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Interleukin-8 production induced by the endozepine triakontatetraneuropeptide in human neutrophils: role of calcium and pharmacological investigation of signal transduction pathways

Interleukin-8 production induced by the endozepine triakontatetraneuropeptide in human neutrophils: role of calcium and pharmacological investigation of signal transduction pathways
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  Interleukin-8 production induced by the endozepinetriakontatetraneuropeptide in human neutrophils: role of calcium and pharmacological investigation of signal transduction pathways Franca Marino a  , Marco Cosentino a, *, Anna Maria Fietta  b , Marco Ferrari a  , Simona Cattaneo a  ,Giuseppina Frigo a  , Sergio Lecchini a  , Gian Mario Frigo c a   Laboratory of Pharmacology, Faculty of Medicine, University of Insubria, Via Ottorino Rossi n. 9, 21100 Varese, VA, Italy  b  Department of Hematological, Pneumological and Cardiovascular Sciences, Faculty of Medicine, University of Pavia, Pavia, Italy c  Department of Internal Medicine and Therapeutics, Faculty of Medicine, University of Pavia, Pavia, Italy Received 31 May 2002; accepted 4 November 2002 Abstract The endozepine triakontatetraneuropeptide (TTN) induces intracellular calcium ([Ca 2 + ] i ) changes and is chemotactic for humanneutrophils (PMNs). Because interleukin-8 (IL-8) production is Ca 2 + dependent and can be induced by chemotactic stimuli, we haveinvestigated the ability of TTN to induce IL-8 production in PMNs, as well as the signal transduction mechanisms involved. Our results showthat TTN increases IL-8 release and IL-8 mRNA expression in a concentration- and time-dependent fashion, and these effects are prevented by the Ca 2 + chelator BAPTA-AM. TTN-induced [Ca 2 + ] i  changes and IL-8 mRNA expression are sensitive to pertussis toxin, to the phospholipase C (PLC) inhibitor U73122 (but not to its inactive analogue U73343) and to the protein kinase C (PKC) inhibitor calphostin C.It is therefore suggested that TTN-induced IL-8 production in human PMNs results from a G protein-operated, PLC-activated [Ca 2 + ] i  rise,and PKC contributes to this effect. These findings further support the possible role of TTN in the modulation of the inflammatory processes. D  2002 Elsevier Science Inc. All rights reserved.  Keywords:  Endozepines; TTN; Intracellular calcium; IL-8; IL-8 mRNA 1. Introduction Triakontatetraneuropeptide [diazepam-binding inhibitor (DBI) 17–50, TTN] is one of the major endogenouslyoccurring metabolites derived from the proteolytic cleavageof the 11-kDa neuropeptide DBI, also known as acyl-CoA- binding protein [1]. DBI-derived peptides are collectivelyknown as endozepines and are widely distributed both in theCNS and in peripheral organs [2,3] where they induceseveral effects, including stimulation of mitochondrial ste-roid biosynthesis in adrenortical and glial cells [4], stim- ulation of corticosteroid secretion in frog adrenal gland [5],inhibition of glucose-induced insulin release from pancre-atic islets [6], stimulation of cholecystokinin secretion from the intestine and STC-1 cells [7].DBI and DBI mRNA can be found in immune tissues [8], and evidence exists that DBI and DBI-derived peptides,including TTN and its related peptides octadecaneuropeptide(DBI 33–50, ODN) and eiksoneuropeptide (DBI 51–70,ENP), can affect host immune defences. In particular, TTNand ODN stimulate the production of tumour necrosis factor alpha, interleukin (IL)-1 beta, IL-6, IL-8 and granulocyte/ macrophage colony-stimulating