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Regulation of endothelin-1 synthesis in cultured guinea pig airway epithelial cells by various cytokines
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1594-1599
August 14, 1992
REGULATION OF ENDOTHELIN-1 SYNTHESIS IN CULTURED GUINEA PIG AIRWAY EPITHELIAL CELLS BY VARIOUS CYTOKINES Takeo ENDO*t, Yoshiyuki UCHIDAt, Hirokazu MATSUMOTO~, Nobuhiro SUZUKg, Akihiro NOMURAt, Fusao HIRATAt § and Shizuo HASEGAWA* *Division of Pulmonary Medicine, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki 305 Japan +Tsukuba Research Laboratories, Takeda Chemical Industries Ltd., Tsukuba, Ibaraki 300-42 Japan t Departments of Pharmaceutical Sciences and Pharmacology, and Institute of Chemical Toxicology, Wayne State University, Detroit, MI 48202 Received July 9, 1992
To study regulatory mechanisms influencing the synthesis and release of ET-1, a potent bronchoconstrictor, epithelial cells from guinea pig tracheas were cultured to test various cytokines for the synthesis of ET-1 and its precursor, big ET-1. Cytokines tested were divided into 4 groups, based on their potential modes of action. IL-8, TNF~ and TGFB transiently increased the synthesis of ET-1, while EGF, PDGF and GM/CSF promoted proliferation of ET-1 synthesizing cells. IL-1 enhanced the synthesis of ET-1 precursor without mitogenesis, whereas IL-2, IL-6 and IGF-1 induced both the synthesis of big ET1 and mitogenesis. These observations suggest that cytokines involved in damage, inflammation and repair of the airway epithelial layer regulate the synthesis and release of ET-1 by multiple mechanisms, thereby influencing airway muscle tone. ®1992Aoademo Press, Inc.
Endothelin(ET)-I is a member of a novel peptide family with a potent constrictive action on both vascular and nonvascular smooth muscle (1, 2). In the respiratory system, ET-1 is synthesized not only in vascular endothelial cells but also in airway epithelial cells, and is proposed to act on nearby smooth muscle in a paracrine fashion (3, 4). Since the ET-1 level in bronchoalveolar lavage fluids from asthmatic patients parallels the severity of symptoms, it has been postulated that ET-1 plays an important role in tuning airway smooth muscle tone (5). In order to further understand the pathophysiological role of ET in airway diseases, elucidation of the hormonal regulation of ET synthesis and release would be essential. In this report, we tested the effect of various cytokines including growth factors and proiuflammatory cytokines on the §To whom correspondence should be addressed at Department of Pharmaceutical Sciences, Wayne State University, 528 Shapero Hall, Detroit, MI 48202.
1594
0006-291X/92 $4.00 Copyright © 1992 by Academic Press, Inc. All rights" of reproduction in any form reserved.
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
synthesis of ET-1 and its precursor, big ET-1, in cultured airway epithelial cells of guinea pigs. We found that IGF-1, IL-2 and IL-6 stimulate both the synthesis of ET-1 and cell proliferation, while IL-1 increases the synthesis of ET-1 without stimulating mitogenesis. Further, GM/CSF, EGF and PDGF enhanced the proliferation of ET-1 containing cells, whereas IL-8, TNFa and TGFB transiently increased the synthesis of ET-1. Our present observations suggest that ET-1 synthesis in airway epithelial cells, which are vulnerable to various chemical and immunological injury, is regulated in various ways by cytokines involved in injury, inflammation and repair processes of the epithelial layer. MATERIALS AND METHODS Epithelial cell culture: Guinea pig tracheal epithelial cells were isolated as described previously (6), and were cultured in Dulbecco's modified Eagles' medium(DMEM)/F12 (Ham) medium (1/1, v/v), supplemented with 5% FCS in collagen-coated 24 well (16 mm diameter) plates (Nunc, Roskilde, Denmark). Airway epithelial cells were seeded at 2.5 x 105 cells/well, and after 5 days culture, almost confluent dishes were used for all experiments. The medium was then changed to a DMEM/F12(Ham) mixture (1/1, v/v) supplemented with 50 U/ml penicillin, 50 ~g/ml streptomycin, 2.5 #g/ml amphotericin B and 50 ug/ml gentamicin without serum, and cells were cultured for another 3 days to obtain a steady basal level of ET-1 synthesis. Epithelial cells were, then, cultured with various cytokines for 24 hr, and after the medium was separated, cells were washed twice with phosphate buffered saline (10 mM sodium phosphate buffer, pH 7.4, containing 150 mM NaC1 ; PBS). Cells were detached by 10 min digestion at room temperature with a 0.25% Trypsin/1 mM EDTA solution. An equal volume of DMEM/F12 medium containing 