Vol.
176,
May
15, 1991
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
Pages
RECEPTOR-OPERATED ACTIVATION OF POLYMORPHONUCLEAR DIFFERENT EFFECTS OF NAP-l/IL-S AND fMET-LEU-PHE
Urs Wirthmueller*‘l
March
972-978
LEUKOCYTES: OR C5a
, Marco Baggiolini #, Alain L. de Week” and Clemens A. Dahinden*
*Institute of Clinical Immunology,
Received
COMMUNICATIONS
20,
and #Theodor Kocher Institute, University of Bern, 3010 Bern, Switzerland
1991
Summary - We examined the production
of PAF and LTB4 by PMN in response to NAP~/IL-8 alone, or after preincubation with GM-CSF, which has been shown to enhance PMN responsiveness and to prime PMN for production of those bioactive lipids. NAP-~/IL-~ does not induce the synthesis of PAF and LTB4 from endogenous phospholipid precursors, even after preincubation with GM-CSF. In addition and again in contrast to fMLP and C5a, NAP-l/IL-8 fails to induce an enhanced oxidative burst in GM-CSF primed PMN. Exogenously added PAF or LTB4 can mimic the priming effect of GM-CSF for an enhanced oxidative burst in response to all examined chemotactic peptides including NAPl/IL-g. Our data reveal a possible autocrine role of PAF and LTB, in the enhanced responsiveness of GM-CSF primed PMN towards fMLP or CSa, but not NAP-l/IL-g. 0 1991AcademicPress, Inc.
Human neutrophil-activating peptide (NAP-~/IL-~) is a novel cytokine which was originally isolated from culture fluids of stimulated human monocytes (1 - 3), and was subsequently shown to be produced by a variety of different cell types (reviewed in 4). Like the bacterial product analogue fMLP and the anaphylatoxin C5a, NAP-I/IL-~ activates human PMN through specific receptors, inducing chemotaxis, shape change, transient rise in cytosolic free calcium concentration, granule release, adherence, and respiratory burst (4 - 6). It has been shown that NAP-~/IL-~ activates PMN via a signal transduction process that involves G-proteins, phospholipase C and protein kinase C, and which is therefore similar to that known to operate for other chemotactic peptide agonists (4,5). Platelet1 Address correspondence to Urs Wirthmueller,
Dept. of Biochemical Genetics, Memorial Cancer Center, P.O. Box 86, 1275 York Avenue, New York, NY 10021. Abbreviations used; C5a: 74 amino acid cleavage product from the 5th complement component; fMLP: N-formyl-methionyl-leucyl-phenylalanine; LTB4: leukotriene B4, 5(S),12(R)-dihydroxy-6,14-cis-8,1O-frans-eicosatetraenoic acid; NAP-l/IL-S: recombinant human neutrophil-activating peptide; PAF: platelet-activating factor, l-Ohexadecyl/octadecyl-2-acetyl-sn-glycero-3-phosphocholine; PBS/AG: Dulbecco’s PBS containing BSA and glucose; PLA2: phospholipase AZ; PMN polymorphonuclear leukocyte; GM-CSF: recombinant human granulocyte-macrophage colony-stimulatingfactor. Sloan-Kettering
0006-291X/91 Copyright All rights
$1.50
0 I991 by Academic Press, of reproduction in any form
Inc. reserved.
972
Vol.
176,
No.
3, 1991
BIOCHEMICAL
activating factor (PAF) and leukotriene which participate
RESEARCH
COMMUNICATIONS
bioactive lipids
reactions (7). It is well known
that PMN
amounts of PAF and LTB4 in response to calcium ionophores
there is only limited information
response to receptor-operated granulocyte-macrophage
BIOPHYSICAL
B4 (LTB4) are potent cell-derived
in allergic and inflammatory
produce considerable However,
AND
activation
on the production
(8).
of these bioactive lipids in
(8,9). Previous studies have shown that
colony-stimulating
factor (GM-CSF)
does not induce the synthesis
of PAF and LTB, by itself, but renders PMN able to generate those bioactive lipids in response to peptide chemotactic
agonists which are otherwise
inactive (10,ll).
