- Email: [email protected]
Tumor necrosis factor attenuates β agonist-evoked Cl− secretion in canine tracheal epithelium
Respiration Physiology, 84 (1991) 379-387
379
© 1991 Elsevier Science Publishers B.V. 0034-5687/91/$03.50 RESP 01781
Tumor necrosis factor attenuates fl agonist-evoked CI- secretion in canine tracheal epithelium M. Satoh, T. Sasaki, S. Shimura, H. Sasaki and T. Takishima First Department of Internal Medicine. Tohoku University School of Medicine, Sendai. Japan (Accepted I1 February 1991) Abstract. We examined the effect of human recombinant TNF0t 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/mi) produced no significant alterations in the basal PD or SCC values. Pretreatment with luminal TNF significantly reduced isoproterenoi (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/~ agonist-evoked increases in chloride secretion across airway epithelium.
Adrenoceptors, in tracheal epithelium; Chloride, secretion in tracheal epithelium; Epithelium, tracheal and tumor necrosis factor', Tracheal epithelium, electrical properties; Tumor necrosis factor, and tracheal epithelium
Tumor necrosis factor (TNF) is known to be one of the cytokines produced by macrophages activated by a variety of stimuli. Although bacterial iipopolysaccharide is by far the most potent, Sendai virus, influenza virus, and certain gram-positive organisms including staphylococci are also capable of inducing TNF secretion from macrophages (Aderka et al., 1986; Beutler et ai., 1986). Furthermore, TNF mRNA production is stimulated in cultured macrophage cell lines by granulocyte macrophage colony stimulating factor (Cannistra et ai., 1987). Several cells other than macrophages have also been shown to produce TNF. We have observed TNF production both from mastocytoma cells (Ohno et al., 1990a) and from basophilic leukemia cell line (Ohno et al., 1990b) and a fraction of the cytotoxic activity produced by mast cells is thought
Correspondence to: T. Takishima, First Department of Internal Medicine, Tohoku University School of Medicine, I-1 Seiryo-machi, Aoba-ku, Sendai 980, Japan.
380
M. SATOH et at.
to be TNF. Also, T-lymphocytes produce abundant quantities of TNF mRNA and protein when incubated with the calcium ionophore A23187 in conjunction with phorbol diester (Cuturi et al., 1987). Macrophages are known to be abundant not only in the alveolar regions, but also in the mucus layers of the airways (Brain et al., 1984). Mast cells, monocytes and lymphocytes are also present, both in the mucus layers and airway walls. These migrating cells accumulate in the airway of patients suffering from various diseases, including bronchial asthma and chronic bronchitis. Therefore, it is possible that these cells in the airway produce a significant amount of TNF, resulting in the alteration of some airway functions, especially when inflammation is present. In fact, an experimental airway infection with Legionella pneumophila induced a significant amount of TNF in bronchoalveolar lavage fluid (Blanchard et aL, 1987). Ion with water transport across airway mucosa plays a primary role in mucociliary clearance and thus in the defense mechanisms against inhaled particles and pathogens. To our knowledge, however, the effect of TNF on ion or water transport in the airways has not been investigated. Although airway epithelial ion transport has been studied in a variety of species, it has been best characterized in canine tracheal epithelium, a tissue that actively secretes chloride and absorbs sodium. Chloride secretion is stimulated by the activation of receptors at the basolaterai membranes (submucosal) and fl-adrenergic stimulation is most potent in chloride secretion across canine tracheal epithelium. Sodium absorption via Na + channels in the apical membranes (luminal) is blocked by an Na ~' channel blocker, amiloride. Transport of both ions can lead to the induction of a transepithelial potential difference (PD) and short circuit current (SCC) across the epithelium which is associated with water movement into airway lumen (Olver et at, 1975; AI-Bazzaz et al., 1981). In the present study, therefore, we examined the effect of hunlan recombinant TNF ~ on PD and SCC in canine trachea in order to learn whether or not TNF influences ion and fluid transport across the airway epithelium (Satoh et al., 1989).
Methods Tracheas were removed from 20 adult mongrel dogs of both sexes (10-20 kg in body weight) under anesthesia with 30 mg per kg of intravenous thiopental, cleared of dissected adjacent tissue, and placed in oxygenated Krebs Ringer buffer (KRB) solution until used. The KRB solution contained NaCI, 125 raM; KCI, 5 raM; MgCI2, 1.2 raM; CaCI2, 1.0 raM; NaHCO3, 25 raM; NaH2PO4, 1.2 mM and glucose, 11 mM. From the posterior membranous portion of the trachea, the outmost layer and smooth muscle layer were dissected away, and then a sheet consisting of submucosai and epithelial layer was mounted in an Ussing chamber (Ikeda et al., 1990). Each experimental chamber was 10 ml in volume with an exposed area of 0.5 cm 2 and was bathed with KRB solution at 37 ° C, gassed with 95 ~o and 5 o~ CO2 at pH 7.4. KRB solution in the experimental chamber was circulated by the driving pressure of 95 O/o02-5 % CO2, mixing the added agents uniformly in the KRB solution. To avoid variations among animals, in each
TNF AND BIOELECTRICAL PROPERTIES OF AIRWAY EPITHEI.IUM
381
experiment, control paired samples were obtained from the same animal. To measure the electrical properties of the tracheal tissues, we used 2 pairs of agar bridges. PD across the tissues was measured with a pair of polyethylene bridges filled with 3 ~o agar in 3 M KCi solution. One end of each bridge was placed 3 mm from either side of the tissue. The other end was connected to a calomel half-cell, which was in turn connected to a voltmeter. A second pair of bridges, filled with 3 ~o agar in 0.9 % NaCI solution, was placed 3 cm from either side of the tissue, and was used to pass sufficient current across the tissue membranes to bring the PD to zero. This current (short-circuit current, SCC) is equivalent to the sum of the transepithelial ion movements. The PD was recorded for 1 min and then clamped at zero volts for 10 or 1 min to allow monitoring of SCC throughout the experimental period (automatic voltage clamp). Tissue conductance (G in m S . c m - 2 ) was calculated by dividing the SCC (/~A.cm -2) by the open-circuit PD (mV). Before initiating the experiments, tissues were allowed to equilibrate for a few hours to establish stable electrical properties. After this time, tissues with PD of less than 10 mV or G greater than 5 mS .cm-2 were presumed to be damaged and were discarded. Isoproterenol hydrochloride and amiloride were purchased from Sigma Chemical Co., MO and polyvalent rabbit antiserum against recombinant mouse TNF was purchased from Genzyme Co., Boston. Human recombinant TNFa was kindly provided by Asahi-Kasei Co., Tokyo.