5% FCS was added, and cells were collected by centrifugation at 1,000 x g for 10 min at 4° C. Cell viability was more than 97% as determined by trypan blue exclusion test. In order to measure thymidine(TdR) uptake, i uCi/ml fH]TdR was added to a culture medium together with cytokines, and cells were harvested 24 hr later. After washing 3 times with ice cold PBS, cells were treated with ice cold 5% trichloroacetic acid(TCA). After washing cells 3 times with 5% TCA, the cells were solubilized with 0.5 N NaOH, and were neutralized with 0.5 N HC1, for radioactivity measurement. Enzyme linked immunoassays of ET-I: Cell pellets were sonicated in 0.5 ml of 50% acetonitrile containing 0.1% trifluoroacetic acid(TFA) for 30 sec and were centrifuged at 1,000 x g at 4°C for 10 min. A medium (1 ml each) was centrifuged at 10,000 x g at 4° C for 10 min and supernatants were used for the assay. Samples were analyzed by the enzyme linked immunoassay for ET-1 and big ET-1 as described previously (7). The minimal sensitivity for ET-1 or big ET-1 detection was 0.2 pg/well. RESULTS Effects of various cytokines on the extracellular level of ET-I: Cultured guinea pig tracheal epithelial cells which were nearly confluent, linearly released ET-1 up to 24 hr. Cytokines tested (human recombinant IL-I~ and g, IL-2, IL-6, IL-8, TNFa, TGFB, PDGF, IGF-1, EGF, and GM/CSF) increased the total amount of ET-1 released in the medium after the 24 hr culture of tracheal epithelial cells. When the amount of ET-1 in a well was corrected for the number of cells (105 cells), IL-1B, IL-6, IL-2 and IGF-1 1595
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
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Figure 1. Effect of various cytokines on release of ET-1 from cultured guinea pig tracheal epithelial cells. Guinea pig tracheal epithelial cells were cultured in serum free DMEM/F12(Ham)(1/1, v/v) medium for 3 days, and various cytokines were added to 6 wells each at a final concentration of 10 ng/ml. After 24 hr of culture, a medium was separated and ET-1 content was measured as described in the text. (* P < 0.05 vs control; ** P < 0.01 vs control.) Figure 2. Effect of various cytokines on cell number of cultured epithelial cells. Guinea pig tracheal epithelial cells were cultured and treated as described in the legend for Fig. 1. After isolation of cells by digestion with a Trypsin-EDTA solution, cells were counted in a hemocytometer. Cell viability was more than 97% by trypan blue exclusion test. TdR uptake was measured as described in the text. (* P < 0.05; ** P < 0.01.)
were found to increase the release of ET-1 (Fig. 1). IL-8, TNF~ and T G F g transiently increased the secretion of ET-1 for up to 6 hr of culture, but thereafter, ET-1 release declined (data not shown). Thus, the total amount of ET-1 released by these cytokines was not significantly increased after 24 hr culture. EGF, P D G F and G M / C S F rather decreased the amounts of ET-1 released per 10~ cells, suggesting that the increased release of ET-1 by these cytokines was more attributable to the increased cell number. The cellular proliferation by these cytokines was further substantiated by the incorporation of [~H]TdR into cells (Fig. 2). In addition, IGF-1, IL-2 and IL-6 also increased TdR uptake under the present experimental conditions. Effects of various cytokines on the synthesis of big ET-I: In order to study the synthesis of ET-1, we measured the level of big ET-1, a precursor of ET-1, in cultured epithelial cells after the 24 hr treatment with various cytokines (Fig. 3). A large increase in the cellular level of big ET-1 was observed with GM/CSF, IGF-1, EGF, IL-1, IL-2, IL-6 or IL-8. Since the levels of big ET-1 in the medium were not significantly altered (1.47 _+ 0.04 pg/ml), these observations suggest either that these cytokines stimulated the synthesis of ET-1 or that they inhibited the conversion of big ET-1 to ET-1. The stimulatory effect of TNFa, TGFI3 or P D G F on the level of big ET-1 was also transient (data not shown). Effects of various cytokines on intracellular level of ET-I: All cytokines tested decreased the intracellular level Of ET-1 (Fig. 4). A large decrease was observed with G M / 1596
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
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Figure 3. Effect of various cytokines on synthesis of big ET-1. Big ET-1 contents of cell pellets obtained as described in the legend for Fig. 2 were measured by the enzymeliked immunoassay with an antipeptide rabbit antibody specific for big ET-1 but not for ET-1. Level of big ET-1 in the medium was less than 1.5 pg/ml. (* P < 0.05; ** P <
0.01.)