We have now compared the effects of NAP-~/IL-~ in PMN to those of the classical chemotactic peptides fMLP and C5a. Surprisingly, neither PAF nor LTB, were produced in response to NAP-~/IL-~ alone or in combination with GM-CSF. Furthermore, the respiratory burst induced by NAP-~/IL-~ was not enhanced by pretreatment with GM-CSF. These observations reveal a clear qualitative difference in the response of PMN to NAP~/IL-8 on one hand and to fMLP or C5a on the other. Materials
and Methods
Reagents - The sodium salt of [JH]acetic acid (3.45 Ci/mmol), [3H]PAF (l-0[3H]alkyl-2sn-glycero-3-phosphocholine, 81 Ci/mmol) and RIA kits for determination of LTB4 were from Amersham International (Buckinghamshire, England). Unlabeled CX6-PAF was purchased from Boehringer (Mannheim, Germany). fMLP was from Bachem AG (Bubendorf, Switzerland) and fatty acid free/low level endotoxin BSA (fraction V) was from Pierce (Oud-Beijerland, The Netherlands). C5a was isolated from human serum after complement activation in the presence of carboxypeptidase inhibitor (12). Recombinant human GM-CSF and recombinant human NAP-I/IL-~ (13) were generously provided by the Sandoz Research Institute, Vienna, Austria. All other reagents were from Sigma Chemical Co. (St. Louis, MO). Isolation of PMN - PMN were purified from freshly drawn blood (14), and suspended in Dulbecco’s PBS containing 1 mM [Caz+], 0.5 mg/ml BSA and 1% glucose (PBS-AG). The preparations contained less than 1% monocytes (10). Detection of bioactive lipids - Lipids were extracted from PMN as shown previously (15). Purification, quantification and characterization of PAF and 5-lipoxygenase products and metabolites were performed as described elsewhere (10, 11). Superoxide production - Superoxide production was measured at 37 oC as the superoxidedismutase sensitive reduction of ferricytochrome c (16). Absorbance changes of preequilibrated assay mixtures were recorded continuously during 10 minutes in a HewlettPackard Co. (Palo Alto, CA) 8452A diode array spectrophotometer equipped with a thermostated seven-place cuvette exchanger. The assay mixture (800 ~1) consisted of 1 x 106 cells/ml PBS-AG containing 85 PM cytochrome c. Results PAF production
- The production
of [3H]PAF
by the acetyltransferase
pathway in PMN
with or without pretreatment with GM-CSF is illustrated in figure 1. The amount of GMCSF applied and the duration of the preincubation with GM-CSF were chosen to give a maximal response in [SHIPAl! production from stimulated PMN. Significant production of [3H]PAF was obtained upon stimulation with fMLP. The effect was already substantial in 973
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fMLP
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C5a
RESEARCH
COMMUNICATIONS
NAP-i/118
Figure 1. [3H]PAF production by stimulated PMN. PMN (5 x 106/ml PBS-AG) were incubated at 370C in a shaking water bath for 2 h with or without 1 pmol GM-CSF and then exposed for 5 minutes to buffer alone (control) or 0.1 PM of a chemotactic peptide (fMLP, CSa or NAP-~/IL-~). [“HIAcetate (25 &i) was added 10 minutes before addition of the stimuli. Total lipids were extracted and PAP was purified (11). Columns represent the mean ? SD of triplicate determinations from three different donors.
untreated cells and was markedly increased by GM-CSF
priming. By contrast, no induction
of [3H]PAF production over control levels was obtained with an equimolar concentration of NAP-Z/IL-~ even in the presence of GM-CSF. Different preincubation periods (10 minutes to 3 h) and alterations
in the amount of added GM-CSF
not lead to an increase in NAP-I/IL-~
(1 nmol to 100 pmol) did
induced [SHJPAP production
(data not shown).
No
significant production of [JH]PAP was obtained by NAP-~/IL-~ concentrations ranging from 1 nM to 10 PM and at time points from 1 to 30 minutes after stimulation (data not shown).
While all PMN preparations
tested so far (n = 23) showed [3H]PAF
production
upon pretreatment with GM-CSF and stimulation with fMLP or C5a, only some responded to fMLP alone (11). The results obtained from such a preparation are shown in figure 1 to demonstrate Production
that NAP-I/IL-~ of arachidonate
is ineffective metaholites
even in cells which respond to fMLP alone.
- The generation of 5-lipoxygenase
products was
examined under conditions similar to those adopted for [3H]PAP production. Figure 2 shows representative chromatograms of PMN extracts fractionated by RP-HPLC. As indicated by the upper tracings (figure 2A), when the cells were primed by GM-CSF and subsequently stimulated with fMLP, considerable amounts of different 5-lipoxygenase products were obtained. By contrast, only minor peaks were observed in parallel experiments using NAP-I/IL-~ as stimulus (figure 2B). None of the peaks had the spectroscopic characteristics of conjugated trienes or dienes, and no LTB, was detected by RIA in the expected elution position of this compound (data not shown). In contrast to [3H]PAP production, stimulation of non-primed cells with either fMLP or C5a alone never resulted in generation of 5-lipoxygenase products. In fact, the chromatograms of control cells, and cells stimulated with NAP-~/IL-~, fMLP or C5a alone where undistinguishable from those shown in figure 2B (data not shown). 974
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i
F. --T-4
Retention
22
Time
j /,..:,o
: ..I
1421
-23
-I
?!4, 30
31
32
(minuted
Figure 2. RP-HPLC analysis of Wipoxygenase products and metabolites from stimulated PMN. PMN (4 x 107 in 2 ml of PBS-AG) were incubatedin a shakingwater bath at 37-C
for 2 h with or without 4 pmol GM-CSF and then exposedfor 2.5 minutesto buffer alone (control) or a chemotacticpeptide. (A), PMN primedwith GM-CSF andstimulatedwith 0.1PM fMLP. Peak 1: 20-COOH-LTB4 (.5(S),12(R)-dihydroxy-20-carboxy-6,14-cis-8,10trans-eicosatetraen-1,20-dioic acid); peak2: 20-OH-LTB, (S(S),12(R),20-trihydroxy-6,14cis-$lO-fauns-eicosatetraenoic acid); peak3: 20-OH SS-12S-DiHETE(S(S),12(S),20trihydroxy-8,14-cis-6,10-frans-eicosatetraenoic acid); peak 4: 6-trans-LTB4(S(S),12(R)dihydroxy-14-cis-6,8,10-truns-eicosatetraenoic acid); peak 5: 12-epi-6-tram-LTB4 (S(S),12(S)-dihydroxy-14-cis-6,8,1O-frans-eicosatetraenoic acid); peak 6: LTB,; peak 7: SSXX-diHETE (S(S),12(S)-dihydroxy-8,14-cis-6,1O-frans-eicosatetraenoic acid); peak 8: SHETE (S(S)-hydroxy-6-cis-8,10,14-frans-eicosatetraenoic acid). (B), PMN primedwith GMCSFand stimulatedwith 0.1PM NAP-~/IL-S. The peaks,two of which are alsopresentin the upper tracings,lackedthe UV propertiesindicative of dienesor trienes.
