Pergamon Press
PHARMACOLOGY LETTERS Accelerated Communication
PRIMING EFFECTS OF MAIR~ALIANTACHYKININS ON HUMANNEUTROPHILS 1 * Sandra Brunel]eschi , Sandra Tarli, Alberto G i o t t i and Roberto Fantozzi Dept. Pharmacology, Univ. Florence, V.le G.B.Morgagni, 65, 50134 Firenze, I t a l y ; * I n s t . Pharmacology, Univ. Ferrara, Via Fossato di Mortara, 64B, 44100 Ferrara, I t a l y . (Submitted August 28, 1990; accepted October 16, 1990; received in final form November 6, 1990)
Abstract. The undecapeptide substance P (SP) is known to activate different cell types involved in inflan~natory and immune processes. By evaluating primed stimulation of human neutrophils, we now demonstrate that SP (10 nM - 0.1 mH) dose-dependently enhances superoxide anion production from cells stimulated by the phospholipid mediator Platelet Activating Factor (PAF). We also provide evidence that neurokinin A (NKA), which is released, as we]] as SP, from C fibers of sensory nerves, potentiates PAF-evoked superoxide anion generation, while neurokinin B (NKB) is ineffective. Introduction The neuropeptide substance P (SP), which is widely distributed throughout the centre] and peripheral nervous system, is a mediator of neurogenic inflamation end can activate cell types involved in inflamatory and immune processes (1-3). I t degranulates rat mast cells, stimulates DNA and protein synthesis from human T lymphocytes, evokes cytokine release from human monocytes, induces oxy-radical production and prostanoid release from guineapig peritoneal and alveo]ar macrophages, promotes neutrophil activation (3-7). However, the concentrations at which SP stimu]ates human neutrophils in v i t r o great]y vary, depending on the functional parameter evaluated. Nanomelar concentrations of SP induce phagocytosis and locomotion of human neutrophils, whereas micromolar concentrations are required to evoke oxy-radical production and lysosomal enzyme re]ease (6-9). Moreover, the N-terminal sequence of the peptide is required to trigger locomotion and phagocytosis, while the respiratory burst and lysosomal enzyme release depend on the C-terminal portion of SP (6-9). The enhanced respiratory burst to a given stimulus, which is observed after neutrophil exposure to a different agonist, has been referred to as "priming" by different authors (10-12). Recent evidence suggests that SP is able to prime neutrophi]s challenged with the bacterial chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (FMLP) or the complement fraction CSa (11-12). However, no efforts have been made, to our knowledge, to v e r i f y the priming effects of other mammalian tachykinins and to ascertain the interaction between SP end another mediator of i n f l a ~ a t i o n , such as Platelet Activating Factor (PAF). PAF was reported to activate human neutrophils by i t s e l f (13). Therefore, we decided to evaluate whether SP, 1 Corresponding author: Sandra Brunelleschi, Department of Pharmacology, University of Florence, V. le G.B. Morgagni, 65, 50134 FIRENZE ( I t a l y ) . 0024-3205/91 $3.00 + .00 Copyright (c) 1991 Pergamon Press plc
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neurokinin A (NKA) and neurokinin B (NKB) could act as priming agents for neutrophils stimulated by PAF. Methods
Human neutrophils were isolated from heparinized venous blood of healthy donors by standard techniques of dextran sedimentation (dextran T-500, Pharmacia), Ficoll-Paque (Pharmacia) gradient centrifugation and hypotonic lysis of erythrocytes, as previously described (14). The cells were suspended in a buffered salt solution (NaCl 138 mM, KCl 2.7 mM, Na~HPO A 8.1 mM, KH~PO~ 1.5 n~4, MgClo 1 mM, CaCl~ I mM; pH 7.4) supplemented withlmg/]hl gJucose.