Figure 4. Effect of various cytoldnes on synthesis of ET-1 in cultured epithelial cells. Guinea pig tracheal epithelial cells were cultured and treated as described in the legend for Fig. 2, ET-1 content of cell pellets was measured as described in the text. (* P < 0.05; ** P < 0.01.)
CSF, EGF, or PDGF, while IL-1, IL-8 or IGF-1 moderately decreased the intracellular level of ET-1. TNF~, TGFI3, IL-6 and IL-2 did not significantly change the intracellular level of ET-1. A decrease in the intracellular level of ET-1 did not account for an increased amount of ET-1 released into the media, suggesting that ET-1 is not stored within cells (data not shown). DISCUSSION In this communication, we demonstrated that various cytokines can affect the synthesis and release of ET-1 in guinea pig cultured airway epithelial cells by multiple mechanisms. IL-1 and IL-6, typical proinflammatory cytokines (8, 9), stimulated the big E T synthesis, thus releasing ET-1 into the medium. On the other hand, GM/CSF, EGF, PDGF, IGF-1, IL-2 and IL-6 were mitogenic in airway epithelial cells as judged by increased cell number or TdR uptake. Among these cytokines, EGF, G M / C S F and P D G F are thought to be competence factors, while IGF-1 is a progression factor (10, 11). Since the effect of IL-2 and ILo6 on epithelial cells was similar to that of IGF-1, these cytokines may act as progression factors for airway epithelial cells. The precise mechanism(s) of action of these cytokines on epithelial proliferation is currently under investigation. Interestingly, competence factors stimulated the release of ET-1 mainly by increasing number of ET-1 producing cells, Since GM/CSF, and E G F markedly decreased the intracellular content of ET-1 and increased the big ET-1 content, it is likely that the conversion of big ET-1 to ET-1 becomes rate limiting in cells treated with 1597
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
these cytokines. On the other hand, progression factors had a modest mitogenic activity on airway epithelials, and increased big ET-1 content without much decrease in the intracellular level of ET-1, suggesting that these cytokines enhance the synthesis of big ET-1 followed by the release of ET-1. TGFg and TNFa have been demonstrated to stimulate the transcription of ET-1 gene in amnion cells and endothelial cells (12, 13). However, the increased synthesis of ET-1 induced by these cytokines including IL-8 was transient in airway epithelial cells. This suggests that these cytoldnes might destabilize the ET-1 message (12). Recent analysis of human ET-1 gene promoter in endothelial cells has revealed that two regions, A and B, are important for constitutive expression of this gene (13, 14, 15). Region A contains GATA motifs, while Region B contains an AP-1 like sequence. These two regions of the ET-1 promoter permit tissue specific expression. Therefore, the regulation of ET-1 gene expression in epithelial cells by various cytokines may be attributable to the expression of nucleic factors which cytokines activate within the cells. Our results suggest that the four cytokine groups may regulate ET-1 gene expression via different nucleic factors, which bind to different sites of the promoter regions. How these factors interact with each others will facilitate comprehension of the mechanism(s) for regulation of ET-1 synthesis in tracheal epithelial cells. The status of epithelial layers affects control of the bronchial tonus. Damage and injury of epithelial layers are followed by inflammation and repair (10). All these processes are regulated by complicated cytokine networks. Since the cytokines tested in this study are proposed to be involved in these processes, the enhanced synthesis and release of ET-1 in epithelial cells by these c3,tokines may take place in vivo. Since ET-1 is one of the most potent bronchoconstrictors (1, 2), ET-1 may be partly, if not totally, responsible for the bronchoconstriction often seen after epithelial damage elicited by exposure to chemicals and antigens (1, 2). In order to substantiate our interpretation, we are now investigating the in vivo synthesis of ET-1 in tracheal epithelial cells of animals, following exposure to aerosolized antigen. Our preliminary results demonstrate that newly regenerated epithelial cells following epithelial damage by immunochallenge are strongly positive by the immunochemical staining of ET-1.