Superoxide production - It has been demonstrated that exogenously added PAF enhances the respiratory burst of PMN in responseto fMLP, C5a and NAP-I/IL-~
(17 - 19). In dose
responsestudies the maximal effective concentration of the different chemotactic peptides for the induction of superoxide production with and without PAF was determined to be > 100 nM (data not shown). In figure 3 we compared the effect of prolonged incubation with GM-CSF to that of exogenously added PAF upon the ability of PMN to produce superoxide in response to the different chemotactic agonists. In agreement with previous reports, GM-CSF enhanced the respiratory burst in responseto fMLP and C5a. By contrast, no enhancement of NAP-~/IL-~ induced superoxide production was observed after priming the cells with GM-CSF. However, exogenously PAF enhanced the respiratory burst in response to NAP-I/IL-~ although to a lesserextent, but still significant and comparable to that elicited by the other chemotactic peptides fMLP and C5a. Similar stimulation patterns were obtained when PAF was substituted by LTB, (data not shown). 975
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C5a
fMLP
RESEARCH
COMMUNICATIONS
NAP-l/IL8
production by stimulated PMN. PMN (5 x 106/mlPBS-AG) were incubatedin a shakingwater bath at 370Cfor 2 h with or without 1pmol GM-CSF. Superoxideproductionwasmeasuredasdescribedundermaterial and methods. Time delay betweenaddition of PAF (1 PM) and fMLP (0.1PM), NAP-I/IL-~ (0.1 PM) or C5a(0.1 PM) was1.5minutes.Columnsrepresentthe mean? SD of three separatesamplesfrom one experimentthat is representativeof two others. Figure 3. Superoxide
When PAF was added together with the chemotactic peptides in GM-CSF primed cells the
enhancement of superoxide release in response to NAP-~/IL-S was still observed, demonstrating that GM-CSF did not prevent the ability of PAF to augment NAP-~/IL-S responsiveness(data not shown). Discussion
NAP-~/IL-~ is known to activate PMN in vitro and to induce massive neutrophilic infiltration in vivo (l-5). In all PMN responses examined so far, the effects of NAP-~/IL-~ were qualitatively similar to those induced by two established chemotactic peptides, fMLP and C5a. This study shows that NAP-I/IL-~ does not induce the production of bioactive lipids, indicating a clear qualitative difference between NAP-I/IL-~ and the classical chemotactic peptides. This observation supports the hypothesis that parts of the signal transduction process for NAP-I/IL-~ differ from the ones of other chemotactic agonists. Our findings are related to an independent recent study, demonstrating that NAP~/IL-S induces synthesis of S-HETE, LTB,, 20-OH-LTB4 and 20-COOH-LTB4 upon supply of exogenous arachidonic acid (20). This observation was not surprising, since similar results were reported previously for PMN activated by fMLP or C5a (21). The initial
enzymatic step in the generation of leukotrienes from arachidonic acid is catalyzed by 5lipoxygenase, an enzyme which is calcium dependent and thus likely to be activated by stimuli which increase the cytosolic free calcium concentration, such as fMLP, C5a or NAP~/IL-8 (22). However, previous studies showed that chemotactic agonistsalone do not
induce leukotriene
production from endogenous arachidonic acid (9, 10,20), indicating
that a rise in intracellular calcium levels is not sufficient for an enhanced production of bioactive lipids. Thus the inability of NAP-~/IL-~ to induce leukotriene production by itself 976
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3, 1991
BIOCHEMICAL
AND
does not distinguish it from other chemotactic amounts of arachidonic
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
peptides. By contrast, relatively large
acid are released (23) and bioactive lipids are produced (9, 11) in
response to fMLP and C5a when the cells are preincubated
with GM-CSF.