Data analysis. All values are expressed as mean + S E. For mean comparison, one-way analysis of variance and two-tailed paired Student's t-test were used and P < 0.05 was considered to be statistically significant.
Results PD and SCC gradually increased in magnitude, reaching a steady state 2 to 4 h after mounting. After the electrical parameters reached the steady state of 28 + 2 mV in PD and 33 _+ 3 #A/cm 2 in SCC (n = 44), each agent was added to the submucosal or luminal solution. The addition of TNF to submucosal or luminal solution did not produce any significant alterations in baseline SCC or PD values. For example, 30 min after the addition of 200 U/ml TNF to the luminal solution, SCC, PD and G were 101 + 1%, 99 + 3% and 102 + 4~!/,, of the baseline control values (n = 3), respectively. However, pretreatment with TNF in the luminal solution significantly reduced isoproterenol (ISOP)-induced SCC and PD with a decrease in G, in a dose-dependent fashion; i.e., 23770 ofbaseline values, 18270, 142% and 134~o in SCC at 10 - 6 M alone, l0 -6 M ISOP + 50 U/ml TNF, l0 - 6 M ISOP + 100 U/ml TNF and 10 - 6 M ISOP + 200 U/ml TNF, respectively. As shown in fig. 1, pretreatment with 200 U/ml TNF in the luminal solution 30 min prior to the addition of 10 - 6 M ISOP to the submucosal solution significantly reduced the peak values of SCC and PD induced by ISOP from
382
M. SATOH et ai. % of control 240 220 -
~--
T
200 180 -160 140 -
.~..,~. •~...~.
120 !00 -
Isop alone
TNF 4Isop
SCC
Isop alone
TNF
4-
Isop alone
TNF
4-
Isop
PD
Isop
G
Fig. I. Effect of TNF pretreatment on short circuit current (SCC), potential difference (PD) and conductance (G) in response to isoproterenol (ISOP) in canine tracheal epithelium. TNF significantly decreased SCC and PD values induced by ISOP with a significant decrease. SCC, PD and G values are expressed as % of baseline control. Open columns indicated ISOP (10-6M) alone and hatched columns ISOP (10 ¢' M) + TNF (200 U/ml) pretreatment (n = 4). **P < 0.01, compared to ISOP alone.
237 + 25 ~, to 134 + 3% 01 = 4) ofbaseline control values in SCC (P < 0.01), and from 191 + 17% to 127 + 6% (n - 4 ) o f baseline control values in P D ( P < 0.01) (fig. 1). G significantly decreased from 121 + 3 °'0 to 105 + 2% of baseline control values (P < 0.01) (fig. 1). After washing the luminal solution in fresh KRB solution, ISOP (10 -¢' M) produced responses of 210 + 30~o and 181 + 18% in SCC and PD, respectively, (n - 4, P < 0.01, each); similar to those before T N F treatment. When 200 U/ml TNF was added to the submucosal solution, TNF did not produce any significant alterations in the values of SCC, PD or G induced by 10- 6 M ISOP in the submucosal solution (n -- 3). The inhibitory effect of luminal TNF on 10 -6 M ISOP-induced SCC, PD and G was abolished by pretreatment with a polyvalent rabbit antiserum against recombinant mTNF~ in the luminal solution (n = 4). In contrast, pretreatment with luminal amiloride (10-4 M) did not produce any alteration in the inhibitory effect of TNF on ISOP-evoked SCC and PD. Namely, 10-4 M amiloride in the luminal solution reduced both SCC and PD up to 70?/0 of basal values and thereafter ISOP (10 -6 M) produced increases of 302 + 30% and 234 + 21% in SCC and PD, respectively (n = 6). Pretreatment with 200 U/ml TNF significantly reduced ISOP (10 -6 M)-evoked SCC, PD and G to 148 + 47~o of basal values, 135 + 6% and 112 + 3% (n = 5, P < 0.01), respectively. Next, we examined the effect ofluminal TNF on SCC and PD that had been elevated by ISOP in order to learn whether or not TNF attenuates the responses after stimulation of fl-receptors. ISOP induced dose-dependent increases in SCC, PD and G, reaching
TNF AND BIOELECTRICAL PROPERTIES OF AIRWAY EPITHELIUM
383
%SCC tOO9590858075-
.L
Fig. 2. Effect of TNF on isoproterenol (ISOP: 10-6 M)-evoked short circuit current (SCC). TNF was added to the luminal solution after the maximum response to ISOP in the submucosal solution and measured at 10 rain-intervals for up to 60 rain after submucosai ISOP stimulation. SCC is expressed as % of the maximum response to ISOP. TNF induced a significant decrease compared to ISOP alone (O) in dose-dependent fashion. Data are expressed as mean + SE for 4 to 8 experiments. *P < 0.05, **P < 0.01, compared to ISOP alone, x , ISOP + TNF (50 U/ml); • ; ISOP + TNF (100 U/ml); I , ISOP + TNF (200 U/ml).
a peak at 10 -6 M; i.e., 113% ofbasal values, 144%, 220%, 252% and 203% in SCC at 10 - 9 M, 10 - SM, 10- 7 M, 10- 6 M and 10- 5 M, respectively. After reaching a plateau (5-10 min after stimulation by the addition of 10- 6 M ISOP to the submucosal solution), T N F was added to the luminal solution and PD and SCC were measured at 10-rain intervals for up to 60 rain after submucosal ISOP stimulation. Luminal T N F produced significant reductions in ISOP-evoked SCC and PD when compared to ISOP alone in a dose-dependent fashion as shown in fig. 2 and 200 U/ml TNF produced responses of 79 + 5% of 10 - 6 M ISOP alone, 83 + 8% and 96 + 3% (n = 4) in SCC, PD and G, respectively, 60 rain after ISOP stimulation. However, the reduction in G did not reach statistical significance perhaps because of the large variations, except at 10 min after T N F administration. Pretreatment with 10 -6 M amiloride in the luminal solution did not produce any significant alteration in the T N F inhibitory effect on ISOP-evoked SCC and PD values after the maximum response to ISOP (10 -6 M) (n = 5). Antiserum against T N F abolished to inhibitory effect of T N F on ISOP-evoked SCC and PD values after the maximum response induced by ISOP (10 -6 M) (n - 4).
