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December 14, 1990

E N D O T H E L I N - 1 ENHANCES SUPEROXIDE G E N E R A T I O N OF HUMAN N E U T R O P H I L S S T I M U L A T E D BY T H E C H E M O T A C T I C P E P T I D E N-FORMYL-METHIONYL-LEUCYL-PHENYLALANINE Kiyoshi Ishida, Koichiro Takeshige and Shigeki Minakami Department of Biochemistry, Kyushu University School of Medicine, Fukuoka 812, Japan Received October 24, 1990 SUMMARY : Endothelin-1 (ET-1) by itself was not an effective stimulus for inducing the superoxide (02-) generation of human neutrophils, but it enhanced the 0 2- generation stimulated by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine(FMLP) about 2-fold when the cells had been preincubated with ET-1 for 10 min at 37 °C. The concentration at which ET-1 was 50% effective was l x l 0 -10 M, and the maximal effect was obtained at l x l 0 -8 M. The enhancement was observed over the range of the effective concentrations of FMLP (10-810 -6 M). ET-1 did not promote the mobilization of intracellular calcium ions and the enhancing effect of ET-1 did not change when calcium ions were depleted. These findings indicate that ET-1 is a potent modulator of human neutrophils and may thus contribute to the inflammatory process. 0 1 9 9 0 Academic Press, Inc.

Neutrophils participate in the inflammation reactions by generating superoxide anions (02-) when stimulated by platelet activating factor, tumor necrosis factor and chemotactic factors such as C5a and leukotriene B4 (1,2). Endothelin-1 (ET-1) is a potent vasoconstrictor peptide which was originally isolated from the culture supematant of porcine aortic endothelial cells (3). It is also produced in many other tissues, and it has numerous other bioactivities such as promoting the proliferation of vascular smooth muscle cells and mesangial cells and the inhibition of the renin release from isolated rat glomemli (4-6). Inflammatory mediators, thrombin and interleukin-1, have been shown to stimulate the endothelial biosynthesis of the peptide (7,8) and the neutrophil adhesion to the endothelium (9-12). We anticipated its effect on neutrophil functions including the 0 2- generation from these observations. In this paper, we show the effect of ET-1 on the 0 2- generation of human neutrophils stimulated by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine. ET-1 did not induce 0 2- generation by itself but enhanced the generation induced by the chemotactic peptide. Abbreviations used : ET-1, endothelin-1 ; FMLP, N-formyl-methionyl-leucyl-phenylalanine ; HEPES, N-2-hydroxythylpiperazine-N'-2-ethanesulfonic acid ; EGTA, ethyleneglycol bis(Baminoethyl ether)-N, N, N',N'-tetraacetic acid. 0006-291X/90 $1.50 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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M A T E R I A L S AND M E T H O D S

Chemicals : Endothelin-1 (ET-1) was purchased from Peptide Institute (Japan). Calciumionophores A23187 and ionomycin were obtakned from Calbiochem-Behring, San Diego, CA. Nformyl-methionyl-leucyl-pheny!alanine (FMLP) and ferricytochrome c were from Sigma Chemical Co., St. Louis. Other chemicals were of analytical grade. Preparation of neutrophiIs : Human neutrophils were isolated from fresh citrated blood by dextran sedimentation followed by Conray-Ficoll gradient centfifugation and hypotonic lysis of erythrocytes (13). The cells were suspended in a HEPES buffer containing 131 mM NaC1, 4.7 mM KC1, 5 mM glucose, and 20 mM HEPES (pH 7.4) and stored on ice until use. Ca 2+depleted cells were prepared as previously described (13). Neutrophils (lxl06 cells/ml) suspended in a divalent-cation-free modified Hanks salt solution were treated with 1 btM A 23187 and 0.1 mM EGTA for 5 min at 37 °C and then washed twice with the HEPES buffer. Measurement ofsuperoxide generation : The 02- production was monitored continuously by the superoxide dismutase-inhibitable reduction of ferricytochrome c at 550-540 nm using a dualwavelength spectrophotometer (Hitachi 557) as previously described (13). The neutrophils(lxl06 celts/ml) were preincubated in the HEPES buffer containing 1 mM CaC12 for 5 min at 37 oC and then treated with ET-1 for 10 min prior to the addition of FMLP. The production of 02- was calculated from reduced ferricytochrome c for 10 rain after the addition of FMLP.