Former
observations, showing the simultaneous production of arachidonic acid metabolites and of PAF by the acetyltransferase pathway, indicate that in GM-CSF primed PMN, fMLP or C5a are able to activate phospholipase AZ, an enzyme which is responsible liberation of arachidonic acid from endogenous lipid pools (10, 11,23,24). observations
are further supported by the inhibitory
for the These
effects of different agonists of
phospholipase A2 on the production of both PAF and LTB4 from endogenous arachidonic acid pools (25). PMN stimulation with NAP-~/IL-~ results in activation of 5lipoxygenase (20) but does not lead to LTB4 and PAF synthesis in GM-CSF primed cells, as shown by the present study. The difference with respect to fMLP and C5a could therefore be taken to indicate that NAP-l/II-8
is not able to activate phospholipase
A,.
It is well established that besides its function as a growth factor for myeloid progenitor cells, GM-CSF also enhances several effector functions in a variety of mature cells (26 - 28). In fact, the respiratory burst induced by all neutrophil chemotactic agonists examined so far was found to be enhanced in GM-CSF
treated cells. It was therefore
particularly interesting to find that the respiratory burst elicited by NAP-~/IL-~ was not affected by GM-CSF, thus uncovering another qualitative difference between these chemotactic
peptides. These results are consistent with our data demonstrating
that NAP-
~/IL-8 does not induce production of bioactive lipids from endogenous arachidonic acid but that PAF or LTB, added together with NAP-~/IL-~ enhance the oxidative burst in PMN. The data further support the hypothesis that PAF or LTB4 may act in an autocrine manner on PMN (10, 11,24,25,28), and - at least in part - be responsible for the enhancement of functional responses like superoxide anion release (27) or cytotoxicity (28) of the primed cells. Since PAF and LTB, are both products from and agonists for PMN they may act as autocrine response amplifiers for these cells. It is therefore difficult to distinguish whether induction of second messengers
in PMN primed with GM-CSF
and C5a are the reason for the enhanced responsiveness
and stimulated with fMLP
(29), or rather a consequence of
production of bioactive lipids. Further investigations using NAP-~/IL-~ as a triggering agent, that is not able to stimulate production of bioactive lipids, may answer such questions. The lack of production of bioactive lipids by NAP-~/IL-~ activated PMN could be related to the function of this novel mediator in vivo. There is evidence suggesting that NAP-~/IL-~ may act as a tissue derived stimulus of neutrophil recruitment involved in pathological as well as physiological processes (4), where autocrine stimulus amplification by bioactive lipids may be undesired.
Acknowledgments. This work was supported by the Swiss National grants # 3.058-0.87, and 31.25700.88. 977
Science Foundation
Vol.
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References 1. Yoshimura, T., Matsushima, K., Tanaka, S., Robinson, E. A., Appella, E., Oppenheim, J. J., and Leonard, E. J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 9233-9237. 2. Walz, A., Peveri, P., Aschauer, H., and Baggiolini, M.(1987) Biochem. Biophys. Res. Commun. 149,755761. 3. Van Damme, J., Van Beeumen, J., Opdenakker, G., and Billiau, A. (1988) J. Exp. Med. 167, 1364-1376. 4. Baggiolini, M., Waiz, A., and Kunkei, S. L. (1989) J. Clin. Invest. 84, 10451049. 5. Thelen, M., Peveri, P., Kernen, P., von Tscharner, V., Walz, A., and Baggiolini, M. (1988) FASEB J. 2,2702-2706. 6. Carveth, H. J., Bohnsack, J. F., McIntyre, T. M., Baggiolini, M., Prescott, S. M., and Zimmerman, G. A. (1989) Biochem. Biophys. Res. Commun. 162,387-393. 7. Samuelsson, B., Borgeat, P., Hammarstrom, S., and Murphy, R. C. (1980) Adv. Prostaglandin Thromboxane Res. 6, l-18. 8. Sisson, J. H., Prescott, S. M., McIntyre, T. M., and Zimmerman, G. A. (1987) J. Immunol. 138,3918-3926. 9. Haines, K. A., Giedd, K. N., Rich, A. M., Korchak, H. M., and Weissmann, G. (1987) Biochem. J. 241,55-62. 10. Dahinden, C. A., Zingg, J., Maly, F. E., and de Week, A. L. (1988) J. Exp. Med. 167, 1281-1295. 11. Wirthmueller, U., de Week, A. L., and Dahinden, C. A. (1989) J. Immunol. 142,32133218. 12. Hugli, T. E., Gerard, C., Kawahara, M., Scheetz, II, M. E., Barton, R., Briggs, S., Koppel, G., and Russel, S. (1981) Mol. Cell. Biochem. 41,59-66. 13. Lindley, I., Aschauer, H., Seifert, J. M., Lam, C., Brunowsky, W., Kownatzki, E., Thelen, M., Peveri, P., von Tscharner, V., and Baggiolini, M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 9199-9203. 