Discussion
The present study revealed that r TNF0c has an inhibitory action on fl-adrenergic agonist-induced PD and SCC increases in canine tracheal epithelium, suggesting that ion transport stimulated by fl-agonist is reduced by the presence of TNF.
384
M. SATOH etaL
We can speculate on some of the possibilities concerning the mechanisms of the inhibitory action of TNF on ISOP-evoked SCC and PD. Firstly, there is the possibility that TNF decreases ISOP action by direct interaction between these two drugs, TNF and ISOP. However, this is unlikely since no inhibitory action was observed when both TNF and ISOP were added to the submucosal solution. Secondly, it may be that TNF acts by stimulating cells other than the epithelial cells, resulting in the release of a substance which in turn decreases SCC and PD. However, no factors or substances released from the migrating cells (macrophages, monoeytes, lymphocytes or neutrophils) or constituent cells of the airway wall are known to inhibit bioelectrical phenomena of airway epithelium despite the presence of many stimulating substances or factors. Thirdly, TNF may directly suppress the opening of ion channels present at the apical cell membrane of epithelial cells. G decreased after TNF pretreatment in ISOP-evoked SCC and PD, suggesting a decrease in the opening ofion channels. TNF has little or no effects on Na + channels at the apical membrane, since amiloride did not alter the inhibitory action on ISOP-induced SCC and PD. However, the problem remains unresolved that the TNF-evoked inhibition on the opening of Na + channels was too small to be detected by the present method. Fourthly, TNF may directly affect cellular ion pumps or cotransporters which exist at the basolateral cell membrane without any alterations to the opening of ion channels at the apical cell membrane of airway epithelial cells, resulting in no changes in G. This possibility seems unlikely since G significantly decreased after TNF pretreatment in the ISOP-evoked experiment. In addition, it seems unlikely that TNF, with its high molecular weight, would penetrate the tight junction between epithelial cells and reach the ion pumps or cotransporters at the basolateral membrane. This also argues against an interaction between fl-adrenoreceptors at the basolateral membrane and TNF-receptors which must be on the apical membrane if the findings in the present study are correct. An interaction between fl-adrenergic and TNF receptors is also unlikely because TNF attenuated the increases in SCC and PD evoked by fl-agonist even after the maximum response by fl-agonist. Finally, this attenuation after the maximum response and the lack of any inhibitory action by amiloride suggests that TNF interacts with the intracellular process of CIsecretion by epithelial cells. A number of findings which indicate a receptor-mediated action of TNF and showing some inhibitory action have been reported, especially in tumor cells (Kull et al., 1985; Baglioni et al., 1985: Wise et al., 1990). For example, a recent experiment (Wise et al., 1990) indicated that TNF decreases surfactant secretion in a pulmonary adenocarcinoma cell, and that the inhibitory effects are associated with decreased surfactant apoprotein A - mRNA. Some interactions between the two major second messenger systems, cAMP and Ca 2 ÷, are known in other tissues and the presence of an antagonistic control in the interaction between the two second messengers may be responsible for the inhibitory action of TNF in the present study. Johnson and Baglioni (1988) have reported that the receptor-mediated activity of TNF is downregulated by activators of protein kinase C (Ca 2 +/phospholipid-dependent enzyme) in human HeLa cells and murine L(s)cells. The chloride secretion of airway epithelial cells is mediated by cyclic AMP as a second messenger and fl-agonists simulate CI - secretion
TNF AND BIOELECTRICAL PROPERTIES OF AIRWAY EPITHELIUM
385
by rising intracellular cAMP, which is degraded by the enzyme diphosphoesterase. It is possible that TNF activates this enzyme, resulting in faster degradation ofintracellular cyclic AMP. Of course, the cytotoxic action of TNF which is known in tumor cells is not responsible for the inhibitory: action obse~ed in the present study since the response to ISOP remained unchanged even after washing TNF out of the mucosal solution. A recent study (Mullin et ai., 1990) indicated that TNF affects the tight junction between cultured epithelial cells from pig kidney (LLC-PK 1 cells) with decreases in both transepithelial resistance and PD, thereby increasing the flow of solute between cells. The mechanism of TNF action in LLC-PK I cells appears to differ from that in canine tracheal epithelium since TNF produced a significant decrease in G (an increase in resistance) in the latter. In addition, TNF did not alter the baseline values of SCC, PD or G, suggesting that the cytotoxic action is not responsible for the inhibitory action of TNF observed in the present study. In conclusion, an intracellular interaction between TNF and r-stimulant is the most likely explanation of these observations although a direct inhibitory action against the opening of CI- channels may" in part play a role. It is possible that airway inflammation, the main aspect of chronic bronchitis (Thompson et al., 1989) or bronchial asthma, induces TNF production in the airways. For example, we have obtained some results which indicated TNF production by mast cells and basophiis with IgE receptor triggering (Ohno et al., 1990a,b). Since basophils and mast cells are thought to play a central role in the allergic reaction in bronchial asthma TNF production by these cells may be involved in the pathogenesJs of asthma. Mast cells and basophils are thought to initiate a bronchial response by releasing chemical mediators through lgE receptor triggering via inhaled antigen (Abraham et al., 1988). Further, ~-adrenergic agonists are widely used for airway dilatation in patients with bronchial asthma and chronic obstructive pulmonary disease. Beta-adrenergic stimulation is well known not only for its ability to dilate airways but also as a potent stimulus ofCi - secretion across the airway mucosa. Airway epithelia show considerable differences in ion transport between species and human epithelium is less potent in Cisecretion in response to ~-adrenergic stimulation than canine tracheal epithelium (Knowles et al., 1984; Widdicombe et al., 1985). In spite ofthis limitation in the present study, taken together with the findings described above, it can be proposed that the inflammation associated with bronchial asthma or chronic bronchitis induces the release of TNF, resulting in the attenuation ofthe effect of fl-agonist on chloride secretion with water into airway lumen, and in a decrease of mucociliary clearance. A recent report (isawa et ai., 1990) has described how inhalation of fl2-stimulant neither accelerates mucus transport nor facilitates mucociliary clearance in patients with various diseases including bronchial asthma. Meanwhile, a number of in vitro experiments (Iravani et al., 1974, Van As, 1974; Verdugo et al., 1980) have indicated that fl 2-stimulants accelerate mucociliary clearance. Our present finding that TNF attenuates //-stimulant-induced ion transport may be an explanation for the lack of any significant effect on mucociliary clearance in vivo in diseased lungs.