factor in human monocytes[9,10],and ODN enhances theLPS-inducedsecretion ofIL-6in human peripheral blood mononuclear cells [11]. We have recently shown that in isolated human neutrophils (PMNs),TTN and ENP induce intracellular calcium ([Ca 2 + ] i )changes. In these cells, TTN also stimulates chemotaxis,O 2  generation and phagocytosis, suggesting its possibleinvolvement in the modulation of the development andregulation of the inflammatory process [12].The chemokine IL-8 is a potent chemoattractant that  promotes the mobilization of PMNs and their recruitment to inflammatory sites [13]. Furthermore, IL-8 upregulates PMN expression of integrins [14], adherence to endothelial 0898-6568/02/$ - see front matter   D  2002 Elsevier Science Inc. All rights reserved.doi:10.1016\S0898-6568(02)00134-1* Corresponding author. Tel./fax: +39-332-811601.  E-mail address: (M. Cosentino) Signalling 15 (2003) 511–517  cells [15] and exocytosis of cytoplasmic granules [16]. Evidence suggests that IL-8 production by PMNs could alsorepresent a paracrine mechanism for the recr uitment of other immune cells to the inflammatory sites [17]. Production and release of IL-8 by PMNs is effectively induced by chemo-tactic stimuli such as LPS or f MLP [18] and occurs through Ca 2 + -dependent mechanisms [19].Because the endozepine TTN is chemotactic for humanPMNs and can mobilize [Ca 2 + ] i  [12], in the present study,we have tested the ability of TTN to trigger IL-8 productionin these cells and by use of a pharmacological approach, wehave investigated TTN-activated signalling pathwaysinvolved in this effect. Our findings show that TTN stim-ulation of human PMNs results in increased synthesis andrelease of IL-8. This effect is Ca 2 + dependent and requiresthe activation of a pertussis toxin (PTX)-sensitive G protein positively coupled to phospholipase C (PLC). Protein kinaseC (PKC) activation seems to contribute to this effect. 2. Materials and methods 2.1. Drugs and chemicals TTN was purchased from Neosystem (Strasbourg,France); Fura-2/AM was obtained from Calbiochem-Nova- biochem (La Jolla, CA, USA). Dextran and Ficoll-PaquePlus were from Pharmacia Biotech (Uppsala, Sweden).Bovine serum albumin (BSA), HEPES, EDTA, EGTA,TRIZMA Base, PTX, 1-[6-([17 h -3-methoxyestra-1,3,5(10)-trien-17-yl]amino)hexyl]-1  H  -pyrrole-2,5-dione (U73122),1-[6-([17 h -3-methoxyestra-1,3,5(10)-trien-17-yl]amino)-hexyl]-2,5-dione (U73343), carbonyl cyanide  p -trifluorome-thoxyphenyl-hydrazone (FCCP) and thapsigargin were fromSigma (St. Louis, MO, USA). All other reagents and solventswere from Merck (Darmstadt, Germany). All solutions werefreshly prepared daily from stock solutions stored at    20 j C until use. 2.2. Cell preparation PMNs were isolated from venous blood obtained fromhealthyvolunteersusing heparinized tubes.Whole bloodwasallowed to sediment on dextran at 37  j C for 30 min. Super-natant was recovered and PMNs were isolated by Ficoll-Paque Plus density-gradient centrifugation as described[20]. Contaminating erythrocytes were eliminated by 10min hypotonic lysis in distilled water with added (g/l): NH 4 Cl 8.25, KHCO 3  1.00, EDTA 0.04. Cells were thenwashed three times in NaCl 0.15 M and resuspended in1 ml Ca 2 + /Mg 2 + -free PBS (composition as follows [g/l]: NaHPO 4  H 2 O 17.80, Na 2 HPO 4  H 2 O 13.80, NaCl 8.80)with added BSA 0.25%. Purity and viability of PMN preparations were always >95% and no platelets or eryth-rocytes could be detected by either light microscopic exami-nation or flow cytometric analysis. 