14. Dahinden, C. A., Galanos, C., and Fehr, J. (1983) J. Immunol. 130,857-862. 15. Bligh, E. G., and Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, 911-918. 16. Markert, M., Andrews P. C., and Babior, B. M. (1984) Methods Enzymol. 105,358-365. 17. Dewald, B., and Baggiolini, M. (1985) Biochem. Biophys. Res. Commun. 128,297-304. 18. Baggiolini, M., and Dewald, B. (1986) Pharmacol. Res. Commun. 18, 51-59. 19. Gay. J. C., Beckman, J. K., Zaboy, K. A., and Lukens, J. N. (1986) Blood 67,931-936. 20. Schroder, J.-M. (1989) J. Exp. Med. 170,847-863. 21. Clancy, R. M., Dahinden, C. A., and Hugli, T. E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 7200-7204. 22. Rouzer, C. A., Rands, E., Kargman, S., Jones, R. E., Register, R. B., and Dixon, R. A. (1988) J. Biol. Chem. 263,10135-10140. 23. DiPersio, J. F., Billing, P., Williams, R., and Gasson, J. C. (1988) J. Immunol. 140, 43154322. 24. Dahinden, C. A., Kurimoto, Y., and Wirthmueller, U. (1990) J. Lipid Mediators 129, 169-175. 25. Wirthmueller, U., de Week, A. L., and Dahinden, C. A. (1990) Biochem. Biophys. Res. Commun. 170,556-562. 26. Silberstein, D. S., Owen, W F., Gasson, J. C., DiPersio, J. F., Golde, D. W., Bina, J. C., Soberman, R., Austen, K. F., and David, J. R. (1986) J. Immunol. 137, 3290-3294. 27. Weisbart, R. H., Golde, D. W., Clark, S. C., Wong, G. G., and Gasson, J. C. (1985) Nature 314,361-363. 28. Moqbel, R., Sass-Kuhn, S. P., Goetzl, E. J., and Kay, A. B. (1983) Clin. Exp. Immunol. 52,519-527. 29. Naccache, P. H., Faucher, N. Borgeat, P., Gasson, J. C., and DiPersio, J. F. (1988) J. Immunol. 140, 3541-3546. 978
176,
May
15, 1991
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
Pages
RECEPTOR-OPERATED ACTIVATION OF POLYMORPHONUCLEAR DIFFERENT EFFECTS OF NAP-l/IL-S AND fMET-LEU-PHE
Urs Wirthmueller*‘l
March
972-978
LEUKOCYTES: OR C5a
, Marco Baggiolini #, Alain L. de Week” and Clemens A. Dahinden*
*Institute of Clinical Immunology,
Received
COMMUNICATIONS
20,
and #Theodor Kocher Institute, University of Bern, 3010 Bern, Switzerland
1991
Summary - We examined the production
of PAF and LTB4 by PMN in response to NAP~/IL-8 alone, or after preincubation with GM-CSF, which has been shown to enhance PMN responsiveness and to prime PMN for production of those bioactive lipids. NAP-~/IL-~ does not induce the synthesis of PAF and LTB4 from endogenous phospholipid precursors, even after preincubation with GM-CSF. In addition and again in contrast to fMLP and C5a, NAP-l/IL-8 fails to induce an enhanced oxidative burst in GM-CSF primed PMN. Exogenously added PAF or LTB4 can mimic the priming effect of GM-CSF for an enhanced oxidative burst in response to all examined chemotactic peptides including NAPl/IL-g. Our data reveal a possible autocrine role of PAF and LTB, in the enhanced responsiveness of GM-CSF primed PMN towards fMLP or CSa, but not NAP-l/IL-g. 0 1991AcademicPress, Inc.
Human neutrophil-activating peptide (NAP-~/IL-~) is a novel cytokine which was originally isolated from culture fluids of stimulated human monocytes (1 - 3), and was subsequently shown to be produced by a variety of different cell types (reviewed in 4). Like the bacterial product analogue fMLP and the anaphylatoxin C5a, NAP-I/IL-~ activates human PMN through specific receptors, inducing chemotaxis, shape change, transient rise in cytosolic free calcium concentration, granule release, adherence, and respiratory burst (4 - 6). It has been shown that NAP-~/IL-~ activates PMN via a signal transduction process that involves G-proteins, phospholipase C and protein kinase C, and which is therefore similar to that known to operate for other chemotactic peptide agonists (4,5). Platelet1 Address correspondence to Urs Wirthmueller,
Dept. of Biochemical Genetics, Memorial Cancer Center, P.O. Box 86, 1275 York Avenue, New York, NY 10021. Abbreviations used; C5a: 74 amino acid cleavage product from the 5th complement component; fMLP: N-formyl-methionyl-leucyl-phenylalanine; LTB4: leukotriene B4, 5(S),12(R)-dihydroxy-6,14-cis-8,1O-frans-eicosatetraenoic acid; NAP-l/IL-S: recombinant human neutrophil-activating peptide; PAF: platelet-activating factor, l-Ohexadecyl/octadecyl-2-acetyl-sn-glycero-3-phosphocholine; PBS/AG: Dulbecco’s PBS containing BSA and glucose; PLA2: phospholipase AZ; PMN polymorphonuclear leukocyte; GM-CSF: recombinant human granulocyte-macrophage colony-stimulatingfactor. Sloan-Kettering
0006-291X/91 Copyright All rights
$1.50
0 I991 by Academic Press, of reproduction in any form
Inc. reserved.
