Aggregation of human polymorphonuclear leukocytes by endothelin: role of platelet-activating factor Dulcenombrc
G6mez-Garre,
Manuel
Carlos Lahoruforio
de Nefrologia.
Guerra,
Caramelo,
Fwrdacidtr Jimk
Eva Gonzilez,
Jestis Escanero
Diaz. U~rirwsidad
A fttbwrmt
Received 2X July lYY2. accepled
Antonio
Lbpcz-FarrC,
Amparo
Riesco.
and Jestis Egido
dc Madrid, Ar wit/u RQ-CS Cardlims 2 SW0
Madrid, Spcrirr
I September 1902
The mechanisms by which cndolhclin-I (ET-l) acts on polymorphonuclcar Isukocytcs (PMN) arc insufficiently known. In this that ET-l is a PMN-aggregating agent, and that platelet-activating factor (PAF) is the principal mediator of ET-l-induced PMN aggregation. ET-I induced doserclatcd PMN aggregation. which started I min after ET-l exposure. Two diffcrcnt specific PAF rct’cptor antagonists blocked the cffcct of ET-I on PMN aggregation. In addition, study. WC asscsscd the hypothcscs
FT.1
ifl&cpd
I! qjyifirant
inrresr
in !hc prrr&~!+
nf Ph.!:
+
PMN
rclcasc from PMN rather than accumulation. This PAF prodiction
nftrr
2 !n 5 mip
was dcpcndcnt
-f
FT-!
on intra-
inr!~h+ir>n
FT-1
and cxtraccllular
;-A.--A .::ciii Li ?4F
Ca’*.
In this regard, the PAF rcccptor xntsgonists significantly blunted the ET-I-induced peak in cytosolic free Ca’ ’ ([Cn” Ii). Our results. thcrcforc, indicate that ET-I is cffcctivc in causing aggregation of human PMN and that its action appears tcr hc mediated by PAF production via a Ca”+=dcpcndcnt mechanism. Endothclin; Aggregation: Polymorphonuclcar leukocytes; PAF (platclct-acaivalirig factor, PAF-accthcr)
1. Introduction Inflammation is characterized by the local accumulation of polymorphonuclear leukocytes CPMN), plasma pro!eins and fluid at the site of injury or infection (Pobcr and Cotran, 1990). The aggregation and adhesion of PMN to cndothclial cells are early steps in inflammation. Furthermore. cndothelial cells exposed to inflammatory mediators, such as thrombin, tumor necrosis factor (TNF) and interleukin-I, reacts by synthesizing platelet-activating factor (PAF) and cndothclin-t (ET-11 CKon and Bade, 1991; Zimmerman et al., 1990; Braquet et al., 19871. ET-I is a potent vasoconstrictor peptide which was first purified from the culture supernatant of porcine aortic cndothelial cells (Yanagisawa et al., 19881, although several other cell types can synthesize it after appropriate stimuli (Ken and Badr, 1991). As mentioned above, the concentration of ET-l increases significantly at sites of inflammatory injury (Ken and Badr, 1991). However, the putative role of ET-1 as a coactivator of inflammatory cells, e.g. PMN, has not been sufficiently explored. Recent experiments have
shown that ET-I is capable of eliciting a transient increase in cytosolic free Ca”+ ([Ca’+]liI in PMN (L6pcz-FarrC ct al., 1991b,c) and cnhanccs supcroxidc anion production (lshida et al., 1990). Howcvcr, the functional consequences of the cffr;ct of ET-I on PMN were not asscsscd in cithcr of thcsc studies (LBpezFarr6 et al., 199lb,c; Ishida et al., 1990). The first aim of the present study was therefore to examine the ability of ET-l to activate PMN, asscsscd by PMN aggregation. In a second part, the possible role of PAF as a mediator of ET-l-induced PMN aggregation was inv&Wigatrd by studying P.AF production and the effect of two types of PAF receptor antagonists. PAF is a phosphoiipid with a wide range of biolugical activities and plays a pivotal role in inflammation. It is also a key molecule in PMN activation, causing chcmotaxis, aggregation, supcroxidc rcleast and dcgranulation (Braquct ct al., I987).