RESULTS Endothelin-1 (ET-1) by itself did not induce the superoxide (02-) production of human neutrophils. However ET-1 enhanced the 02- production induced by the chemotactic peptide N-fonm.yi-methionyl-leucyl-phenylalanine (FMLP) when the neutrophils were preincubated with ET-1 for 10 rain at 37 °(2. Figure 1 shows the representative time courses of the FMLP-induced 02 - production. The initial rate and the total amounts of the 02 - production were both enhanced about 2-fold by ET-1. The production induced by phorbol 12-myristate 13-acetate was not affected by ET-1. The enhancement of the 0 2- production by ET-1 was dependent on the preincubation time and the preincubation of the cells with ET-1 for 10 min was necessary to obtain the maximal effect (Fig.2). ET-1 enhanced the 02- production induced by FMLP in a dosedependent manner, as shown in Fig.3. The maximal enhancement of the 02- production was observed at l x l 0 -8 M of ET-1, and the half effective concentration was l x l 0 -10 M. The

endothelin

+

endothelin

-

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H Fig.1. Time course of the FMLP-induced 02- production by human neutrophils pretreated with or without endothelin-1. Neutrophils (lxl06 cells/ml) were preincubated with ET-1 (lx 10-8 M) for 10 min at 37 °C and then l~eated with FMLP (lxl0 -7 M). The 02- production was measured as described in Materials and Methods. 497

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8

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Fig.2. The FMLP-induced 0 2- production of human neutrophils as a function of preincubation time with ET-1. Neutrophils (lxl06 cells/ml) were preincubated with ( • ) or without ( O ) ET-1 ( l x l 0 -8 M) for the indicated time at 37 oC and then treated with FMLP (lxl0 -7 M). The O 2- production was measured as described in Materials and Methods. Fig.3. Dose-dependence of ET-1 enhancement of FMLP-induced 02- production. Neutrophils (lxl06 cells/ml) were preincubated with ET-1 at various concentrations for 10 min at 37 °C and then treated with FMLP (lxl0 -7 M). The 02- production was measured as described in Materials and Methods. concentrations were essentially the same as those for the ET-1 effect on vascular contraction (3). Figure 4 shows the dose-response curves of F M L P for the 0 2- production of neutrophils pretreated with or without ET-1. ET-1 enhanced 0 2- production over the whole range of effective F M L P concentrations (10-8-10 -6 M). 8

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Fig.4. Dose-dependence on FMLP of the 02- production of neutrophils pretreated with ( • ) or without ( O ) ET-1. Neutrophils (lx106 cells/ml) were preincubated with or without ET-1 (lxl0 -8 M) for 10 min at 37 °C and then treated with FMLP at various concentrations. Fig.5. Effect of ET-1 on the FMLP-induced 0 2- production by Ca2+-depleted neutrophils. After preincubation of the Ca2+-depleted cells with or without ET-1 ( l x l 0 -8 M) for 10 min at 37 °C in the reaction mixture without Ca2+, the reaction was started by the addition of FMLP (lxl0 -7 M) and CaC12 (1 raM) (upper trace) or FMLP alone (lower trace). 498

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Because calcium ions are considered to be involved in the stimulation and regulation of neutrophil 0 2- production (1), neutrophils were depleted of calcium ions by treating the cells with the calcium-ionophore A 23187 in the presence of EGTA, and the effect of ET-1 on the 02production of the Ca2+-depleted cells induced by FMLP was investigated (Fig.5). The stimulation of the cells was performed by the addition of FMLP and lmM CaC12. Preincubation of the depleted cells with ET-1 also enhanced the 02- production about 2-fold, indicating that calcium ions are not necessary for the effect of ET-1. No 0 2- production was observed when the depleted cells were stimulated by FMLP without CaC12 in accord with the previous result (13). Because preincubation of the neutrophils with the calcium-ionophore ionomycin at a suboptimal concentration (10 nM) in the presence of extracellular calcium ions markediy enhances the 0 2production induced by FMLP (14), ET-1 and ionomycin may act in different ways. This is supported by the finding that the enhancement of the FMLP-induced 0 2- production by preincubation with both ET-1 and ionomycin essentially equaled the sum of the enhancement produced by ET-1 alone and that produced by ionophore alone (results not shown). When the intracellular free calcium concentration ([Ca2+]i) of neutrophils was measured by using a fluorescent probe, fura-2 (15), in the presence of lmM CaC12, ET-1 neither promoted changes in [Ca2+]i by itself nor enhanced the increase in [Ca2+]i induced by FMLP (results not shown).