972
Vol.
176,
No.
3, 1991
BIOCHEMICAL
activating factor (PAF) and leukotriene which participate
RESEARCH
COMMUNICATIONS
bioactive lipids
reactions (7). It is well known
that PMN
amounts of PAF and LTB4 in response to calcium ionophores
there is only limited information
response to receptor-operated granulocyte-macrophage
BIOPHYSICAL
B4 (LTB4) are potent cell-derived
in allergic and inflammatory
produce considerable However,
AND
activation
on the production
(8).
of these bioactive lipids in
(8,9). Previous studies have shown that
colony-stimulating
factor (GM-CSF)
does not induce the synthesis
of PAF and LTB, by itself, but renders PMN able to generate those bioactive lipids in response to peptide chemotactic
agonists which are otherwise
inactive (10,ll).
We have now compared the effects of NAP-~/IL-~ in PMN to those of the classical chemotactic peptides fMLP and C5a. Surprisingly, neither PAF nor LTB, were produced in response to NAP-~/IL-~ alone or in combination with GM-CSF. Furthermore, the respiratory burst induced by NAP-~/IL-~ was not enhanced by pretreatment with GM-CSF. These observations reveal a clear qualitative difference in the response of PMN to NAP~/IL-8 on one hand and to fMLP or C5a on the other. Materials
and Methods
Reagents - The sodium salt of [JH]acetic acid (3.45 Ci/mmol), [3H]PAF (l-0[3H]alkyl-2sn-glycero-3-phosphocholine, 81 Ci/mmol) and RIA kits for determination of LTB4 were from Amersham International (Buckinghamshire, England). Unlabeled CX6-PAF was purchased from Boehringer (Mannheim, Germany). fMLP was from Bachem AG (Bubendorf, Switzerland) and fatty acid free/low level endotoxin BSA (fraction V) was from Pierce (Oud-Beijerland, The Netherlands). C5a was isolated from human serum after complement activation in the presence of carboxypeptidase inhibitor (12). Recombinant human GM-CSF and recombinant human NAP-I/IL-~ (13) were generously provided by the Sandoz Research Institute, Vienna, Austria. All other reagents were from Sigma Chemical Co. (St. Louis, MO). Isolation of PMN - PMN were purified from freshly drawn blood (14), and suspended in Dulbecco’s PBS containing 1 mM [Caz+], 0.5 mg/ml BSA and 1% glucose (PBS-AG). The preparations contained less than 1% monocytes (10). Detection of bioactive lipids - Lipids were extracted from PMN as shown previously (15). Purification, quantification and characterization of PAF and 5-lipoxygenase products and metabolites were performed as described elsewhere (10, 11). Superoxide production - Superoxide production was measured at 37 oC as the superoxidedismutase sensitive reduction of ferricytochrome c (16). Absorbance changes of preequilibrated assay mixtures were recorded continuously during 10 minutes in a HewlettPackard Co. (Palo Alto, CA) 8452A diode array spectrophotometer equipped with a thermostated seven-place cuvette exchanger. The assay mixture (800 ~1) consisted of 1 x 106 cells/ml PBS-AG containing 85 PM cytochrome c. Results PAF production
- The production
of [3H]PAF
by the acetyltransferase
pathway in PMN
with or without pretreatment with GM-CSF is illustrated in figure 1. The amount of GMCSF applied and the duration of the preincubation with GM-CSF were chosen to give a maximal response in [SHIPAl! production from stimulated PMN. Significant production of [3H]PAF was obtained upon stimulation with fMLP. The effect was already substantial in 973
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Figure 1. [3H]PAF production by stimulated PMN. PMN (5 x 106/ml PBS-AG) were incubated at 370C in a shaking water bath for 2 h with or without 1 pmol GM-CSF and then exposed for 5 minutes to buffer alone (control) or 0.1 PM of a chemotactic peptide (fMLP, CSa or NAP-~/IL-~). [“HIAcetate (25 &i) was added 10 minutes before addition of the stimuli. Total lipids were extracted and PAP was purified (11). Columns represent the mean ? SD of triplicate determinations from three different donors.
untreated cells and was markedly increased by GM-CSF
priming. By contrast, no induction
of [3H]PAF production over control levels was obtained with an equimolar concentration of NAP-Z/IL-~ even in the presence of GM-CSF. Different preincubation periods (10 minutes to 3 h) and alterations
in the amount of added GM-CSF
not lead to an increase in NAP-I/IL-~
(1 nmol to 100 pmol) did
induced [SHJPAP production
(data not shown).
