclonal antibody to GM-CSF and indomethacin inhibited these epithelial-cell-mediated effects, and GM-CSF and PGE2 were shown to prolong eosinophil survival. These findings indicate that airway epithelial cells can enhance eosinophil survival. It is suggested that the mechanism may involve GM-CSF and PGE2 generation.

We examined the effect of human recombinant TNFα on the potential difference (PD) and short circuit current (SCC) of canine tracheal epithelium using an Ussing chamber. Luminal or submucosal TNF (2 to 200 U/ml) produced no significant alterations in the basal PD or SSC values. Pretreatment with luminal TNF significantly reduced isoproterenol (ISOP, 10 −6 M)-evoked increases in SCC and PD to 57% and 66% of that with ISOP alone, respectively, with a significant decrease in conductance (G) to 87% of that with ISOP alone in a dose-dependent fashion, from 10 to 200 U/ml. Even after ISOP (10 −6 M)-evoked PD and SCC had reached a plateau, TNF produced significant decreases in PD and SCC up to 79% and 83% of that with ISOP alone, respectively, in a dose-dependent fashion, from 50 to 200 U/ml. Amiloride did not alter the inhibitory action of TNF on ISOP-evoked SCC and PD values. Antiserum against TNF abolished the inhibitory action of TNF on ISOP-evoked response. In contrast, submucosal TNF did not alter PD, SCC or G. These findings indicate that TNF attenuates β agonist-evoked increases in chloride secretion across airway epithelium.

In order to determine the prognosis of patients with chronic idiopathic pulmonary fibrosis (IPF), we evaluated clinical, laboratory, and bronchoalveolar lavage (BAL) data at the onset of IPF in 25 patients who survived beyond 1 yr (nine women and 16 men, 59 +/- 3 yr of age, mean +/- SE). When the patients were divided into two groups according to whether they had or did not have mucous hypersecretion, 11 patients with hypersecretion (Group A) had a poorer survival rate (6 yr) than did 14 patients without hypersecretion (Group B) (10 yr) (p less than 0.01). Further, there was a significant negative correlation between sputum volume and the duration of survival in 25 patients (r = -0.55, p less than 0.01). Before glucocorticoid treatment, we also found significantly larger numbers of neutrophils (17%) and eosinophils (5%) in differential cell counts of bronchoalveolar lavage fluid (BALF) in Group A than in Group B (neutrophils, 1%; eosinophils, 0.6%) (p less than 0.05 each). Chest radiographic findings and other laboratory data including pulmonary function tests did not correlate with the survival rate. These findings suggest that mucous hypersecretion as well as neutrophils and eosinophils in BALF are among the determinants of prognosis in patients with chronic IPF.

We measured the intracellular free calcium ion concentration [( Ca2+]i) of acinar cells in isolated feline tracheal submucosal glands in response to secretagogues using the Ca2(+)-sensitive fluorescent dye fura-2. The secretagogues included cholinergic, adrenergic agonists, substance P (SP), and vasoactive intestinal polypeptide (VIP) which induce mucus glycoprotein secretion from feline tracheal submucosal glands. Methacholine (MCh) produced a significant increase in [Ca2+]i of up to 9.8 times that of control in a dose-dependent fashion at concentrations of 10(-8) to 10(-3) M. [Ca2+]i increase by MCh reached a peak within 30 s after stimulation and thereafter showed a sustained rise. In Ca2(+)-free medium, MCh produced an initial transient rise, which was less than 30% of that in a Ca2(+)-containing solution, and which lasted for 60 s with no prolonged sustained rise in [Ca2+]i. Atropine abolished MCh-evoked [Ca2+]i increase. Phenylephrine and SP produced a prolonged increase in [Ca2+]i without an initial transient increase. Phenylephrine (up to 10(-4) M) evoked an increase in [Ca2+]i by up to 240% that of control, which was abolished by prazosin. SP (up to 10(-4) M) also evoked an increase in [Ca2+]i by 155% that of control, which was abolished by atropine. By contrast, both isoproterenol (up to 10(-5) M) and VIP (up to 10(-5) M) failed to alter [Ca2+]i. These findings indicate that the mucus glycoprotein secretion evoked by muscarinic cholinergic, alpha-adrenergic agonist or SP can be mediated by intracellular Ca2+, whereas that by beta-adrenergic agonists or VIP cannot.