2.3. IL-8 assay PMNs were resuspended at the concentration of 1  10 7 cells/ml in RPMI and incubated at 37  j C for up to 5 h. After incubation, the cells were centrifuged(600   g  , 5 min, 20  j C) and the supernatant was har-vested for IL-8 assay. IL-8 levels in PMN supernatantswere quantified using a sandwich-type enzyme-linkedimmunosorbent assay (ELISA). Antibody pairs were usedaccording to the protocols suggested by the supplier firm(ImmunoKontact, Frankfurt am Main, Germany) as fol-lows: IL-8 MoAb clone 3 IL-8-H10 at 1  A g/ml, detectingMoAb clone I8S2 at 50 ng/ml. The detection limit of theassay was 1 pg/ml. Fig. 1. (A) Stimulatory effect of TTN on IL-8 release in human PMNsincubated for 1 h (squares), 3 h (triangles), or 5 h (circles). Each point is themean F S.D. of at least four separate experiments. *=  P  <.05 and**=  P  <.01 vs. respective control. (B) Effect of 45 min preincubation withthe intracellular Ca 2 + chelator BAPTA-AM on TTN-induced IL-8 release inhuman PMNs incubated for 1 h (empty columns), 3 h (hatched columns), or 5 h (filled columns). Data are expressed as percentage of control values.Each column is the mean F S.D. of at least four separate experiments.*=  P  <.05 vs. respective control.  F. Marino et al. / Cellular Signalling 15 (2003) 511–517  512  2.4. IL-8 mRNA analysis Total RNA was extracted from 1  10 6 PMNs byPerfect RNA Eukaryotic Mini kit (Eppendorf, Hamburg,Germany). The kit utilizes a chaotropic guanidinium iso-thiocyanate solution for cell lysis and rapid inactivation of cellular RNAses. RNA is subsequently bound to the matrixof the column, washed to remove contaminants and theneluted with molecular biology grade water. In the present study, the amount of extracted RNA was estimated byspectrophotometry at 260 nm. Total RNA was reversetranscribed and cDNA was amplified using a one-stepRT-PCR reaction kit (Finnzymes, Espoo, Finland). Briefly,1  A g total RNA was added to a reaction mixture consistingof 5  A l RT-10   reaction buffer, 1  A l MgCl 2  50 mM, 1  A ldeoxynucleotide triphosphate mixture (10 nM each), 1  A lspecific primer (Invitrogen, San Giuliano Milanese, MI,Italy), 1  A l avian myeloblastosis virus RT 5 U/ml, 1  A lthermostable DNA polymerase (DYNazyme II DNA poly-merase) 1 U/ml. Diethylpyrocarbonate-treated water wasadded up to a final volume of 50  A l. Thirty cycles of PCR were then performed according to the following steps:48  j C, 30 min (once); 94  j C, 30 s; 65  j C, 45 s; 72  j C, 45 s.At the end, the reaction mixture was kept for 15 min at 72  j Cand finally chilled at    4  j C until analysis, which was performed by electrophoretical separation of a 10- A l aliquot of the PCR product on a 2% agarose gel and subsequent visualization by ethidium bromide staining (Bio-Rad, Her-cules, CA, USA). For selection of the primers, we referred tothe National Center for Biotechnology Information database.The primers’sequences were as follows: human IL-8 (sense:5  V -CCACCCATGGCAAATCCAT-GGC-3  V ); antisense: 5  V -TCTCAGCCCTCTTCAAAAACTTCTC-3  V ) [21], and hu- man glyceraldehyde-3-phosphate dehydrogenase (GADPH,‘‘housekeeping’’ gene, sense: 5  V -CCACCCATGGCAAATC-CAT-GGC-3  V ; antisense: 5  V -TCTAGACGGCAGGTCAGGT-CCACC-3  V ) [22]. In the presence of IL-8 mRNA, a 289-base  pair cDNA fragment was expected to be amplified. For thesemiquantitative evaluation of PCR data, mRNA expressionof GADPH was used as a control. Optical density of indi-vidual bands was evaluated by Kodak Camera and Kodak 1D LE 3.5.L software. 