2. Materials and methods
Blood was drawn from healthy human donors and PMN wcrc isolated using density-gradient ccntrifugation, scdimcntation and hypotonic lysis of rrythrocytcs (Hcnson ct al., 1072). After separation, the PMN wcrc
suspcndcd in incubated medium. The PMN prcparation was more than Y5% pure (with a contamination of platclcts Iowcr than 3’i 1 and more than YS’ii viahlc hilSl3.l on tepitfl hluc exclusion.
USA) or IO-H M fMLP was added. The incubation was continued for different times at 37°C. Incubations were stopped by the ;rddition of 3 ml I N HCI in methanol.
PMN wcrc resuspcndcd in Krcbs-Hcnsclcit buffer containing, in mM: 11X.4 NaCI, 4.7 KCI. 2.5 CaCI,, 1.2 5 glucose and M&O,, 1.2 KH ZPO,. 25 NaHCO,, 0.2%; bovine strum albumin. pH 7.4. A suspension of PMN (10’ cells/ml) was incuhatcd at 37°C for 2 min in ;I Payton dual-channel aggrcgomctcr with 1000 rpm continuous stirring at then stimulated with ET-I (human. porcine, Bachcm Chcm. Bubcndorf. Switzcrlandl, PAF f l-O-alkyl-2-O-acctyl-sn-~lyccryl-3-ph~~sph~9rylcholine. Sigma Chcm. St. Lc9uis, MO, USA) of fMLP 1N-formyl-mcthionyl-Icucyl-phcnylalaninc, Sigma chcm. St. k9uis, MO. USA). Results arc cxprcsscd as a pcrccntago ci.angc in light transmission with rcspcct to . &;;&;: ;j&i;\bii di’* ;{;i;,; !. iii xmiC ii@Ii iiilfthiitkiiiil;.
matography on precoatcd plates of silica gel 60 in propionic : propanol : water : chloroform (2 : 2 : 1: 1I. The silica was scrapped off in narrow bands hascd on their comi~rati~~nwith PAF standard. The distribution of PAF in the incubation buffer and in the cells was mcasurcd by incubating PMN as dcscribcd and centrifuging the cell suspension tl20t_lO rpm for 1 min) at the end of the agonist incubation period. The cell-free supcrnatant was rcmovcd, the pellet was rcsuspcndcd, and parallel extractions wcrc complctcd on the two fractions.
cxpcriments, PMN wcrc incuba~cd in a Caz+-free medium containing 2.5 mM ctl~ylcnc~lyc~9l~bis-~ pamino-ethyl-cthcr)-N.N ‘-tctraacctic acid (EGTA, Sigma Chcm.. St. Louis. MO, USA)) or prcincubatcd ItI min with IO ’ M YI-!N.N-dictytamino)-octyi-3,4.5tr~mcth~9xybenz~?atc hyd-ochloridc fTMB8, Sigma Chcm., St. Lewis, MO, USA) or the PAF rcccptor antagonists. 5 * iOF’ M BNS202l (Institute Hcnri Beaufour. LC Plcssis-Robinson. France) and !i - 10-“ M WEBZOt(h (Bochringcr Ingclhcim. Germany).
PMN wcrc loaded with fura- as dcscribcd prcviously (Hcnson ct al., lY72). Cells pcllctcd by low-speed ccntrifugation wcrc washed twice and rcsuspcndcd t I.5 x ltl”/mlJ in PSS buffer containing, in mM: 140 NaCl, 4.6 KCI, 1 MgCl,, 2 CaCl?, IO glucose, It) HEPES, pH 7.4 and incubated for 30 min at 37°C. Tr~lnsmissi~)nwas measured at 37°C in a spcctrofluorometer (LS 50 Perkin Elmer) with a stirring cuvettc containing 8tOOr;ll of PSS buffer and 200 ~1 of PMN suspcncion. [Ca”‘li was calculated by using standard equations (Grynkicwicz ct al., IYXS; Okada ct al., IYYO~. assuming a K,r of furafor [Ca” li of 224 nm (L&cz-Farrc c\ al., lYYlb,c; Grynkicwicz ct al., 1985; Okada ct al.. IYYO). F,,,,,, and F,,,,, wcrc dctcrmincd in each oxpcrimcnt by the addition of Triton X-100 (tl.lp/i.) arid 10 mM EGTA. rcspcctivcly.