386
M. SATOH et al.
Acknowledgements. We gratefully acknowledge Professor A. Nishiyama and Dr. N. Saitoh for discussion a,~ld suggestion, Dr. Ronald Scott for reading, and Ms Yumi Saitoh for typing the manuscript. This study was supported by a Grant-in-Aid for General Scientific Research (No. 01570422) from the Japanese Ministry of Education, Science and Culture.
References Abraham, W. M., J. S. Stevenson, M. Eldridge, R. Gasrrido and L. Nieves (1988). Nedocromii sodium in allergen induced bronchial responses and airway hyperresponsiveness in allergic sheep. J. Appl. Physiol. 65: 1062-1068. Aderka, D., H. Holtmann, L. Toker, T. Hahn and D. Wailach (1986). Tumor necrosis factor induction by Sendai virus. J. Immunol. 136: 2938-2942. AI-Bazzaz, F., Y.P. Yadava and C. Westenfelder (1981). Modification of Na and C! transport in canine tracheal mucosa by prostaglandins. Am. J. Physiol. 240: Fl01-105. Baglioni, C., S. McCandless, J. Tavernier and W. Fiefs (1985). Binding ofhuman tumor factor to high affinity receptors on HeLa and iymphoblastoid cells sensitive to growth inhibition. J. Biol. Chem. 260: 13395-13397. Beutler, B., N. Krochin, I.W. Milsark, A. Goldberg and A. Cerami (1986). Induction of cachectin (tumor necrosis factor) synthesis by influenza virus: deficient production by endotoxin-resistant (C3H/HEJ) rnacrophages. Clin. Res. 34: 491A. Bianchard, D.K., J.Y. Djeu, T.W. Klein, H. Friedman and W.E. Stewart II (1987). Induction of tumor necrosis factor by Legionella pneumophila. Infect. lmmun. 55: 433-437. Brain, J. D., P. Gehr and R.l. Kavet (1984). Airway macrophages. The importance of the fixation method. Am. Rev. Respir. Dis. 129: 823-826. Cannistra, S.A., A. Rambaldi, D.R. Spriggs, F. Herrmann, D. Kufe and J.D. Griffin (1987). Human granulocyte-ma,~rophage colony-stimulating factor indllces expression ofthe tumor necrosis factor gene by the U937 cell line and by normal human monocytes. J. Clin. Invest. 79: 1720-1728. Cuturi, M. C., M. Murphy, M. P. Costa-Giomi, R. Weinmann, B. Perussia and G. Trinchieri (1987). Independent regulation of tumor necrosis factor and lymphotoxin production by human peripheral blood lymphocytes. J. Exp. M¢d. 165: 1581-1594. lkeda, K., T. Sasaki, S. Shimura, M. Satoh, H. lshihara, H. Sasaki, T. rakis|,.i,,a, ¥. Saitoh and A. Nishiyama (1990). Effect of surfactant on bioelectric properties of canine tracheal epithelium. Respir. Physiol. 81: 41-49. lravani, J. and G.N. Melville (1974). Mucociliary function of the respiratory tract as influenced by drugs. Respiration 31: 350-357. Isawa, T., T. Teshima, T. Hirano, Y. Anazawa, M. Miki, K. Konno and M. Motomiya (1990). Does a //2-stimulator really facilitate mucociliary transport in the auman lungs in vitro'?.A study with procaterol. Am. Rev. Respir. Dis. 141: 715-720. Johnson, S. E. and C. Baglioni (1988). Tumor necrosis factor receptors and cytocidai activity are down-regulated by activators of protein kinase C. J. Biol. Chem. 263: 5686-5692. Knowles, M., G. Murray, J. Shallal, F. Askin, V. Ranga, J. Gatzy and R. Boucher (1984). Bioelectric properties and ion flow across excised human bronchi. J. Appl. Physiol. 56: 368-877. Kull, F.C., S. Jacobs dnd P. Cuatrecasas (1985). Cellular receptor for ~251-1abeled tumor necrosis factor: Specific binding, affinity labeling, and relationship to sensit;-tity. Proc. Natl. Acad. Sci. USA 82: 5756-5760. Mullin, J.M. and K.V. Snook (1990). Effect of tumor necrosis factor on epithelial tight junctio.ns and transepithelial permeability. Cancer Res. 50:2172-2176. Ohno, !., T. Tanno, Y. Yamauchi and T. Takishima (1990a). Production of turnout necrosis factor by mastocytoma P815 cells. Immm~ology 69: 312-315. Ohno, I., T. Tanno, K. Yamauchi and T. Takishima (1990b). Gene expression and production of tumor
TNF AND BIOELECTRICAL PROPERTIES OF AIRWAY EPITHELIUM
387
necrosis factor by a rat basophilic leukemia cell line {RBL-2H3) with lgE receptor triggering. Immunology 70: 88-93. Olver, R.E., B. Davis, M.G. Marin and J.A. Nadei (1975). Active transport of Na ÷ and Cl- across the canine tracheal epithelium in vitro. Am. Rev. Respir. D/s. 122:811-815. Satoh, M., T. Sasaki, S. Shimura, I. Ohno, Y. Tanno, H. $asaki and T. Takishima (1989). Regulatory effect of TNF on bioelectrical properties of canine tracheal epithelium (abstract). Am. Rev. Respir. D/s. 139: A476. Thompson, A.B., D. Daughton, R.A. Robbins, M.A. Ghafouri, M. Oehlerking and S.I. Rennard (1989). intraluminai airway inflammation in chronic bronchitis: Characterization and correlation with clinical parameters. Am. Rev. Respir. Dis. 140: 1527-1537. Van As, A. (1974). The role of selective beta2-adrenoreceptor stimulants in the control of ciliary activity. Respiration 31 : 146-15 !. Verdugo, P., N. T. Johnson and P. Y. Taur (1980). Beta-adrenergic stimulation of respiratory ciliary activity. J. Appl. Physiol. 48: 868-871. Widdicombe, J.H., D.L. Coleman, W.E. Finkbeiner and I.K. Tuet (1985). Electrical properties of monolayers cultured from cells of human tracheal mucosa. J. Appl. Physiol. 58: 1729-1735. Wise, J., J. Clark, W. Hull, R. Holtzman and J. Whitsett (1990). Tumor necrosis factor • inhibits surfactantassociated protein A expression (abstract). Am. Rev. Respir. Dis. 141: A692.