Cyclic ADP-ribose (cADPR), a putative Ca2+-mobilizing second messenger, has been reported to operate in several mammalian cells. To investigate whether cADPR is involved in electrolyte secretion from airway glands, we used a patch-clamp technique, the measurement of microsomal Ca2+release, quantification of cellular cADPR, and RT-PCR for CD38 mRNA in human and feline tracheal glands. cADPR (>6 μM), infused into the cell via the patch pipette, caused ionic currents dependent on cellular Ca2+. Infusions of lower concentrations (2–4 μM) of cADPR or inositol 1,4,5-trisphosphate (IP3) alone were without effect on the baseline current, but a combined application of cADPR and IP3mimicked the cellular response to low concentrations of acetylcholine (ACh). Microsomes derived from the isolated glands released Ca2+in response to both IP3and cADPR. cADPR released Ca2+from microsomes desensitized to IP3or those treated with heparin. The mRNA for CD38, an enzyme protein involved in cADPR metabolism, was detected in human tissues, including tracheal glands, and the cellular content of cADPR was increased with physiologically relevant concentrations of ACh. We conclude that cADPR, in concert with IP3, operates in airway gland acinar cells to mobilize Ca2+, resulting in Cl−secretion.

Bronchorrhea, defined as watery sputum of 100 ml or more per day, was seen in a 52-year-old female patient with diffuse lymphangitic metastasis of colon carcinoma to the lung. For 5 months before the visit to our clinic, she complained of progressive worsening of the cough, watery sputum, and shortness of breath. On admission to our hospital, she expectorated large amounts of nonpurulent watery sputum (150 to 300 ml/d), and showed diffuse reticular and linear shadows in both lungs on chest radiograph and severe obstructive impairment (FEV1 percent, 35 percent) in lung function tests. Histologic findings obtained from both surgical specimens at abdominal operation for ileus and lungs at the autopsy revealed lymphangitic metastasis of ascending colon carcinoma to the lung. At autopsy, histologically the lungs showed diffuse infiltrations of mucus-secreting adenocarcinoma cells to both lung parenchyma and airway submucosa.

Diffuse panbronchiolitis (DPB) in Japan is a chronic bronchitis observed in nonsmoking adults, with severe obstruction and poor prognosis. DPB shares pathologic and clinical characteristics with mild adult cystic fibrosis (CF), except that CF is frequent in whites (Europeans and Americans of European descent) but not in Japanese. Recently, the cystic fibrosis transmembrane conductance regulator (CFTR) gene was identified, and a 3-base pair deletion (delta F508) was confirmed as a major mutation responsible for CF. We extracted genomic DNA from white blood cells of 17 DPB patients and from paraffin-embedded tissues of 4 DPB patients at autopsy. Two polymerase chain reaction (PCR) primers were made in exon 10 of the CFTR gene so that a three-base shorter segment of 78 base pairs was amplified from the CFTR gene with the delta F508 mutation; the DNA segment amplified from the normal gene contains an F508 area with 81 base pairs. Every DNA segment amplified from DPB patients showed a normal 81-base pair length, indicating no DNA sample contained the delta F508 mutation. These results based on delta F508 mutation analysis in the CF gene indicate that DPB may represent a disease different from CF.

Bronchial glands, which consist of mucous and serous cells, are abundant in human airways, playing a major role in the airway secretion. Cl(-) secretion is accompanied by water transport to the lumen in the acinar cells of bronchial glands. Agonists that increase [Ca(2+)]i induce the Cl(-) secretion in bronchial glands. Ca(2+) release from a IP(3)-sensitive Ca(2+) pool at the apical portion stimulates and opens Ca(2+)-sensitive Cl(-) channels at the apical membrane, producing Cl(-) secretion in bronchial glands. K(+) channels at the basolateral membranes are Ca(2+)-sensitive and activated by Ca(2+) release from a cADPribose-sensitive Ca(2+) pool, maintaining the Cl(-) secretion in bronchial glands. Further, cADP ribose in concert with IP(3) induce [Ca(2+)]i oscillation, inducing Cl(-) secretion in bronchial glands. Some tyrosine kinases are involved in the Cl(-) secretion in bronchial glands. Mucous and serous cells in bronchial glands take part in mucin secretion and the secretion of defensive substances (glycoconjugates), respectively. [Ca(2+)]i oscillations are shown to play a central role in the exocytosis of secretory granules in serous cells of bronchial glands. Other signal transductions of mucin and glycoconjugates in airway gland cells remain to be studied, although agonists which increase [cAMP]i are also well known to induce mucin and glycoconjugate secretion from airway glands.