Lipids wcrc cxtractcd from the methanol phase as dcscribcd by Biigh and Dyer ( 1YiYj. Samplss were dried under N,, dissolved in 200 ~1 of chloroform: methanol (1 :!I) and separated by thin-layer chro-
PAF activity was dctcrmincd by the rclcesc of [ “H~scrstonIr; (Nzw England Nuclear, Boston, MA, Us&j l&in prcioaocd i&&it piareicis in rhc prebcncc of IO’” M indomcthacin, as d~scribcd previously tSunchcz-Crcspo et al.. lY83l. In parallel cxpcrimcnts, PAF production by stimulatcd PMN was mcasurcd as [jH]acctatc (New Eng land Nuclear, Boston, MA, USA) incorporation into PAF. The i~cu~iti~)n prot~~c~)lwas the same as dcscribed above cxccpt that 25 FGi of [‘Hfacctatc was added to 1 ml of PMN suspension. After the silica WBS scrapped off, the radioactivity was dctcrmincd by liquid scintillation spcctromciry (Sisson ct al., 1987). The results arc cxpresscd as the pcrccntage of [*‘H]acctatc inc~)rp~~~tcd by PMN under basal coi~dit~ons{t~kcn as itlt~~~) for caeh cxpcrimcnt. In order to bc ccrtnin that the matcriai cxtractcd and mcasurcd was really PAF, selcctcd samples wcrc analyzed by HPLC using a dual pump Kontron Model 420 (Konrron lnstrumcnt, Zurich, Switzerland) with 2W x 4.t9 mm Sphcri-5 silica c~~lurnn, as dcscrihed prcviousiy (Blank and Snyder, lYK3t. The rctcntion time of PMN [‘I-!lPAF was idcntical to a synthctir labclcd PAF (Cl&CIX mixture, Amcrsham, 5uckinghamshirc, UK).
All values arc mcnns f S&M, C~jrnpar~s~~~sbctwccn means of multiple groups wcrc analyzed by one-way analysis of variaracc and Schcffd’s multiple comparison test. 3, Results
Isolated PNN wcrc rcsuspcndod in Krcbs-Hcnselcit buffer (5.10” PMN/mli and after IO min IO”’ M ET-l, 10 @M A23187 (Sigma Chcm. St. Louis, MO,
TABLE
1
Changes in agpr~gatiun in EGTA- or TMUK-incuhatrd humian PMN dmulatcd by ET-I. Pk4N WCI’Cstimufated for 111min with III-’ M ET-I in a f’;t’ ‘-fret medium cnntaining 2.5 mM EGTA (ad&d at the same rime as ET-I) (n = 5) or pr~~n~u~~~l~~for if1 min with 10 ’ M TMBK in = 5). hggregt;ticm is cxprcssed us fi change in light !rrmsmission.
it P < ft.lF~with respect to PMN stimulated hy ET-I.