DISCUSSION The results presented here show that ET-1 acts on neutrophils as well as vascular smooth muscle cells (3) and kidney ceils (5,6). The enhancement of the 02- production by ET-1 seems to be achieved by modulating the signal transduction pathway for the 0 2- production at a level between the FMLP-receptors and the protein kinase C because ET-1 enhanced the production induced by FMLP, a receptor-mediated agonist, without affecting changes in [Ca2+]i but did not enhance that by phorbol 12-myristate 13-acetate, an agonist that bypasses the receptors and directly activates the protein kinase (1). Kitagawa et al. (16) and Weisbart et al. (17) have reported similar priming effects of granulocyte and granulocyte-macrophage colony-stimulating factors on human neutrophils and the latter factor enhances the FMLP-induced 02- production without effects on changes in [Ca2+] i but causes an enhanced production of diacylglycerol (18), an activator of protein kinase C, which is mediated by phospholipase D (19). The enhancement of the 02- production of neutrophils by ET-1 suggests that the peptide participates in inflammation reactions. Nagata et al. have reported that ET-1 stimulates the release of 15-hydroxyeicosatetraenoic acid and oxygen radicals in the distal rat lung (20). Functions of other inflammatory cells such as monocytes (21) and platelets (22) are not affected by ET-1.

ACKNOWLEDGMENT This work was supported by a grant from the Ministry of Education, Science and Culture, Japan. 499

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REFERENCES 1. Rossi, F. (1986) Biochim. Biophys. Acta. 853, 65-89 2. Yuo, A., Kitagawa, S., Suzuki, I., Urabe, A., Okabe, T., Saito, M. and Takaku, F. (1989) J. ImmunoI. 142, 1678-1684 3. Yanagisawa,M., Kurihara,H., Kimura,Y., Tomobe,Y., Kobayashi,M., Mitsui,Y., Yazaki,Y., Goto,K.and Masaki,T.(1988) Nature. 332,411-415 4. MacCumber, M. W., Ross, C. A., Glaser, B. M. (1989) Proc. Natl. Acad. Sci. USA 86, 7285-7289 5. Yanagisawa, M. and Masaki, T. (1989) Biochem. Pharmacol. 38, 1877-1883 6. Yanagisawa, M. and Masaki, T. (1989) Trends. Pharmacol. Sci. 10, 374-378 7. Moon, D. G., Horgan, M. J., Andersen, T. T., Krystek, Jr., S. R., Fenton, II, J. W. and Malic, A. B. (1989) Proc. Natl. Acad. Sci. USA 86, 9529-9533 8. Yoshizumi, M., Kurihara, H., Morita, T., Yamashita, T., Oh-hashi, Y., Sugiyama, T., Takaku, F., Yanagisawa, M., Masaki, T. and Yazaki, Y. (1990) Biochem. Biophys. Res. Commun. 166, 324-329 9. Zimmerman, G. A., Mclntyre, T. M. and Prescott, S. M. (1985) J. Clin. Invest. 76, 22352246 10. Toothill, V. J., Mourik, J. A. V., Niewenhuis, H. K., Metzelaar, M. J. and Pearson, J. D. (1990) J. Immunol. 145, 283-291 11. Bevilacqua, M. P., S. Stengelin, M. A. Gimbrone,Jr. and b. Seed. (1989) Science. 243, 1160-1163 12. Osborn, L. (1990) Cell. 62, 3-6 13. Nakagawara, M., Takeshige, K., Sumimoto, H., Yoshitake, J. and Minakami, S. (1984) Biochim. Biophys. Acta. 805, 97-103 14. Finkel, T. H., Pabst, M. J., Suzuki, H., Guthrie, L. A., Forehand, J. R., Phillips, W. A. and Johnson, R. B. (1987) J. Biol. Chem. 262, 12589-12596 15. Kuroki, M., Takeshige, K. and Minakami, S. (1989) Biochim. Biophys. Acta. 1012, 103106 16. Kitagawa, S., Yuo, A., Souza, L. M., Saito, M., Miura, Y. and Takaku. (1987) Biochem. Biophys. Res. Commun. 144, 1143-1146 17. Weisbart, R. H., Golde, D. W., Clark, S. C., Wong, G. G. and Gasson, J. C. (1985) Nature. 314, 361-363 18. Tyagi, S. R., Winton, E. F. and Lambeth, J. D. (1989) FEBS Lett. 257, 191-195 19. Bourgoin, S., Plante, E., Gaudry, M., Maccache, P. H., Borgeat, P. and Poubelle, P. E. (1990) J. Exp. Med. 172, 767-777 20. Nagase, T., Fukuchi, Y., Jo, C., Teramoto, S., Uejima, N., Ishida, K., Shimizu, T. and O1imo, H. (1990) Biochem. Biophys. Res. Commun. 168, 485-489 21. Bath, PM. W., Mayston, S. A. and Martin, J. F. (1990) Exp. Cell. Res. 187, 339-342 22. Ohlstein, E. H., Storer, B., Nambi, P., Given, M. and Lippton, H. (1990) Thromb. Res. 57,967-974

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