ACKNOWLEDGMENT The authors express their gratitude to Ms. Aiko Hirata for typing and editing the manuscript. They also thank to Dr. Robert T. Louis-Ferdinand for reviewing the manuscript. This study was supported in part by grants from NIH (ES 04802 to F.H.) and the Ministry of Education, Science and Culture of Japan (to S.H.). 1598
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
Simonson, M. S., and Dunn, M. J. (1990) FASEB J. 4, 2989-3000. Uchida, Y., Ninomiya, H., Saotome, M., Nomura, A., Ohtsuka, M., Yanagisawa, M., Goto, K., Masaki, T., and Hasegawa, S, (1988) Eur. J. Pharmacol. 154, 227228. MacCumber, M. W., Ross, C. A., Glaser, B. M., and Snyder, S. H. (1989) Proc. Natl. Acad. Sci. USA 86, 7285-7289. Mattoli, S., Mezzetti, M., Riva, G., Allegra, L., and Fasoli, A. (1990) Am. J. Respir. Cell Mol. Biol. 3, 145-151. Nomura, A., Uchida, Y., Kameyama, M., Saotome, M., Oki, K., and Hasegawa, S. (1989) The Lancet, 747-748. Wu, R. C., and Smith, D. (1982) In Vitro 18, 800-812. Suzuki, N., Matsumoto, H., Kitada, C., Masaki, T., and Fujino, M. (1989) J. Immunol. Method 118, 245-250. Kelley, J. (1990) Am. Rev. Respir. Dis. 141, 765-772. Hamblin, A. S. (1991) Ann. NY Acad. Sci. 629, 250-261. Jetten, A. M., Vollberg, T. M., Nervi, C., and George, M. D. (1990) Am. Rev. Respir. Dis. 142, $36-$39. Aaronson, S. A., Rubin, J. S., Finch, P. W., Wong, J., Marchese, C., Falco, J., Taylor, W. G., and Kraus, M. H. (1990) Am. Rev. Respir. Dis. 142, $7-S10. Casey, M. L., Word, R. A., and MacDonald, P. C. (1991) J. Biol. Chem. 266, 5762-5768. Lee, M.- E., Dhadly, M. S., Temizer, D. H., Clifford, J. A., Yoshizumi, M., and Quertermous, T. (1991) J. Biol. Chem. 266, 19034-19039. Lee, M.-E., Bloch, K. D., Clifford, J. A., and Quertermous, T. (1990) J. Biol. Chem. 265, 10446-10450. Oliver, F.J., de la Rubia, G., Feener, E. P., Lee, M.-E., Loeken, M. R., Shiba, T., Quertermous, T., and King, G. L. (1991) J. Biol. Chem. 266, 23251-23256.
1599
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1594-1599
August 14, 1992
REGULATION OF ENDOTHELIN-1 SYNTHESIS IN CULTURED GUINEA PIG AIRWAY EPITHELIAL CELLS BY VARIOUS CYTOKINES Takeo ENDO*t, Yoshiyuki UCHIDAt, Hirokazu MATSUMOTO~, Nobuhiro SUZUKg, Akihiro NOMURAt, Fusao HIRATAt § and Shizuo HASEGAWA* *Division of Pulmonary Medicine, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki 305 Japan +Tsukuba Research Laboratories, Takeda Chemical Industries Ltd., Tsukuba, Ibaraki 300-42 Japan t Departments of Pharmaceutical Sciences and Pharmacology, and Institute of Chemical Toxicology, Wayne State University, Detroit, MI 48202 Received July 9, 1992
To study regulatory mechanisms influencing the synthesis and release of ET-1, a potent bronchoconstrictor, epithelial cells from guinea pig tracheas were cultured to test various cytokines for the synthesis of ET-1 and its precursor, big ET-1. Cytokines tested were divided into 4 groups, based on their potential modes of action. IL-8, TNF~ and TGFB transiently increased the synthesis of ET-1, while EGF, PDGF and GM/CSF promoted proliferation of ET-1 synthesizing cells. IL-1 enhanced the synthesis of ET-1 precursor without mitogenesis, whereas IL-2, IL-6 and IGF-1 induced both the synthesis of big ET1 and mitogenesis. These observations suggest that cytokines involved in damage, inflammation and repair of the airway epithelial layer regulate the synthesis and release of ET-1 by multiple mechanisms, thereby influencing airway muscle tone. ®1992Aoademo Press, Inc.