aggregated 1 min after ET-1 exposure ifig. II3 Umcline, 2 + I; 10-’ M ET-l, 23 + 5% light t~~~sm~~s~on, P < 0.051, with only a small further increase from 1 to IQ min. The aggregation obtained with ltYx M ET-I was equivslcnt to that produced by lO’-’ M PAF and was significantly elevated compared to that elicited by the ~hemota~ti~ peptide fMLP (IO”” Mf (fig. 18). in an at&S?pt 16 anaiyze the raie of ionized calcium as the intra~e~~u~ar mediator of PMN aggregation induced by ET-l, additional ertpcriments were pcrformed. PMN aggregation induced by ET-l in Ca’+-free mcd~um suntanning 2.5 mM ECTA was signif~~ntly decreased c@mpiarcd to that induced by ET-I in the prrrscncc of Ca”+ (table 1I, Furthermore, ET- I-induced PMN aggregation wats lower in PMN prcincubated with !.O-.e M TME38, an inhibitor of C$.” rclcasc from int~a~~llul~~rstores (table 11. h a second set of experiments, we i~vest~g~lted the role of PAF as a possibtc mediator of ET-l-induced PNN aggr~~ti~9n. As shown in fig. 2, PMN ~~~~re~tiun induced by ET-t was significantly inhibited af!cr 10 min of prcirrcubation with two different specific PAF rl3ccptar ~l?~go~~sts, 5 * fC1m’5M BN52021 or 5 9
IO‘- M WEB2086. Nevertheless, no significant effect of 5 * lQ_” M BN52021 was noted on the PMN aggregation induced by III-^’ M fMII_+P (lUek M WLp, 22 -t 5; ICl-x M tMLP + BNX%21,26 & 5% light transmission). E3NS2tI21(5 * ii)-’ M) by itself did net a&g PMrJ aggr~~tion*
ET-1 enhanced [Ca”+& of PMN in a dose-dcpendent manner, and both Ca”-free medium and TMB8 significantly inhibited the ET-I-induced transient increase in [@a”]i, as recently described by our group (LtSpez-FarrG et al., f99lb,cI. The increase in fCa’+]i induced by IO-’ M ET-l was s~~n~fi~~ntlydiminished by preir?cubating the PMN with 5 1 10e5 M BN52021 for 10 min (fig. 3). As expected, the same results were obtained when cells were stimulated with lo-” M PAF after preincubtition with BN52021. However, no effect of BNS2021 was noted on the increase in fCa’+& irmU3.i @jriii I;.M tfviii-, IIol‘was any efiect oj 3 i i$j .: 111BNS2021 on PMN aggregation.
ET-1 and fMLP i~~du~~da si~~~if~~antincrease in total PAF production by human PMN after 2 min of in~~l~~at~on and the effect was maintained up to 15 rnin (fig. 4). The: amount of PAF produced by ALP-stimulated PMN ~3s lower than produced by ET-l activated PMN* Several ~eport~ indicwtc that the PAF syntllesi~ed by PMN is retained by the cells. WC malyzcd this issue in ~T-~-stim~~l~~tedPMN by n~ca~ur~ng PAF in both the incubation buffm and the ceils. As shown in fig. 5, the ET- {-jljduced release of PAF into the incubatiuln buffer accounted for 72 & 8% of the total amount of PAF
oyI_;___L_u---___u_L_~
15
6
lime
0 _-lO‘* Y El-l
ET-l*BNSWZl
ET-l’WEB2066
Fig. 2. Effect of two specific PAF receptor antagonists on PMN aggregation ind, ted by IO-& M ET-I. PMN were preincuhated for 10 min with S.lOY” M BNS2021 or S.lO-” M WEBZOX6 hefore stimulation with IO-’ M ET-l. Aggregation is expressed as % change in light transmission. Data are meansfS.E.M., n = 5. P < 0.05 with respect io basal conditions. * P < 0.05 with respect lo PMN stimularion with ET-l.
l
Fig. 5. Percentage of PAF released by human PMN at different times after stimulation with IO-’ M ET-l or IO-’ M fMLP. Data are P < 0.05 with respect to baseline values. means* S.E.M.. n = 8. * P < 0.05 with respect to ET-I values. ( ) IO-” M ET-I. (0) IO-”
M IMLP.
produced in 2 min, reaching 91 f 6% 5 min after ET-1 activation. PAF synthesized in response to fMLP stimulation was detected mainly in PMN and not in the Irrr!!hatin!! rn~dl!!rn ‘fig. 51. To confirm the effect of ET-l on PAF production, additional experiments were carried out in which [“HIacetate incorporation into PAF was measured at a fixed time of 5 min. Thus, lOen M ET-1 stimulated the incorporation of [3H]acetate into PAF (212 f 40% with respect to PMN stimulated under basal conditions, P < 0.05). Pretreatment of human PMN with lO-” M TMB8 or the use of a medium containing 2.5 mM EGTA inhibited PAF production induced by ET-I (table 21.