Polycationic proteins, e.g., major basic protein from eosinophils or cathepsin G from neutrophils, have been shown to increase nonspecific airway responsiveness. Along with several indirect manners of action, polycations were reported to contract smooth-muscle strips and to raise the cellular Ca(2+) concentration as a direct action on airway smooth muscle. However, the mechanistic basis for the direct behavior remains to be elucidated. To address this issue, we examined the effects of synthetic cationic polypeptides poly-L-arginine and poly-L-lysine on fresh single smooth-muscle cells from bovine trachea using a patch-clamp technique. Both of the polycations significantly depolarized the membrane from a baseline of about -40 to -20 mV in a dose-dependent manner. The polycations also suppressed whole-cell spontaneous transient outward currents as well as both the conductance (from a baseline of about 130 to 70 pS) and open-state probability (about 25% of control values) of large-conductance Ca(2+)-dependent K(+) channel (maxi-K channel) on excised outside-out patch membranes. The polycations were without effect on the whole-cell Ca(2+) currents induced by depolarizing voltage pulses. We concluded that the synthetic polycations had at least two sites of action; one is the delayed rectifier K(+) channel that is responsible for the membrane depolarization that increases Ca(2+) influx, and the other is the maxi-K channel the suppression of which inhibits muscle relaxation. These results may explain the direct contractile action and, therefore, one of the mechanisms underlying the airway hyperresponsiveness induced by various polycationic proteins.

Pulmonary alveolar proteinosis (PAP), a disease characterised by accumulation of surfactant in alveoli, is diagnosed on the basis of invasive biopsy procedures. We have measured apoprotein A (SP-A) concentrations in sputum to see if this is useful for the diagnosis of PAP. Sputum samples from three patients with PAP and twenty patients with other pulmonary disease were assayed using monoclonal antibodies to SP-A. SP-A concentrations were 400 times higher in patients with PAP than in the controls, suggestions that this measurement is useful for the diagnosis of PAP, especially where lung biopsy is contraindicated.

A female patient, 69 years old, was hospitalized because of a 2-year history of recurrent hemoptyses resulting in severe anemia. X-ray examination of the chest showed a mass lesion in the right lower lung field, which had grown over the preceding 2 years. Bronchographic, arteriographic and CT examinations excluded the possibilities of bronchiectasis, pulmonary A-V fistula or sequestration. Histological examination following right lower lobectomy revealed no evidence of neoplasms or tuberculosis, fungal and parasitic infections but showed a predominant mononuclear cell infiltration and abundant small vessels in the affected small bronchi, and peribronchiolar and adjacent alveolar regions. To our knowledge, no case with chronic bronchopneumonia accompanied by such massive hemoptyses, as seen in this case, has been reported to date.

To clarify the changes in bronchial cartilage in diseased airways, we performed morphometric analysis of airways in autopsied lungs of 16 patients with chronic bronchitis (Group CB), pulmonary emphysema (Group PE), and bronchial asthma (Group BA), and in control patients without respiratory diseases (Group CN). Although degeneration of bronchial cartilage was clearly observed in airways from all groups except Group CN, the most extreme change was seen in Group CB. Increased perichondrial fibrosis was observed in both Groups CB and BA, and the more extreme change was seen in Group BA. Both the area proportions of degenerated cartilage (Deg%) and perichondrial fibrosis (Fib%) to total cartilage in bronchi (3 to 8 mm in diameter), cut vertically in the cross-section profile, were measured with a digitizing tablet coupled to a computer. No significant differences in the area proportion of cartilage to bronchial wall were observed among the four study groups. The Deg% values of Groups CB (mean: 15.4%), BA (mean: 12.9%), and PE (mean: 9.6%) were significantly higher than those of Group CN (mean: 1.0%) (p < 0.01 in each case). The Deg% values correlated significantly with the number of neutrophils in the bronchial walls (r = 0.63, p < 0. 01). Both Group CB (mean: 28.5%) and Group BA (mean: 33.6%) showed significantly higher values of Fib% than did Group CN (mean: 18.5%) (p < 0.01, each), and the value for Group PE (mean: 21.8%) was slightly increased (p < 0.05). The values of Fib% correlated significantly with the number of eosinophils in the bronchial walls (r = 0.51, p < 0.05), thickness of basement membrane (r = 0.77, p < 0.0002), bronchial gland area (r = 0.56, p < 0.02), and goblet-cell area (r = 0.55, p < 0.02). Further, the values of Deg% correlated significantly with those of Fib% (r = 0.64, p < 0.01). These findings indicate that airways in chronic obstructive pulmonary disease and bronchial asthma have both degenerative changes in the cartilage (chondrocytes) and increased perichondrial fibrosis, and that these alterations in bronchial cartilage may differ in chronic bronchitis and bronchial asthma.