2.5. Measurement of [Ca 2+  ] i PMNs were incubated at 37  j C with 5  A M FURA-2/AM(stock solution, 1 mM in dimethyl sulfoxide). After 30 min,cells were washed thrice by gentle centrifugation (300   g  , 5min, 20  j C) and finally resuspended at the concentration of 2  10 6 cells/ml in PBS supplemented with 10 mM HEPES,10 mM glucose, 0.25% BSA and 1 mM CaCl 2  and placed ina thermostatically controlled (37  j C) cuvette equipped witha cuvette stirrer for [Ca 2 + ] i  measurement. Fluorescencemeasurements were performed using a Perkin-Elmer LS-50B spectrofluorimeter. Excitation of FURA-2 was per-formed at 340 and 380 nm. Excitation band widths wereset at 5 nm. The ratio of fluorescence signals emitted at 510 nm was used to calculate the [Ca 2 + ] i  according to Fig. 2. Effect of TTN on the expression of IL-8 mRNA in human PMNs. (A) Time course of IL-8 mRNA levels in control (C) and in TTN (100  A M)-treated (T)cells; (B) concentration–effect relationship for TTN-induced IL-8 mRNA expression; (C) effect of TTN (100  A M) alone and in the presence of BAPTA-AM(10  A M, 45 min) on IL-8 mRNA expression at 5 h. Results shown are from one representative of at least three separate experiments. H=GADPH(housekeeping gene) mRNA; N=negative control (no RNA); M=molecular weight markers.  F. Marino et al. / Cellular Signalling 15 (2003) 511–517   513  Grynkiewicz et al. [23].  R max  and  R min  were obtained after addition of 0.5% Triton X-100 and 1 mM CaCl 2  or 45 mMTris and 50 mM EGTA/Tris, respectively. [Ca 2 + ] i  changeswere calculated as the difference ( D ) between the highest values (peak levels) reached after addition of a given agent and the mean 1-min pretreatment values (resting levels) ineach experiment. 2.6. Statistical analysis Results are presented as means F S.D. of the mean. Thestatistical significance of the differences was determined byone-way analysis of variance followed by Dunnett or Bon-ferroni post test, as appropriate. Concentration–responserelationships were analysed by nonlinear regression using acommercial software (Prism 2.0, GraphPad Software, SanDiego, CA, USA), and a sigmoidal concentration–responsecurve was fitted to find the mean value of the EC 50  (i.e., theconcentration which elicited 50% of the maximal response)together with its 95% confidence interval (CI). 3. Results 3.1. Effect of TTN on IL-8 release and IL-8 mRNAexpression in human PMNs In our experimental conditions, PMNs spontaneouslyreleased IL-8 in a time-dependent fashion with a maximum Fig. 3. Effect of TTN on the expression of IL-8 mRNA in human PMNs.(A) Time course of IL-8 mRNA levels in control (open columns) and inTTN (100  A M)-treated (filled columns) cells; (B) concentration–effect relationship for TTN-induced IL-8 mRNA expression. Optical densityvalues are normalized relatively to the values of the housekeeper GADPHin each experiment. Columns are the means of at least three separateexperiments with bars showing S.D. *=  P  <.05 and **=  P  <.01 vs. control.Fig. 4. Representative tracings showing the effect of TTN (100  A M, added at arrow) on [Ca 2 + ] i  in human PMNs loaded with FURA-2 under standardconditions and in the presence of PTX (4  A g/ml, 4 h), U73122 (3  A M, 60 s), U73343 (3  A M, 60 s), calphostin C (1  A M, 5 min).Fig. 5. Effect of different pharmacological agents on TTN-induced [Ca 2 + ] i rise in human PMNs. Data are expressed as percentage of the effect of TTNalone. Each column is the mean of three to five separate experiments with bars showing S.D. *=  P  <.05 and **=  P  <.01 vs. TTN alone.  