BN62021
Fig. 3. Effect of thr PAF receptor antagonist, 5~10-s M BN52021. on the [Ca” I, transient induced by IO-’ M ET-I, lO1n M PAF or 10 -” M fMLP. The rise in (Ca’ ’ 1,was calculated as the increase in [Ca’* 1,relative to baseline for each individual experiment. Data arc meansfS.E.M., n = 5. * P < 0.05 with respect to stimuli without 1 I\!-* M ET-l, (a) 10~’ M PhF, (a) 10KH M NLP.
250
(mid
fl
4. Discussion Our result?; demonstrate that ET-I is an effective aggregating agent of human PMN and that this effect is mediated, in a significant proportion, through PAF production. In this regard, BN52021 and WEB2086, two potent specific PAF receptor antagonists (Braquct et al., 1985; Casals-Stenzels ct al., 1983, blocked ETI-induced PMN aggregation. Upcz-FarrC et al. (1991a)
*
TABLE
2
Effect of EGTA or TMBH on PAF production hy PMN stimulated with IO-’ M ET-I. PMN were stimulated 5 min with 10~’ M ET-I in a medium containing 2.5 mM EGTA (added iIt the same time as ET-I) (n = 4) or preincubatcd with IO’.’ M TMBH (n = 4). 2
6
15
Time (min) Fig. 4. PAF production hy human PMN at different times afkr stimulation with IO -” M ET-I, IO yM A23lX7 or IO ” M fMLP. Data are means* S.E.M.. n = X. P < 0.05 with rcspcct to hascline ) Baseline. tfs) IO PM A23187, (I#) IO--” M ET-l, to) IOmH M fMLP.
Treatment
pg PAF/S*
Baseline IO-‘” M ET-I ET- I i.2.S mM EGTA ET-I t, IO ’ M TMBH
43* H 17f**40 ” 3Of 3 ” 41)f IO ”
IO” PMN
” P < 0.05 with rcspcct to harclinc values. ” P < 0.05 with rcspcct td PMN stimulalcd with IO ” M ET-I.
171
recently suggested that the effects of ET-I on renal function and on mesangial cell contraction may be related to the incrcasc in PAF synthesis by those cells. Many studies have suggested that Ca’+ influx and the increase in [Ca ‘+j, trigger PMN aggregation induced by several agents (O’Flaherty et al., 1978). The observation that TMBX or Ca2+-fret medium blocked ET-I-induced PMN aggregation further suggests an important role for both extracellular and intracellular Ca2+ in the ET-l effect. ET-I increases the intracellular Ca2+ level in many cells (Ken and Badr, 19911, including human PMN Upez-Farr6 et al., 199lb,c). In the present study, we fo!md that the PAF or ET-l-induced increase in [Ca2+li was inhibited by the PAF receptor antagonists. Neverthelcss, BNS2021 was not able to inhibit the fMLP-induced increase in [Ca’+]i. Thus, the inhibition by BN52021 or WEB2086 of PAP; and ET-l-induced aggregation seems to bc quite specific. To explore further the possibility that PAF production rn?!!sl mrdiqtr !t?P n!=pV+ PffeCtr rf FT-1 nn PMN, we measured PAF synthesis by bioassay and by incorporation of [3H]acetat.e into labeled PAF. Mueller et al. (1984) found previously that human PMN synthesize labeled PAF from t3H]acetate. As shown in this study, PAF production by PMN stimulated with ET-l was demonstrated by both methods. Furthermore, PAF produced as a result of ET-l stimulation was dctectcd in the incubation buffer, whereas, PAF produced as a result of fMLP stimulation was detected mainly in the cell fraction. These data suggcstcd that fMLP stimulates the synthesis but not the rcleasc of PAF from PMN, as reported by other authors (Sisson ct al., 