The coexistence of chronic sinusitis (CS) may deteriorate the clinical condition of lower airway diseases such as bronchial asthma (BA) or chronic bronchitis (CB). However, the bronchial hyperresponsiveness (BH) in CS without any apparent lower airway disease is not fully understood nor are the effects of treatment. We examined lower airway hyperresponsiveness to methacholine (MCh) in 42 subjects with CS but without allergic rhinitis (AR) who had normal lung functions without any pulmonary symptoms, comparing it with that of 50 subjects with stable BA, 50 subjects with simple CB and 40 subjects with AR, and further examined the effect of endoscopic sinus surgery in 7 CS subjects with BH. The BH to MCh was measured in terms of the minimum dose (Dmin), defined as the cumulative dose at the point where respiratory conductance began to decrease. A Dmin

We tested the effects of tetraethylammonium (TEA), acetylcholine (ACh), histamine, and endothelin-1 on single airway smooth-muscle cells from bovine trachea, using the patch-clamp technique. Resting membrane potential was -48 +/- 1 mV (n = 47). Both TEA and ACh significantly depolarized the membrane, by +28 +/- 4 mV (P < 0.001, n = 12) and +21 +/- 2 mV (P < 0.01, n = 7), respectively, in the whole-cell configuration. In contrast, both histamine and endothelin-1 hyperpolarized the membrane, by -21 +/- 6 mV (P < 0.01, n = 8) and -15 +/- 2 mV (P < 0.01, n = 8), respectively. Calcium-dependent large-conductance K+-channels (127 pS) and small-conductance K+ channels (21 pS) were identified in excised patches. The small-conductance K+ channel was inhibited by 4-aminopyridine and activated by both histamine and endothelin-1. Furthermore, TEA did not alter the membrane hyperpolarization by these agonists, suggesting that the small-conductance K+ channel or delayed-rectifier K+ channel was involved in the membrane hyperpolarization. Membrane hyperpolarization by histamine and endothelin-1 suggests that activation of voltage-dependent calcium channels (VDCCs) or of calcium influx does not contribute substantially to the contractile response of airway smooth-muscle contraction to these agonists.

To determine whether human airway submucosal glands produce and secrete surfactant proteins, we examined their protein and gene expression in submucosal glands from trachea and bronchi obtained from operated and autopsied lungs within 4 h of death. Using a monoclonal antibody (PE-10) against surfactant protein A (SP-A), a positive immunoperoxidase stain was observed over serous cells of submucosal glands in histologic sections of airway walls. Measurement of SP-A in culture medium samples using single-step enzyme-linked immunosorbent assay showed a significant secretion of SP-A by isolated submucosal glands (1.2 +/- 0.08 ng/ml/h, SEM, n = 40). In gene expression experiments by reverse transciption-polymerase chain reaction, the SP-A complementary DNA (cDNA) segment was amplified from isolated submucosal glands, indicating the presence of SP-A messenger RNA (mRNA) in airway submucosal glands. Bronchial superficial epithelial cells failed to show the presence of SP-A mRNA. No cDNA segment of SP-B, SP-C, or SP-D cDNA was amplified from isolated submucosal glands or superficial epithelial cells, whereas all were amplified from alveolar tissue. Furthermore, in contrast to the control alveolar tissue, which expressed both SP-A1 and SP-A2 genes, SP-A2 gene transcript alone was detected in isolated submucosal glands by Southern analysis that included the digestion of the amplified SP-A cDNA fragment with the restriction enzyme Apa I. These findings indicate that human airway submucosal gland cells can transcribe the SP-A2 gene and produce SP-A protein in a manner different from peripheral airways and alveoli, playing a role in the airway defense mechanism.

In patients with pulmonary emphysema, emphysematous changes are not uniform and vary from minimum alveolar destruction to advanced bullous formation, depending on the lobe or site in the lungs. However, we have little knowledge on whether or how this nonuniformity or localization affects pulmonary function in PE patients. Therefore, we measured the computed tomography (CT) density of divided sites in lungs with high-resolution CT images from 25 PE patients (FEV1.0%, mean ± SD 36 ± 9%, %DLCO 48 ± 16%, all men, 68 ± 4 years) and compared them to various parameters of pulmonary function. The mean CT density of whole lungs correlated with 12 pulmonary function parameters including FEV1.0 and diffusion capacity. When both lung fields were divided into peripheral, intermediate and central portions, the CT density of the central portion correlated with all pulmonary function parameters with which CT density of whole lungs correlated. In contrast, the CT density of the peripheral portion significantly correlated with only 7 parameters with smaller correlation coefficient values than those of the central portion. When divided into upper, middle and lower portions, the CT densities of upper, middle and lower portions correlated with 6, 8 and 10 of the 12 pulmonary function parameters which correlated with the density of whole lungs, respectively. The delta value of CT densities between the upper and lower portions or between the lateral and medial portions correlated with obstructive impairment (FEV1.0 and FEV1.0%). These findings suggest that (1) central rather than peripheral emphysematous changes affect pulmonary function, and (2) uniformity of emphysematous change correlates with the severity of airway obstruction in PE patients.