Endothelin(ET)-I is a member of a novel peptide family with a potent constrictive action on both vascular and nonvascular smooth muscle (1, 2). In the respiratory system, ET-1 is synthesized not only in vascular endothelial cells but also in airway epithelial cells, and is proposed to act on nearby smooth muscle in a paracrine fashion (3, 4). Since the ET-1 level in bronchoalveolar lavage fluids from asthmatic patients parallels the severity of symptoms, it has been postulated that ET-1 plays an important role in tuning airway smooth muscle tone (5). In order to further understand the pathophysiological role of ET in airway diseases, elucidation of the hormonal regulation of ET synthesis and release would be essential. In this report, we tested the effect of various cytokines including growth factors and proiuflammatory cytokines on the §To whom correspondence should be addressed at Department of Pharmaceutical Sciences, Wayne State University, 528 Shapero Hall, Detroit, MI 48202.
1594
0006-291X/92 $4.00 Copyright © 1992 by Academic Press, Inc. All rights" of reproduction in any form reserved.
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
synthesis of ET-1 and its precursor, big ET-1, in cultured airway epithelial cells of guinea pigs. We found that IGF-1, IL-2 and IL-6 stimulate both the synthesis of ET-1 and cell proliferation, while IL-1 increases the synthesis of ET-1 without stimulating mitogenesis. Further, GM/CSF, EGF and PDGF enhanced the proliferation of ET-1 containing cells, whereas IL-8, TNFa and TGFB transiently increased the synthesis of ET-1. Our present observations suggest that ET-1 synthesis in airway epithelial cells, which are vulnerable to various chemical and immunological injury, is regulated in various ways by cytokines involved in injury, inflammation and repair processes of the epithelial layer. MATERIALS AND METHODS Epithelial cell culture: Guinea pig tracheal epithelial cells were isolated as described previously (6), and were cultured in Dulbecco's modified Eagles' medium(DMEM)/F12 (Ham) medium (1/1, v/v), supplemented with 5% FCS in collagen-coated 24 well (16 mm diameter) plates (Nunc, Roskilde, Denmark). Airway epithelial cells were seeded at 2.5 x 105 cells/well, and after 5 days culture, almost confluent dishes were used for all experiments. The medium was then changed to a DMEM/F12(Ham) mixture (1/1, v/v) supplemented with 50 U/ml penicillin, 50 ~g/ml streptomycin, 2.5 #g/ml amphotericin B and 50 ug/ml gentamicin without serum, and cells were cultured for another 3 days to obtain a steady basal level of ET-1 synthesis. Epithelial cells were, then, cultured with various cytokines for 24 hr, and after the medium was separated, cells were washed twice with phosphate buffered saline (10 mM sodium phosphate buffer, pH 7.4, containing 150 mM NaC1 ; PBS). Cells were detached by 10 min digestion at room temperature with a 0.25% Trypsin/1 mM EDTA solution. An equal volume of DMEM/F12 medium containing 5% FCS was added, and cells were collected by centrifugation at 1,000 x g for 10 min at 4° C. Cell viability was more than 97% as determined by trypan blue exclusion test. In order to measure thymidine(TdR) uptake, i uCi/ml fH]TdR was added to a culture medium together with cytokines, and cells were harvested 24 hr later. After washing 3 times with ice cold PBS, cells were treated with ice cold 5% trichloroacetic acid(TCA). After washing cells 3 times with 5% TCA, the cells were solubilized with 0.5 N NaOH, and were neutralized with 0.5 N HC1, for radioactivity measurement. Enzyme linked immunoassays of ET-I: Cell pellets were sonicated in 0.5 ml of 50% acetonitrile containing 0.1% trifluoroacetic acid(TFA) for 30 sec and were centrifuged at 1,000 x g at 4°C for 10 min. A medium (1 ml each) was centrifuged at 10,000 x g at 4° C for 10 min and supernatants were