379
© 1991 Elsevier Science Publishers B.V. 0034-5687/91/$03.50 RESP 01781
Tumor necrosis factor attenuates fl agonist-evoked CI- secretion in canine tracheal epithelium M. Satoh, T. Sasaki, S. Shimura, H. Sasaki and T. Takishima First Department of Internal Medicine. Tohoku University School of Medicine, Sendai. Japan (Accepted I1 February 1991) Abstract. We examined the effect of human recombinant TNF0t 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/mi) produced no significant alterations in the basal PD or SCC values. Pretreatment with luminal TNF significantly reduced isoproterenoi (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/~ agonist-evoked increases in chloride secretion across airway epithelium.
Adrenoceptors, in tracheal epithelium; Chloride, secretion in tracheal epithelium; Epithelium, tracheal and tumor necrosis factor', Tracheal epithelium, electrical properties; Tumor necrosis factor, and tracheal epithelium
Tumor necrosis factor (TNF) is known to be one of the cytokines produced by macrophages activated by a variety of stimuli. Although bacterial iipopolysaccharide is by far the most potent, Sendai virus, influenza virus, and certain gram-positive organisms including staphylococci are also capable of inducing TNF secretion from macrophages (Aderka et al., 1986; Beutler et ai., 1986). Furthermore, TNF mRNA production is stimulated in cultured macrophage cell lines by granulocyte macrophage colony stimulating factor (Cannistra et ai., 1987). Several cells other than macrophages have also been shown to produce TNF. We have observed TNF production both from mastocytoma cells (Ohno et al., 1990a) and from basophilic leukemia cell line (Ohno et al., 1990b) and a fraction of the cytotoxic activity produced by mast cells is thought
Correspondence to: T. Takishima, First Department of Internal Medicine, Tohoku University School of Medicine, I-1 Seiryo-machi, Aoba-ku, Sendai 980, Japan.
380
M. SATOH et at.
to be TNF. Also, T-lymphocytes produce abundant quantities of TNF mRNA and protein when incubated with the calcium ionophore A23187 in conjunction with phorbol diester (Cuturi et al., 1987). Macrophages are known to be abundant not only in the alveolar regions, but also in the mucus layers of the airways (Brain et al., 1984). Mast cells, monocytes and lymphocytes are also present, both in the mucus layers and airway walls. These migrating cells accumulate in the airway of patients suffering from various diseases, including bronchial asthma and chronic bronchitis. Therefore, it is possible that these cells in the airway produce a significant amount of TNF, resulting in the alteration of some airway functions, especially when inflammation is present. In fact, an experimental airway infection with Legionella pneumophila induced a significant amount of TNF in bronchoalveolar lavage fluid (Blanchard et aL, 1987). Ion with water transport across airway mucosa plays a primary role in mucociliary clearance and thus in the defense mechanisms against inhaled particles and pathogens. To our knowledge, however, the effect of TNF on ion or water transport in the airways has not been investigated. Although airway epithelial ion transport has been studied in a variety of species, it has been best characterized in canine tracheal epithelium, a tissue that actively secretes chloride and absorbs sodium. Chloride secretion is stimulated by the activation of receptors at the basolaterai membranes (submucosal) and fl-adrenergic stimulation is most potent in chloride secretion across canine tracheal epithelium. Sodium absorption via Na + channels in the apical membranes (luminal) is blocked by an Na ~' channel blocker, amiloride. Transport of both ions can lead to the induction of a transepithelial potential difference (PD) and short circuit current (SCC) across the epithelium which is associated with water movement into airway lumen (Olver et at, 1975; AI-Bazzaz et al., 1981). In the present study, therefore, we examined the effect of hunlan recombinant TNF ~ on PD and SCC in canine trachea in order to learn whether or not TNF influences ion and fluid transport across the airway epithelium (Satoh et al., 1989).
Methods Tracheas were removed from 20 adult mongrel dogs of both sexes (10-20 kg in body weight) under anesthesia with 30 mg per kg of intravenous thiopental, cleared of dissected adjacent tissue, and placed in oxygenated Krebs Ringer buffer (KRB) solution until used. The KRB solution contained NaCI, 125 raM; KCI, 5 raM; MgCI2, 1.2 raM; CaCI2, 1.0 raM; NaHCO3, 25 raM; NaH2PO4, 1.2 mM and glucose, 11 mM. From the posterior membranous portion of the trachea, the outmost layer and smooth muscle layer were dissected away, and then a sheet consisting of submucosai and epithelial layer was mounted in an Ussing chamber (Ikeda et al., 1990). Each experimental chamber was 10 ml in volume with an exposed area of 0.5 cm 2 and was bathed with KRB solution at 37 ° C, gassed with 95 ~o and 5 o~ CO2 at pH 7.4. KRB solution in the experimental chamber was circulated by the driving pressure of 95 O/o02-5 % CO2, mixing the added agents uniformly in the KRB solution. To avoid variations among animals, in each
TNF AND BIOELECTRICAL PROPERTIES OF AIRWAY EPITHEI.IUM
381
experiment, control paired samples were obtained from the same animal. To measure the electrical properties of the tracheal tissues, we used 2 pairs of agar bridges. PD across the tissues was measured with a pair of polyethylene bridges filled with 3 ~o agar in 3 M KCi solution. One end of each bridge was placed 3 mm from either side of the tissue. The other end was connected to a calomel half-cell, which was in turn connected to a voltmeter. A second pair of bridges, filled with 3 ~o agar in 0.9 % NaCI solution, was placed 3 cm from either side of the tissue, and was used to pass sufficient current across the tissue membranes to bring the PD to zero. This current (short-circuit current, SCC) is equivalent to the sum of the transepithelial ion movements. The PD was recorded for 1 min and then clamped at zero volts for 10 or 1 min to allow monitoring of SCC throughout the experimental period (automatic voltage clamp). Tissue conductance (G in m S . c m - 2 ) was calculated by dividing the SCC (/~A.cm -2) by the open-circuit PD (mV). Before initiating the experiments, tissues were allowed to equilibrate for a few hours to establish stable electrical properties. After this time, tissues with PD of less than 10 mV or G greater than 5 mS .cm-2 were presumed to be damaged and were discarded. Isoproterenol hydrochloride and amiloride were purchased from Sigma Chemical Co., MO and polyvalent rabbit antiserum against recombinant mouse TNF was purchased from Genzyme Co., Boston. Human recombinant TNFa was kindly provided by Asahi-Kasei Co., Tokyo.