No
significant production of [JH]PAP was obtained by NAP-~/IL-~ concentrations ranging from 1 nM to 10 PM and at time points from 1 to 30 minutes after stimulation (data not shown).
While all PMN preparations
tested so far (n = 23) showed [3H]PAF
production
upon pretreatment with GM-CSF and stimulation with fMLP or C5a, only some responded to fMLP alone (11). The results obtained from such a preparation are shown in figure 1 to demonstrate Production
that NAP-I/IL-~ of arachidonate
is ineffective metaholites
even in cells which respond to fMLP alone.
- The generation of 5-lipoxygenase
products was
examined under conditions similar to those adopted for [3H]PAP production. Figure 2 shows representative chromatograms of PMN extracts fractionated by RP-HPLC. As indicated by the upper tracings (figure 2A), when the cells were primed by GM-CSF and subsequently stimulated with fMLP, considerable amounts of different 5-lipoxygenase products were obtained. By contrast, only minor peaks were observed in parallel experiments using NAP-I/IL-~ as stimulus (figure 2B). None of the peaks had the spectroscopic characteristics of conjugated trienes or dienes, and no LTB, was detected by RIA in the expected elution position of this compound (data not shown). In contrast to [3H]PAP production, stimulation of non-primed cells with either fMLP or C5a alone never resulted in generation of 5-lipoxygenase products. In fact, the chromatograms of control cells, and cells stimulated with NAP-~/IL-~, fMLP or C5a alone where undistinguishable from those shown in figure 2B (data not shown). 974
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i
F. --T-4
Retention
22
Time
j /,..:,o
: ..I
1421
-23
-I
?!4, 30
31
32
(minuted
Figure 2. RP-HPLC analysis of Wipoxygenase products and metabolites from stimulated PMN. PMN (4 x 107 in 2 ml of PBS-AG) were incubatedin a shakingwater bath at 37-C
for 2 h with or without 4 pmol GM-CSF and then exposedfor 2.5 minutesto buffer alone (control) or a chemotacticpeptide. (A), PMN primedwith GM-CSF andstimulatedwith 0.1PM fMLP. Peak 1: 20-COOH-LTB4 (.5(S),12(R)-dihydroxy-20-carboxy-6,14-cis-8,10trans-eicosatetraen-1,20-dioic acid); peak2: 20-OH-LTB, (S(S),12(R),20-trihydroxy-6,14cis-$lO-fauns-eicosatetraenoic acid); peak3: 20-OH SS-12S-DiHETE(S(S),12(S),20trihydroxy-8,14-cis-6,10-frans-eicosatetraenoic acid); peak 4: 6-trans-LTB4(S(S),12(R)dihydroxy-14-cis-6,8,10-truns-eicosatetraenoic acid); peak 5: 12-epi-6-tram-LTB4 (S(S),12(S)-dihydroxy-14-cis-6,8,1O-frans-eicosatetraenoic acid); peak 6: LTB,; peak 7: SSXX-diHETE (S(S),12(S)-dihydroxy-8,14-cis-6,1O-frans-eicosatetraenoic acid); peak 8: SHETE (S(S)-hydroxy-6-cis-8,10,14-frans-eicosatetraenoic acid). (B), PMN primedwith GMCSFand stimulatedwith 0.1PM NAP-~/IL-S. The peaks,two of which are alsopresentin the upper tracings,lackedthe UV propertiesindicative of dienesor trienes.