used for the assay. Samples were analyzed by the enzyme linked immunoassay for ET-1 and big ET-1 as described previously (7). The minimal sensitivity for ET-1 or big ET-1 detection was 0.2 pg/well. RESULTS Effects of various cytokines on the extracellular level of ET-I: Cultured guinea pig tracheal epithelial cells which were nearly confluent, linearly released ET-1 up to 24 hr. Cytokines tested (human recombinant IL-I~ and g, IL-2, IL-6, IL-8, TNFa, TGFB, PDGF, IGF-1, EGF, and GM/CSF) increased the total amount of ET-1 released in the medium after the 24 hr culture of tracheal epithelial cells. When the amount of ET-1 in a well was corrected for the number of cells (105 cells), IL-1B, IL-6, IL-2 and IGF-1 1595
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
250 •
[
20o-
3.0
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Figure 1. Effect of various cytokines on release of ET-1 from cultured guinea pig tracheal epithelial cells. Guinea pig tracheal epithelial cells were cultured in serum free DMEM/F12(Ham)(1/1, v/v) medium for 3 days, and various cytokines were added to 6 wells each at a final concentration of 10 ng/ml. After 24 hr of culture, a medium was separated and ET-1 content was measured as described in the text. (* P < 0.05 vs control; ** P < 0.01 vs control.) Figure 2. Effect of various cytokines on cell number of cultured epithelial cells. Guinea pig tracheal epithelial cells were cultured and treated as described in the legend for Fig. 1. After isolation of cells by digestion with a Trypsin-EDTA solution, cells were counted in a hemocytometer. Cell viability was more than 97% by trypan blue exclusion test. TdR uptake was measured as described in the text. (* P < 0.05; ** P < 0.01.)
were found to increase the release of ET-1 (Fig. 1). IL-8, TNF~ and T G F g transiently increased the secretion of ET-1 for up to 6 hr of culture, but thereafter, ET-1 release declined (data not shown). Thus, the total amount of ET-1 released by these cytokines was not significantly increased after 24 hr culture. EGF, P D G F and G M / C S F rather decreased the amounts of ET-1 released per 10~ cells, suggesting that the increased release of ET-1 by these cytokines was more attributable to the increased cell number. The cellular proliferation by these cytokines was further substantiated by the incorporation of [~H]TdR into cells (Fig. 2). In addition, IGF-1, IL-2 and IL-6 also increased TdR uptake under the present experimental conditions. Effects of various cytokines on the synthesis of big ET-I: In order to study the synthesis of ET-1, we measured the level of big ET-1, a precursor of ET-1, in cultured epithelial cells after the 24 hr treatment with various cytokines (Fig. 3). A large increase in the cellular level of big ET-1 was observed with GM/CSF, IGF-1, EGF, IL-1, IL-2, IL-6 or IL-8. Since the levels of big ET-1 in the medium were not significantly altered (1.47 _+ 0.04 pg/ml), these observations suggest either that these cytokines stimulated the synthesis of ET-1 or that they inhibited the conversion of big ET-1 to ET-1. The stimulatory effect of TNFa, TGFI3 or P D G F on the level of big ET-1 was also transient (data not shown). Effects of various cytokines on intracellular level of ET-I: All cytokines tested decreased the intracellular level Of ET-1 (Fig. 4). A large decrease was observed with G M / 1596
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
6-
80
~,
$,
I-,,,~
o
~iii
60
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Figure 3. Effect of various cytokines on synthesis of big ET-1. Big ET-1 contents of cell pellets obtained as described in the legend for Fig. 2 were measured by the enzymeliked immunoassay with an antipeptide rabbit antibody specific for big ET-1 but not for ET-1. Level of big ET-1 in the medium was less than 1.5 pg/ml. (* P < 0.05; ** P <
0.01.)