Data analysis. All values are expressed as mean + S E. For mean comparison, one-way analysis of variance and two-tailed paired Student's t-test were used and P < 0.05 was considered to be statistically significant.
Results PD and SCC gradually increased in magnitude, reaching a steady state 2 to 4 h after mounting. After the electrical parameters reached the steady state of 28 + 2 mV in PD and 33 _+ 3 #A/cm 2 in SCC (n = 44), each agent was added to the submucosal or luminal solution. The addition of TNF to submucosal or luminal solution did not produce any significant alterations in baseline SCC or PD values. For example, 30 min after the addition of 200 U/ml TNF to the luminal solution, SCC, PD and G were 101 + 1%, 99 + 3% and 102 + 4~!/,, of the baseline control values (n = 3), respectively. However, pretreatment with TNF in the luminal solution significantly reduced isoproterenol (ISOP)-induced SCC and PD with a decrease in G, in a dose-dependent fashion; i.e., 23770 ofbaseline values, 18270, 142% and 134~o in SCC at 10 - 6 M alone, l0 -6 M ISOP + 50 U/ml TNF, l0 - 6 M ISOP + 100 U/ml TNF and 10 - 6 M ISOP + 200 U/ml TNF, respectively. As shown in fig. 1, pretreatment with 200 U/ml TNF in the luminal solution 30 min prior to the addition of 10 - 6 M ISOP to the submucosal solution significantly reduced the peak values of SCC and PD induced by ISOP from
382
M. SATOH et ai. % of control 240 220 -
~--
T
200 180 -160 140 -
.~..,~. •~...~.
120 !00 -
Isop alone
TNF 4Isop
SCC
Isop alone
TNF
4-
Isop alone
TNF
4-
Isop
PD
Isop
G
Fig. I. Effect of TNF pretreatment on short circuit current (SCC), potential difference (PD) and conductance (G) in response to isoproterenol (ISOP) in canine tracheal epithelium. TNF significantly decreased SCC and PD values induced by ISOP with a significant decrease. SCC, PD and G values are expressed as % of baseline control. Open columns indicated ISOP (10-6M) alone and hatched columns ISOP (10 ¢' M) + TNF (200 U/ml) pretreatment (n = 4). **P < 0.01, compared to ISOP alone.
237 + 25 ~, to 134 + 3% 01 = 4) ofbaseline control values in SCC (P < 0.01), and from 191 + 17% to 127 + 6% (n - 4 ) o f baseline control values in P D ( P < 0.01) (fig. 1). G significantly decreased from 121 + 3 °'0 to 105 + 2% of baseline control values (P < 0.01) (fig. 1). After washing the luminal solution in fresh KRB solution, ISOP (10 -¢' M) produced responses of 210 + 30~o and 181 + 18% in SCC and PD, respectively, (n - 4, P < 0.01, each); similar to those before T N F treatment. When 200 U/ml TNF was added to the submucosal solution, TNF did not produce any significant alterations in the values of SCC, PD or G induced by 10- 6 M ISOP in the submucosal solution (n -- 3). The inhibitory effect of luminal TNF on 10 -6 M ISOP-induced SCC, PD and G was abolished by pretreatment with a polyvalent rabbit antiserum against recombinant mTNF~ in the luminal solution (n = 4). In contrast, pretreatment with luminal amiloride (10-4 M) did not produce any alteration in the inhibitory effect of TNF on ISOP-evoked SCC and PD. Namely, 10-4 M amiloride in the luminal solution reduced both SCC and PD up to 70?/0 of basal values and thereafter ISOP (10 -6 M) produced increases of 302 + 30% and 234 + 21% in SCC and PD, respectively (n = 6). Pretreatment with 200 U/ml TNF significantly reduced ISOP (10 -6 M)-evoked SCC, PD and G to 148 + 47~o of basal values, 135 + 6% and 112 + 3% (n = 5, P < 0.01), respectively. Next, we examined the effect ofluminal TNF on SCC and PD that had been elevated by ISOP in order to learn whether or not TNF attenuates the responses after stimulation of fl-receptors. ISOP induced dose-dependent increases in SCC, PD and G, reaching
TNF AND BIOELECTRICAL PROPERTIES OF AIRWAY EPITHELIUM
383
%SCC tOO9590858075-
.L
Fig. 2. Effect of TNF on isoproterenol (ISOP: 10-6 M)-evoked short circuit current (SCC). TNF was added to the luminal solution after the maximum response to ISOP in the submucosal solution and measured at 10 rain-intervals for up to 60 rain after submucosai ISOP stimulation. SCC is expressed as % of the maximum response to ISOP. TNF induced a significant decrease compared to ISOP alone (O) in dose-dependent fashion. Data are expressed as mean + SE for 4 to 8 experiments. *P < 0.05, **P < 0.01, compared to ISOP alone, x , ISOP + TNF (50 U/ml); • ; ISOP + TNF (100 U/ml); I , ISOP + TNF (200 U/ml).
a peak at 10 -6 M; i.e., 113% ofbasal values, 144%, 220%, 252% and 203% in SCC at 10 - 9 M, 10 - SM, 10- 7 M, 10- 6 M and 10- 5 M, respectively. After reaching a plateau (5-10 min after stimulation by the addition of 10- 6 M ISOP to the submucosal solution), T N F was added to the luminal solution and PD and SCC were measured at 10-rain intervals for up to 60 rain after submucosal ISOP stimulation. Luminal T N F produced significant reductions in ISOP-evoked SCC and PD when compared to ISOP alone in a dose-dependent fashion as shown in fig. 2 and 200 U/ml TNF produced responses of 79 + 5% of 10 - 6 M ISOP alone, 83 + 8% and 96 + 3% (n = 4) in SCC, PD and G, respectively, 60 rain after ISOP stimulation. However, the reduction in G did not reach statistical significance perhaps because of the large variations, except at 10 min after T N F administration. Pretreatment with 10 -6 M amiloride in the luminal solution did not produce any significant alteration in the T N F inhibitory effect on ISOP-evoked SCC and PD values after the maximum response to ISOP (10 -6 M) (n = 5). Antiserum against T N F abolished to inhibitory effect of T N F on ISOP-evoked SCC and PD values after the maximum response induced by ISOP (10 -6 M) (n - 4).