F. Marino et al. / Cellular Signalling 15 (2003) 511–517  514  at 3 h followed by a slight decline at 5 h. Incubation withTTN concentration-dependently enhanced IL-8 release at each time interval (Fig. 1, panel A), with the following EC 50 values (with 95% CI): 18.28 (5.50–60.71)  A M at 1 h, 13.58(5.39–34.20)  A M at 3 h and 23.40 (12.80–42.79)  A M at 5 h.Pretreatment with the intracellular Ca 2 + chelator BAPTA-AM (10  A M, 45 min) completely prevented this effect  (Fig.1, panel B). To assess whether TTN-induced IL-8 releaseinvolved increased IL-8 gene expression, the effect of TTNwas also tested on IL-8 mRNA levels. RT-PCR analysisshowed that unstimulated PMNs in the 1- to 5-h time periodexpressed steady-state levels of IL-8 mRNA which rose inthe presence of TTN in a time- and concentration-dependent fashion up to about 4-fold the basal values. As in the case of IL-8 release, BAPTA-AM (10  A M, 45 min) completely prevented the effect of TTN (Figs. 2 and 3). 3.2. Effect of PTX, of PLC and PKC inhibitors and of   pharmacological manipulation of intracellular Ca 2+  storeson TTN-induced [Ca 2+  ] i  changes Because the effect of TTN on IL-8 production wasdependent upon Ca 2 + mobilization, the signal transductionmechanisms underlying TTN-induced [Ca 2 + ] i  changeswere investigated. In agreement with previous observations[12], in human PMNs, TTN concentration-dependentlyinduced a rapid and transient rise of [Ca 2 + ] i , with anEC 50  value (with 95% CI) of 23.97 (15.55–36.95)  A M,and the maximal effect was reached at the concentration of 100  A M. The effect of TTN (100  A M) on [Ca 2 + ] i  was prevented by pretreatment with PTX (4  A g/ml) and con-centration-dependently reduced by the PLC inhibitor U73122 (0.01–3  A M) but not by its inactive analogueU73343 (3  A M). TTN-induced [Ca 2 + ] i  rise was alsosignificantly reduced in the presence of the PKC inhibitor calphostine C, although the effect was incomplete even at the maximal concentration (1  A M) which is selective for the inhibition of PKC (Figs. 4 and 5). The relative contribution of intracellular Ca 2 + stores to the [Ca 2 + ] i  riseinduced by TTN was assessed by using the protonophoreFCCP, which depletes mitochondrial Ca 2 + , and theSERCA pump inhibitor thapsigargine, which inducesCa 2 + release from the endoplasmic reticulum. Typicaltracings showing the effect of the agents per se and onTTN-induced [Ca 2 + ] i  rise are shown in Fig. 6. Addition of  FCCP (5  A M) to the incubation medium evoked a slowrise of [Ca 2 + ] i  ( D =45.01 F 0.57 nM in 20.3 F 2.7 min, n =3). Preincubation with FCCP (5  A M, 20 min), however,had no effect on TTN (100  A M)-induced increases in[Ca 2 + ] i  ( D =305.13 F 15.85 nM with TTN in the presenceof FCCP,  P  >.05 vs.  D =288.62 F 16.53 nM with TTNalone). On the contrary, preincubation with thapsigargine(2  A M, 7 min), which per se induced a rise of [Ca 2 + ] i  upto 100.70 F 23.54 nM over resting values in 5.6 F 0.3 min( n =3), completely prevented the response to TTN (100 A M) ( D =  6.21 F 10.23 nM,  n =3). Fig. 6. Representative tracings showing the effect of pretreatment with FCCP (5  A M) or thapsigargine (2  A M) on TTN (100  A M)-induced [Ca 2 + ] i  rise in humanPMNs loaded with FURA-2.Fig. 7. Effect of different pharmacological agents on TTN-induced IL-8mRNA expression in human PMNs after 5 h incubation. Optical densityvalues are normalized relatively to the values of the housekeeper GADPHin each experiment. Columns are the means of at least three separateexperiments with bars showing S.D. *=  P  <.05 and **=  P  <.01 vs. controland #=  P  <.01 and ##=  P  <.01 vs. TTN alone.  F. Marino et al. / Cellular Signalling 15 (2003) 511–517   515
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