Figure 4. Effect of various cytoldnes on synthesis of ET-1 in cultured epithelial cells. Guinea pig tracheal epithelial cells were cultured and treated as described in the legend for Fig. 2, ET-1 content of cell pellets was measured as described in the text. (* P < 0.05; ** P < 0.01.)
CSF, EGF, or PDGF, while IL-1, IL-8 or IGF-1 moderately decreased the intracellular level of ET-1. TNF~, TGFI3, IL-6 and IL-2 did not significantly change the intracellular level of ET-1. A decrease in the intracellular level of ET-1 did not account for an increased amount of ET-1 released into the media, suggesting that ET-1 is not stored within cells (data not shown). DISCUSSION In this communication, we demonstrated that various cytokines can affect the synthesis and release of ET-1 in guinea pig cultured airway epithelial cells by multiple mechanisms. IL-1 and IL-6, typical proinflammatory cytokines (8, 9), stimulated the big E T synthesis, thus releasing ET-1 into the medium. On the other hand, GM/CSF, EGF, PDGF, IGF-1, IL-2 and IL-6 were mitogenic in airway epithelial cells as judged by increased cell number or TdR uptake. Among these cytokines, EGF, G M / C S F and P D G F are thought to be competence factors, while IGF-1 is a progression factor (10, 11). Since the effect of IL-2 and ILo6 on epithelial cells was similar to that of IGF-1, these cytokines may act as progression factors for airway epithelial cells. The precise mechanism(s) of action of these cytokines on epithelial proliferation is currently under investigation. Interestingly, competence factors stimulated the release of ET-1 mainly by increasing number of ET-1 producing cells, Since GM/CSF, and E G F markedly decreased the intracellular content of ET-1 and increased the big ET-1 content, it is likely that the conversion of big ET-1 to ET-1 becomes rate limiting in cells treated with 1597
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
these cytokines. On the other hand, progression factors had a modest mitogenic activity on airway epithelials, and increased big ET-1 content without much decrease in the intracellular level of ET-1, suggesting that these cytokines enhance the synthesis of big ET-1 followed by the release of ET-1. TGFg and TNFa have been demonstrated to stimulate the transcription of ET-1 gene in amnion cells and endothelial cells (12, 13). However, the increased synthesis of ET-1 induced by these cytokines including IL-8 was transient in airway epithelial cells. This suggests that these cytoldnes might destabilize the ET-1 message (12). Recent analysis of human ET-1 gene promoter in endothelial cells has revealed that two regions, A and B, are important for constitutive expression of this gene (13, 14, 15). Region A contains GATA motifs, while Region B contains an AP-1 like sequence. These two regions of the ET-1 promoter permit tissue specific expression. Therefore, the regulation of ET-1 gene expression in epithelial cells by various cytokines may be attributable to the expression of nucleic factors which cytokines activate within the cells. Our results suggest that the four cytokine groups may regulate ET-1 gene expression via different nucleic factors, which bind to different sites of the promoter regions. How these factors interact with each others will facilitate comprehension of the mechanism(s) for regulation of ET-1 synthesis in tracheal epithelial cells. The status of epithelial layers affects control of the bronchial tonus. Damage and injury of epithelial layers are followed by inflammation and repair (10). All these processes are regulated by complicated cytokine networks. Since the cytokines tested in this study are proposed to be involved in these processes, the enhanced synthesis and release of ET-1 in epithelial cells by these c3,tokines may take place in vivo. Since ET-1 is one of the most potent bronchoconstrictors (1, 2), ET-1 may be partly, if not totally, responsible for the bronchoconstriction often seen after epithelial damage elicited by exposure to chemicals and antigens (1, 2). In order to substantiate our interpretation, we are now investigating the in vivo synthesis of ET-1 in tracheal epithelial cells of animals, following exposure to aerosolized antigen. Our preliminary results demonstrate that newly regenerated epithelial cells following epithelial damage by immunochallenge are strongly positive by the immunochemical staining of ET-1.
ACKNOWLEDGMENT The authors express their gratitude to Ms. Aiko Hirata for typing and editing the manuscript. They also thank to Dr. Robert T. Louis-Ferdinand for reviewing the manuscript. This study was supported in part by grants from NIH (ES 04802 to F.H.) and the Ministry of Education, Science and Culture of Japan (to S.H.). 1598
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
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