Discussion
The present study revealed that r TNF0c has an inhibitory action on fl-adrenergic agonist-induced PD and SCC increases in canine tracheal epithelium, suggesting that ion transport stimulated by fl-agonist is reduced by the presence of TNF.
384
M. SATOH etaL
We can speculate on some of the possibilities concerning the mechanisms of the inhibitory action of TNF on ISOP-evoked SCC and PD. Firstly, there is the possibility that TNF decreases ISOP action by direct interaction between these two drugs, TNF and ISOP. However, this is unlikely since no inhibitory action was observed when both TNF and ISOP were added to the submucosal solution. Secondly, it may be that TNF acts by stimulating cells other than the epithelial cells, resulting in the release of a substance which in turn decreases SCC and PD. However, no factors or substances released from the migrating cells (macrophages, monoeytes, lymphocytes or neutrophils) or constituent cells of the airway wall are known to inhibit bioelectrical phenomena of airway epithelium despite the presence of many stimulating substances or factors. Thirdly, TNF may directly suppress the opening of ion channels present at the apical cell membrane of epithelial cells. G decreased after TNF pretreatment in ISOP-evoked SCC and PD, suggesting a decrease in the opening ofion channels. TNF has little or no effects on Na + channels at the apical membrane, since amiloride did not alter the inhibitory action on ISOP-induced SCC and PD. However, the problem remains unresolved that the TNF-evoked inhibition on the opening of Na + channels was too small to be detected by the present method. Fourthly, TNF may directly affect cellular ion pumps or cotransporters which exist at the basolateral cell membrane without any alterations to the opening of ion channels at the apical cell membrane of airway epithelial cells, resulting in no changes in G. This possibility seems unlikely since G significantly decreased after TNF pretreatment in the ISOP-evoked experiment. In addition, it seems unlikely that TNF, with its high molecular weight, would penetrate the tight junction between epithelial cells and reach the ion pumps or cotransporters at the basolateral membrane. This also argues against an interaction between fl-adrenoreceptors at the basolateral membrane and TNF-receptors which must be on the apical membrane if the findings in the present study are correct. An interaction between fl-adrenergic and TNF receptors is also unlikely because TNF attenuated the increases in SCC and PD evoked by fl-agonist even after the maximum response by fl-agonist. Finally, this attenuation after the maximum response and the lack of any inhibitory action by amiloride suggests that TNF interacts with the intracellular process of CIsecretion by epithelial cells. A number of findings which indicate a receptor-mediated action of TNF and showing some inhibitory action have been reported, especially in tumor cells (Kull et al., 1985; Baglioni et al., 1985: Wise et al., 1990). For example, a recent experiment (Wise et al., 1990) indicated that TNF decreases surfactant secretion in a pulmonary adenocarcinoma cell, and that the inhibitory effects are associated with decreased surfactant apoprotein A - mRNA. Some interactions between the two major second messenger systems, cAMP and Ca 2 ÷, are known in other tissues and the presence of an antagonistic control in the interaction between the two second messengers may be responsible for the inhibitory action of TNF in the present study. Johnson and Baglioni (1988) have reported that the receptor-mediated activity of TNF is downregulated by activators of protein kinase C (Ca 2 +/phospholipid-dependent enzyme) in human HeLa cells and murine L(s)cells. The chloride secretion of airway epithelial cells is mediated by cyclic AMP as a second messenger and fl-agonists simulate CI - secretion
TNF AND BIOELECTRICAL PROPERTIES OF AIRWAY EPITHELIUM
385
by rising intracellular cAMP, which is degraded by the enzyme diphosphoesterase. It is possible that TNF activates this enzyme, resulting in faster degradation ofintracellular cyclic AMP. Of course, the cytotoxic action of TNF which is known in tumor cells is not responsible for the inhibitory: action obse~ed in the present study since the response to ISOP remained unchanged even after washing TNF out of the mucosal solution. A recent study (Mullin et ai., 1990) indicated that TNF affects the tight junction between cultured epithelial cells from pig kidney (LLC-PK 1 cells) with decreases in both transepithelial resistance and PD, thereby increasing the flow of solute between cells. The mechanism of TNF action in LLC-PK I cells appears to differ from that in canine tracheal epithelium since TNF produced a significant decrease in G (an increase in resistance) in the latter. In addition, TNF did not alter the baseline values of SCC, PD or G, suggesting that the cytotoxic action is not responsible for the inhibitory action of TNF observed in the present study. In conclusion, an intracellular interaction between TNF and r-stimulant is the most likely explanation of these observations although a direct inhibitory action against the opening of CI- channels may" in part play a role. It is possible that airway inflammation, the main aspect of chronic bronchitis (Thompson et al., 1989) or bronchial asthma, induces TNF production in the airways. For example, we have obtained some results which indicated TNF production by mast cells and basophiis with IgE receptor triggering (Ohno et al., 1990a,b). Since basophils and mast cells are thought to play a central role in the allergic reaction in bronchial asthma TNF production by these cells may be involved in the pathogenesJs of asthma. Mast cells and basophils are thought to initiate a bronchial response by releasing chemical mediators through lgE receptor triggering via inhaled antigen (Abraham et al., 1988). Further, ~-adrenergic agonists are widely used for airway dilatation in patients with bronchial asthma and chronic obstructive pulmonary disease. Beta-adrenergic stimulation is well known not only for its ability to dilate airways but also as a potent stimulus ofCi - secretion across the airway mucosa. Airway epithelia show considerable differences in ion transport between species and human epithelium is less potent in Cisecretion in response to ~-adrenergic stimulation than canine tracheal epithelium (Knowles et al., 1984; Widdicombe et al., 1985). In spite ofthis limitation in the present study, taken together with the findings described above, it can be proposed that the inflammation associated with bronchial asthma or chronic bronchitis induces the release of TNF, resulting in the attenuation ofthe effect of fl-agonist on chloride secretion with water into airway lumen, and in a decrease of mucociliary clearance. A recent report (isawa et ai., 1990) has described how inhalation of fl2-stimulant neither accelerates mucus transport nor facilitates mucociliary clearance in patients with various diseases including bronchial asthma. Meanwhile, a number of in vitro experiments (Iravani et al., 1974, Van As, 1974; Verdugo et al., 1980) have indicated that fl 2-stimulants accelerate mucociliary clearance. Our present finding that TNF attenuates //-stimulant-induced ion transport may be an explanation for the lack of any significant effect on mucociliary clearance in vivo in diseased lungs.