Superoxide production - It has been demonstrated that exogenously added PAF enhances the respiratory burst of PMN in responseto fMLP, C5a and NAP-I/IL-~
(17 - 19). In dose
responsestudies the maximal effective concentration of the different chemotactic peptides for the induction of superoxide production with and without PAF was determined to be > 100 nM (data not shown). In figure 3 we compared the effect of prolonged incubation with GM-CSF to that of exogenously added PAF upon the ability of PMN to produce superoxide in response to the different chemotactic agonists. In agreement with previous reports, GM-CSF enhanced the respiratory burst in responseto fMLP and C5a. By contrast, no enhancement of NAP-~/IL-~ induced superoxide production was observed after priming the cells with GM-CSF. However, exogenously PAF enhanced the respiratory burst in response to NAP-I/IL-~ although to a lesserextent, but still significant and comparable to that elicited by the other chemotactic peptides fMLP and C5a. Similar stimulation patterns were obtained when PAF was substituted by LTB, (data not shown). 975
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C5a
fMLP
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NAP-l/IL8
production by stimulated PMN. PMN (5 x 106/mlPBS-AG) were incubatedin a shakingwater bath at 370Cfor 2 h with or without 1pmol GM-CSF. Superoxideproductionwasmeasuredasdescribedundermaterial and methods. Time delay betweenaddition of PAF (1 PM) and fMLP (0.1PM), NAP-I/IL-~ (0.1 PM) or C5a(0.1 PM) was1.5minutes.Columnsrepresentthe mean? SD of three separatesamplesfrom one experimentthat is representativeof two others. Figure 3. Superoxide
When PAF was added together with the chemotactic peptides in GM-CSF primed cells the
enhancement of superoxide release in response to NAP-~/IL-S was still observed, demonstrating that GM-CSF did not prevent the ability of PAF to augment NAP-~/IL-S responsiveness(data not shown). Discussion
NAP-~/IL-~ is known to activate PMN in vitro and to induce massive neutrophilic infiltration in vivo (l-5). In all PMN responses examined so far, the effects of NAP-~/IL-~ were qualitatively similar to those induced by two established chemotactic peptides, fMLP and C5a. This study shows that NAP-I/IL-~ does not induce the production of bioactive lipids, indicating a clear qualitative difference between NAP-I/IL-~ and the classical chemotactic peptides. This observation supports the hypothesis that parts of the signal transduction process for NAP-I/IL-~ differ from the ones of other chemotactic agonists. Our findings are related to an independent recent study, demonstrating that NAP~/IL-S induces synthesis of S-HETE, LTB,, 20-OH-LTB4 and 20-COOH-LTB4 upon supply of exogenous arachidonic acid (20). This observation was not surprising, since similar results were reported previously for PMN activated by fMLP or C5a (21). The initial
enzymatic step in the generation of leukotrienes from arachidonic acid is catalyzed by 5lipoxygenase, an enzyme which is calcium dependent and thus likely to be activated by stimuli which increase the cytosolic free calcium concentration, such as fMLP, C5a or NAP~/IL-8 (22). However, previous studies showed that chemotactic agonistsalone do not
induce leukotriene
production from endogenous arachidonic acid (9, 10,20), indicating
that a rise in intracellular calcium levels is not sufficient for an enhanced production of bioactive lipids. Thus the inability of NAP-~/IL-~ to induce leukotriene production by itself 976
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does not distinguish it from other chemotactic amounts of arachidonic
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peptides. By contrast, relatively large
acid are released (23) and bioactive lipids are produced (9, 11) in
response to fMLP and C5a when the cells are preincubated
with GM-CSF.
Former
observations, showing the simultaneous production of arachidonic acid metabolites and of PAF by the acetyltransferase pathway, indicate that in GM-CSF primed PMN, fMLP or C5a are able to activate phospholipase AZ, an enzyme which is responsible liberation of arachidonic acid from endogenous lipid pools (10, 11,23,24). observations
are further supported by the inhibitory
for the These
effects of different agonists of
phospholipase A2 on the production of both PAF and LTB4 from endogenous arachidonic acid pools (25). PMN stimulation with NAP-~/IL-~ results in activation of 5lipoxygenase (20) but does not lead to LTB4 and PAF synthesis in GM-CSF primed cells, as shown by the present study. The difference with respect to fMLP and C5a could therefore be taken to indicate that NAP-l/II-8
is not able to activate phospholipase
A,.
It is well established that besides its function as a growth factor for myeloid progenitor cells, GM-CSF also enhances several effector functions in a variety of mature cells (26 - 28). In fact, the respiratory burst induced by all neutrophil chemotactic agonists examined so far was found to be enhanced in GM-CSF
treated cells. It was therefore
particularly interesting to find that the respiratory burst elicited by NAP-~/IL-~ was not affected by GM-CSF, thus uncovering another qualitative difference between these chemotactic
peptides. These results are consistent with our data demonstrating
that NAP-
~/IL-8 does not induce production of bioactive lipids from endogenous arachidonic acid but that PAF or LTB, added together with NAP-~/IL-~ enhance the oxidative burst in PMN. The data further support the hypothesis that PAF or LTB4 may act in an autocrine manner on PMN (10, 11,24,25,28), and - at least in part - be responsible for the enhancement of functional responses like superoxide anion release (27) or cytotoxicity (28) of the primed cells. Since PAF and LTB, are both products from and agonists for PMN they may act as autocrine response amplifiers for these cells. It is therefore difficult to distinguish whether induction of second messengers
in PMN primed with GM-CSF
and C5a are the reason for the enhanced responsiveness
and stimulated with fMLP
(29), or rather a consequence of
production of bioactive lipids. Further investigations using NAP-~/IL-~ as a triggering agent, that is not able to stimulate production of bioactive lipids, may answer such questions. The lack of production of bioactive lipids by NAP-~/IL-~ activated PMN could be related to the function of this novel mediator in vivo. There is evidence suggesting that NAP-~/IL-~ may act as a tissue derived stimulus of neutrophil recruitment involved in pathological as well as physiological processes (4), where autocrine stimulus amplification by bioactive lipids may be undesired.
Acknowledgments. This work was supported by the Swiss National grants # 3.058-0.87, and 31.25700.88. 977
Science Foundation
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