Wortmannin enhances tumour necrosis factora-stimulated neutrophil apoptosis. JOANNA MURRAY, ALISON M. CONDLIFFE, CHRIS HASLETT and EDWIN R.
80s Biochemical Society Transactlons ( 1 996) 24 Wortmannin enhances tumour necrosis factora-stimulated neutrophil apoptosis. J O A N N A MURRAY, ALISON M. CONDLIFFE, CHRIS HASLETT and EDWIN R. CHILVERS.
Neutrophils cultured in vitro undergo rapid (24-48 hr) and spontaneous apoptosis. This process results in their recognition and uptake by macrophages and is thought to represent a major mechanism for the removal of neutrophils from inflamed sites 111. We have recently shown that TNFa has the unique ability among other activating/priming agents to stimulate neutrophil apoptosis at early time points (4hr) 121. Recent reports have implicated the sphingomyelin-ceramide pathway in mediating the pro-apoptotic effects of TNF in leukaemic cell lines 13, 41, and the phosphatidylinositol 3-kinase (P13K) pathway in mediating nerve growth factor (NGF)-stimulated survival of rat phaeochromocytoma PC-12 cells [51. To investigate the role of these signalling pathways in TNFa-induced neutrophil apoptosis, we have examined the effect of cell permeable ceramide (Cer) analogues, exogenous sphingomyelinase (SMase), and the PI3K inhibitor wortmannin (WM) on neutrophil apoptosis in vitro. Human neutrophils were prepared from peripheral blood of healthy volunteers using dextran sedimentation and plasmaPercoll gradients [I]. Harvested neutrophils were suspended in Iscove's modified Dulbecco's medium (MDM) supplemented with 10% autologous serum and 50 U/ml penicillin and streptomycin, and cultured in a humidified 5% CO2-air incubator. For the Cer studies, 6.75 x lo5 cells were cultured in the presence or absence of 10 pM C2- or Cs-Cer, 200 mU/ml SMase or 12.5 ng/ml TNFa in a final volume of 150 pI in flat bottomed 96 well flexiplates. For the WM investigations, 20 x lo6 cells were cultured in the presence or absence of 100 nM WM and/or 12.5 ng/ml TNFa in teflon bags in a final volume of 2 ml. After 6 hr in culture, neutrophils were harvested and assessed for recovery, viability (trypan blue exclusion) and apoptosis (% of cells on stained cytocentrifuge preparations containing darkly stained pyknotic nuclei). Figure 1 demonstrates the inability of C&er and SMase, both at concentrations known to induce apoptosis in other myeloid models 13, 41, to mimic TNFa in stimulating early neutrophil apoptosis. Identical data were obtained using 10 pM
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Fieure 1. Inabilitv of C6-ceramide and suhinaomvelinase to mimic TNFa-stimulated neutrouhil aDoDtosis. Neutrophils were cultured for 6 hr in serum-supplemented MDM containing (final concentration) : TNFa (12.5 ng/ml), c6Cer (10 pM), or SMase (200 mU/ml). Control cells were cultured either in serum-supplemented MDM alone (TNFa control) or in the same medium containing O.l%ethanol (Cs-Cer control) or PBS/glycerol (0.2%) (SMase control). Data are means f sem (n=9,3 separate experiments).
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Rayne Laboratory, Respiratory Medicine Unit, University of Edinburgh, Medical School, Teviot Place, EH8 9AC, UK.
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FiPure 2. Wortmannin augments TNFa-stimulated but not the basal rate of neutrouhil auoutosis at 6 hr. Neutrophils were cultured either in serum-supplemented MDM alone or in identical medium containing (final concentration) : TN Fa (12.5ng/ml), WM (100 nM) or TNFa + WM. WM was replenished (50 nM) hourly d u e to its instability in aqueous solution. Data are means k sem (n=Y, 3 separate experiments). C2-Cer. In addition, we have previously shown that bacteri'il endotoxin (LPS), that has a strong structural and functional resemblance to Cer [61, inhibits rather than promotes neutrophil apoptosis [71. Furthermore, in agreement with an earlier study 181, Cer and SMase did not niiniic the ability of TNFa to enhance fMLP-stimulated superoxide generation (results not shown). These data suggest that the sphingomyelin-ceraiiiide pathway is not the principal signalling route employed by TNFa in human neutrophils. The fungal metabolite WM is a highly selective P13K inhibitor and abolishes agonist-stimulated superoxidr anion generation in human neutrophils 191. In o u r experiments, WM alone had no effect on the basal rate of apoptosis at 6hr but greatly augmented the ability of TNFa to induce apoptosis (figure 2). Due to the instability of WM in aqueous solutions, this effect was observed only with regular replenishment of WM. Neither WM alone, or WM + TNFa, had any effect on cell viability or recovery. The suggestion that P13K may inhibit or retard TNFa-stimulated apoptosis is supported by data showing that this enzyme plays a crucial role i n NCF and PDGF-mediated survival in PC-12 cells [5]. The inability of WM to influence the basal rate of apoptosis in neutrophils suggests that either P13K is not involved in tonic inhibiton of apoptosis or that a serum factor(s) stimulates a distinct form of P13K that is relatively insensitive to WM. O u r findings indicate that the pro-apoptotic effect of TNFa observed in neutrophils is mediated by a sphingomyelin-Cerindependent, but P13K regulated, pathway, and suggests a potential role for PtdIns(3,4,5)P3 in cell survival. We thank the Wellcome Trust and MRC for grant support. 1. Savill, J.S., Wyllie, A.H., Henson, J.E., Walport, M.J., Henson, P.M. & Haslett, C. (1989) J. Clin. Invest. 83, 865875 2. Murray, J., Condliffe, A.M., Haslett, C. & Chilvers, E.R. (1995) Clin. Sci. 88, 28p 3. Obeid, L.M., Linardic, C.M., Karolak, L.A. &I Hannun, Y.A. (1993) Science 259, 1769-1771 4. Jarvis, W.D., Kolesnick, R.N.. Fornari. F.A.. Travlor. R.S.. Gewirtz, D.A. & Grant, S. (1994) Proc. Nail. Aiad.'Scil ' USA 91, 73-77 5. Yao, R. & Cooper, G.M. (1995) Science 267, 2003-2006 6. Wright, S.D. & Kolesnick, R.N. (1995) Immunol. Today 16, 297-302 7. Lee, A., Whyte, M.K.B., Haslett, C. (1993) J. Leuk. B i d 54, 283-288 8. Yanaga, F. & Watson, S.P. (1994) Biochem. J. 298, 733-738 9. Laudanna, C., Rossi, F. & Berton, G. (1993) Biochem. Biophys. Res. Commun. 190,935-940