386
M. SATOH et al.
Acknowledgements. We gratefully acknowledge Professor A. Nishiyama and Dr. N. Saitoh for discussion a,~ld suggestion, Dr. Ronald Scott for reading, and Ms Yumi Saitoh for typing the manuscript. This study was supported by a Grant-in-Aid for General Scientific Research (No. 01570422) from the Japanese Ministry of Education, Science and Culture.
References Abraham, W. M., J. S. Stevenson, M. Eldridge, R. Gasrrido and L. Nieves (1988). Nedocromii sodium in allergen induced bronchial responses and airway hyperresponsiveness in allergic sheep. J. Appl. Physiol. 65: 1062-1068. Aderka, D., H. Holtmann, L. Toker, T. Hahn and D. Wailach (1986). Tumor necrosis factor induction by Sendai virus. J. Immunol. 136: 2938-2942. AI-Bazzaz, F., Y.P. Yadava and C. Westenfelder (1981). Modification of Na and C! transport in canine tracheal mucosa by prostaglandins. Am. J. Physiol. 240: Fl01-105. Baglioni, C., S. McCandless, J. Tavernier and W. Fiefs (1985). Binding ofhuman tumor factor to high affinity receptors on HeLa and iymphoblastoid cells sensitive to growth inhibition. J. Biol. Chem. 260: 13395-13397. Beutler, B., N. Krochin, I.W. Milsark, A. Goldberg and A. Cerami (1986). Induction of cachectin (tumor necrosis factor) synthesis by influenza virus: deficient production by endotoxin-resistant (C3H/HEJ) rnacrophages. Clin. Res. 34: 491A. Bianchard, D.K., J.Y. Djeu, T.W. Klein, H. Friedman and W.E. Stewart II (1987). Induction of tumor necrosis factor by Legionella pneumophila. Infect. lmmun. 55: 433-437. Brain, J. D., P. Gehr and R.l. Kavet (1984). Airway macrophages. The importance of the fixation method. Am. Rev. Respir. Dis. 129: 823-826. Cannistra, S.A., A. Rambaldi, D.R. Spriggs, F. Herrmann, D. Kufe and J.D. Griffin (1987). Human granulocyte-ma,~rophage colony-stimulating factor indllces expression ofthe tumor necrosis factor gene by the U937 cell line and by normal human monocytes. J. Clin. Invest. 79: 1720-1728. Cuturi, M. C., M. Murphy, M. P. Costa-Giomi, R. Weinmann, B. Perussia and G. Trinchieri (1987). Independent regulation of tumor necrosis factor and lymphotoxin production by human peripheral blood lymphocytes. J. Exp. M¢d. 165: 1581-1594. lkeda, K., T. Sasaki, S. Shimura, M. Satoh, H. lshihara, H. Sasaki, T. rakis|,.i,,a, ¥. Saitoh and A. Nishiyama (1990). Effect of surfactant on bioelectric properties of canine tracheal epithelium. Respir. Physiol. 81: 41-49. lravani, J. and G.N. Melville (1974). Mucociliary function of the respiratory tract as influenced by drugs. Respiration 31: 350-357. Isawa, T., T. Teshima, T. Hirano, Y. Anazawa, M. Miki, K. Konno and M. Motomiya (1990). Does a //2-stimulator really facilitate mucociliary transport in the auman lungs in vitro'?.A study with procaterol. Am. Rev. Respir. Dis. 141: 715-720. Johnson, S. E. and C. Baglioni (1988). Tumor necrosis factor receptors and cytocidai activity are down-regulated by activators of protein kinase C. J. Biol. Chem. 263: 5686-5692. Knowles, M., G. Murray, J. Shallal, F. Askin, V. Ranga, J. Gatzy and R. Boucher (1984). Bioelectric properties and ion flow across excised human bronchi. J. Appl. Physiol. 56: 368-877. Kull, F.C., S. Jacobs dnd P. Cuatrecasas (1985). Cellular receptor for ~251-1abeled tumor necrosis factor: Specific binding, affinity labeling, and relationship to sensit;-tity. Proc. Natl. Acad. Sci. USA 82: 5756-5760. Mullin, J.M. and K.V. Snook (1990). Effect of tumor necrosis factor on epithelial tight junctio.ns and transepithelial permeability. Cancer Res. 50:2172-2176. Ohno, !., T. Tanno, Y. Yamauchi and T. Takishima (1990a). Production of turnout necrosis factor by mastocytoma P815 cells. Immm~ology 69: 312-315. Ohno, I., T. Tanno, K. Yamauchi and T. Takishima (1990b). Gene expression and production of tumor
TNF AND BIOELECTRICAL PROPERTIES OF AIRWAY EPITHELIUM
387
necrosis factor by a rat basophilic leukemia cell line {RBL-2H3) with lgE receptor triggering. Immunology 70: 88-93. Olver, R.E., B. Davis, M.G. Marin and J.A. Nadei (1975). Active transport of Na ÷ and Cl- across the canine tracheal epithelium in vitro. Am. Rev. Respir. D/s. 122:811-815. Satoh, M., T. Sasaki, S. Shimura, I. Ohno, Y. Tanno, H. $asaki and T. Takishima (1989). Regulatory effect of TNF on bioelectrical properties of canine tracheal epithelium (abstract). Am. Rev. Respir. D/s. 139: A476. Thompson, A.B., D. Daughton, R.A. Robbins, M.A. Ghafouri, M. Oehlerking and S.I. Rennard (1989). intraluminai airway inflammation in chronic bronchitis: Characterization and correlation with clinical parameters. Am. Rev. Respir. Dis. 140: 1527-1537. Van As, A. (1974). The role of selective beta2-adrenoreceptor stimulants in the control of ciliary activity. Respiration 31 : 146-15 !. Verdugo, P., N. T. Johnson and P. Y. Taur (1980). Beta-adrenergic stimulation of respiratory ciliary activity. J. Appl. Physiol. 48: 868-871. Widdicombe, J.H., D.L. Coleman, W.E. Finkbeiner and I.K. Tuet (1985). Electrical properties of monolayers cultured from cells of human tracheal mucosa. J. Appl. Physiol. 58: 1729-1735. Wise, J., J. Clark, W. Hull, R. Holtzman and J. Whitsett (1990). Tumor necrosis factor • inhibits surfactantassociated protein A expression (abstract). Am. Rev. Respir. Dis. 141: A692.