~ell v i a b i l i t y (t~ypan blue e~clusion) was > 95~. Neutrophils (I x 10 o cells/m]) were preincubated in the presence or absence of increasing concentrations of mammalian tachykinins for 3 min at 37°C (11) and then stimulated for a further 5 min with submaximal concentrations of PAF (Bachem). In preliminary experiments, in which we evaluated the direct stimulating effects of SP, NKA and NKB, cells were treated for 5 min at 37°Cwith or without cytochalasin B (5 ug/ml; Aldrich) and then challenged with mammalian tachykinins or other known neutrophil stimuli (e.g., PAF, FMLP). Cytochalasin B pretreatment was omitted in priming experiments according to Perianin et a1.(11) and Wozniack et al.(12).Superoxide anion (0~) production, which was continuously monitored spectrophotometrically at 37 C by measuring superoxide dismutase (Boehringer Mannheim)-inhibitable cy~ochrome C (Sigma) reduction, was expressed as nmeles cytochrome C reduced/10v cells/mi~ (15). SP, SP(I-7) and NKA were obtained from Peninsula and dissolved in water; NKB (Peninsula) was dissolved in DMSO and evaluated t i l l 30 uM, in order to obtain a final DMSOconcentration no higher than 0.1~. Results were expressed as means ~ s.e.m. Statistical analysis was carried out by ANOVA. Results We f i r s t determined the a b i l i t y of SP, NKA and NKB to stimulate per se generation from human neutrophils. In the presence of cytochalasin B, SP evoked 05 production in the range 10 uM - 0.1 mM. Whenthe effects of 0.1 mM SP were compared with those of 1 uM FMLPand 10 uM PAF (these concentrations of FMLPand PAF gave maximal activation of human neutrophils), SP was only one f i f t h as active as FMLPand half active as PAF (5.2 ± 0.3, 1.8 + 0.28 and 0.99 + 0.2 nmoles cytochrome C reduced/t06 cells /min for FMLP,-PAF and SP, ~espectively; n = 3-6). In the same experimental conditions the N-terminal heptapeptide SP (1-7) was ineffective, while NKA and NKB showed_ negligible activity (0.15 ± 0.03 and 0.2 +_0.1 nmeles cytochrome C reduced/lO6 cells/min for 0.1 mM NKA and 30 uM NKB, respectively; n = 3). In the absence of cytochalasin B, SP-evoked 05 production was strongly reduced, while FMLPand PAF were s t i l l active (0.2 + 0.08, 3.6 + 0.2 and 1.5 + 0.3 nmoles cytochrome C reduced/t0 6 cells/min for 071 mMSP, 1-uMFMLPand I~ uM PAF, respectively; n = 5).The effects of mammalian tachykinins were unaffected by pretreating the cells with the enkephalinase inhibitor, thiorphan (1-10 uM, 10 mini data not shown). We then evaluated the a b i l i t y of mammalian tachykinins to prime human neutrophils challenged with submaximal concentrations of PAF (0.I uM) and FMLP (10 nM). The experiments were performed in the absence of cytochalasin B. PAF 0.1 uM and FMLP 10 nM produced 0.54 + 0.04 (n=5) and 2.1 + 0.3 (n=3) nmoles cytochrome C reduced/106 cells/min,- respectively. In -these experiments neutrophils were preincubated 3 min in the presence of increasing concentrations of mammalian tachykinins before exposure to PAF. In fact, time course (0-15 min) experiments revealed that the 3-5 min pretreatment period was the optimal time point for priming effects of mammalian tachykinins (data
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not shown).SP dose-dependently enhanced PAF-induced 02 generation, with maximal effects at 0.1 mH SP ( F i g . l ) . At this concentration~ SP enhanced the response to PAF approximately four-fold and the recorded 02 production was 2.08 + 0.23 nmeles cytochrome C reduced/106 cells/mtn, while the N-terminal peptide SP (1-7) was inactive (not reported). NKA was less potent than SP in enhancing PAF-evoked O~ generation, a two-fold enhancement being observed at 0.1 mH NKA (Fig. 1 ) . " On the contrary, NKB did not prime human neutrophtls (Fig. 1). Similar results were obtained when human neutrophils were stimulated by 10 nM FHLP, the same order of a c t i v i t y SP • NKA • NKB (inactive) being observed (data not shown). The enkephalinase inhibitor thiorphan did not s i g n i f i c a n t l y modify tachykinin-evoked priming effects in human neutrophils (data not shown).
2.5
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i 1.5 U
1.0 0.5 -
-7
-6
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log [TACHYKININ]
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(M)
FIG. I Effects of mammalian tachykinins on PAF-evoked superoxide anion production. Neutrophils were prelncubated with tachykinins or medium ~.r 3 min at 37°C and then challenged with 0.I uH P A F . ~ : 0.I uH PAF; + SP; (~: + NKA; m ~ + NKB. Means ± s.e.m, of f~ve experiments. A statistically significant difference between experiments performed in the absence and in the presence of tachyklnins is designated by * (0.05
NKB (inactive) might suggest that NKI receptors are the main mediators of tachykinin-evoked priming effects, although the concentrations at which tachykinins work in our model are higher than those observed in other monoreceptorial systems (16). NKA is co-stored and co-released with SP from peripheral terminals of primary sensory neurons (17). Therefore, the finding that SP and NKA, but not NKB, prime huma~ neutrophils challenged with the phospholipid mediator PAF further stresses the role of these tachykinins in regulating neutrophil activity and suggests that neurogenic stimulation can potentiate biological functions of phagocytes. Acknowledgements This work was supported in part by a MPI grant from the University of Ferrara. References i. 2. 3. 4. 5.
B. PERNOW, Pharmacol. Rev. 35 85-141 (1983). J.C. FOREMANand C.C. JORDAn,Trends Pharmac.Sci.5 116-119 (1984). D.G. PAYAN, Ann. Rev. Med. 40 341-352 (1989). M. LOTZ, J.H. VAUGHANand D~.CARSON, Science 241 1218-1221 (1988). S. BRUNELLESCHI, L. VANNI, F. LEDDA, A. GIOTTI, C.A. MAGGIand R. FANTOZZl, Br. J. Pharmacol. 100 417-420 (1990). 6. Z. BAR-SHAVIT, F. GOLDMAN, Y. STABINSKY, P. GOTTLIEB, M. FRIDKIN, V.I. TEICHBERG and S. BLUMBERG, Biochem. Biophys. Res. Commun. 94 1445-1451 (Ig80). 7. M.C. SERRA, F. BAZZONI, V. DELLA BIANCA, M. GRESKOWIAK and F. ROSSI, J. Immunol. 141 2118-2124 (1988). 8. C.J. WIEDERMANN, F.J. WIEDERMANN, A. APPERL, G.KIESELBACH, G. KONWALINKA and H. BRAUNSTEINER, Naunyn-Schmied.Arch.Pharmacol. 340 185-190 (1989). 9. I. IWAMOTO, H. YAMAZAKI, N. NAKAGAWA, A. KIMURA,H. TOMIOKA and S. YOSHIOA, NeuropeptidesI__6103-107 (1990). IO.J.R. FOREHAND, M.J. PABST, W.A. PHILLIPSand R.B. JOHNSTON, J. Clin. Invest. 83 74-83 (1989). 11.A. PERIANI~ R. SNYDERMANand B. MALFROY,Biochem. Biophys. Res. Commun.
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161 520-524 (1989). 12.A. WOZNIACK, G. MCLENNAN, W.H.BETTS, G.A.MURPHY and R. SCICCHITANO, Immunology 6._.88359-364 (1989). 13.R.J. SMITH, B.J. BOWMANand S.S. IDEN, BiochemPharmacol. 33 973-978 (1984). 14.R. FANTOZZI, S. BRUNELLESCHI, S. CAMBI, P. BLANDINA,E. MASINI and P.F. MANNAIONI, Agents Actions 14 441-450 (1984). 15.J.E. SMOLEN, H.M.KORCHAK~ndG. WEISSMANN,Biochem.Biophys.Acta 677 512520 (1981). 16.D. REGOLI, G. DRAPEAU, S. DION and P. D'ORLEANS-JUSTE, Pharmacology 38 1-35 (1989). 17.A. SARIA, R. GAMSE,J. PETERMANN, J.A. FISCHER, E. THEODORSSON-NORHEIM and J.M.LUNDBERG, Neurosci. Lett. 63 310-314 (1986).