19871, whereas ET-l stimulates both the synthesis and the release of PAF from PMN. This is an important point, since PAF must be released in order to act on the rcccptor localized at the plasma mcmbranc Icvcl. This may explain why PAF antagonists inhibited the PMN aggregation and the intracellular free @a’* increase induced by ET-I but not by fMLP. That this effect was dccrcased by TMB8- or EGTA-containing medium further suggests a pivotal role of both transmembrane Ca”” flux and Ca” rclcasc from intraccllular stores. With regard to the possible mechanism of ET-l-stimulated PAF synthesis, ET-l has been rcported to activate phospholipasc A, in various tissues (DeNucci ct al., 1988) which, if prcscnt in human PMN, may provide substrates for acctyltransferasc and, subsequently, for PAF synthesis. Regarding tl:d possible pathophysiological relcvancc of the action of ET-I on PMN, Ishida ct al. (l990) have recently rcportcd that ET-l primes the enhanccmcnt of supcroxide production by PMN stimulated by the chemotactic pcptide IMLP. In this regard, WC recently rcportcd preliminary results demonstrating that a singlc bolus injection of ET-l into conscious rats induced
a rapid dc*.rcasc in the number of circulating PMN (tipez-FarrC et al., 1991~). In line with this, our present Findings suggest that ET-I may participate in inflamniatory reactions.
Acknowledgements This work was partially supported by grants from Fondo de Investigaciones Sani(arias de la Seguridad Social (F&s) YO/229. 91 /lh2. Y2/972 and Fundacicin Iiiigo Alvarez de Toledo. D.G.C. is ;I fellow of the Fundacicin Conchite Ribago, A.R. is a fellow of FISss. A.L.F. is a postdoctoral fellow of the Fundaci6n JimCnez Diaz and Boehringcr Mannheim. M.G. and J. Escanero are visiting investigators from the University of Zungoza. The authors thank L. Gulliksen for secretarial assistance.
References Blank. M.L. 2nd F. Snyder. lY83. lmprovcd high-performance liquid “i.7,....,. t ,.,.$.,._,.:,. . ... !...Ati_: . ..1..*:.._ _I...“l... ,.-.:..,:t&.. . l.. .- ..c= i..i;_l. -..;-._ .,..a F..,*.,_ ,..~i from oihe:, p~&hdl&db, J: Chromatogr. 273, 415. Bligh. E.G. and W.J. Dyer. IYSY, A rapid method of total lipid extraclion and purification. Con. J. Biochem. Physiol. 37, 01 I. Brnquet, P.. B. Spinncwyn. M. Bruquel. R.H. Bourgain. J.E. Taylor, A. Etienne and K. Duew, lYH5, BNZi12i nnd related compounds: :I IICW series of high specific PAF-acether receptor antagonists isohtled from (iirrkgo bihh. Blood Vessels lb, 559. Bruquet. P.. L. Touqui. T.Y. Shen and B.B. Vorguftig. IYX7, Pcrspeclives in pl:l(clct-activ:ttilIg factor research. Phnrmacol. Rev, 30. 07. Ct~sul~-Stct~~cl~, J.. G. Muucevir nnd K. Wcber, 1087. Phorm;lcologiC:IIactions of WEB 20X0. :I new specific antugonist to plntclct uctivuling fnclor. J. I’ll;NVlilCOl. Exp. Thcr. 241. 947. Dc Nucci. G.. R. Thomus. I’. D’Orlcnns-Juste, E. Anluncs, C. Wnldcr. T.D. Warner :IIKIJ.R. Vtme. IYHH. Prcssor effects ol circulilting cndothclin ilIX limited uy its rcmovul in lhc pulmon;uy circul;itioti iiiid hy rclcasc of pros(ucycliti mid ciiilotlicliuiii-tcrived relnxing fuctor, Proc. Null. Acnd. SCI.‘2s. 0707. Grynkicwicz. G., M. Poenie and R.Y. Tsien. IYXS. A new gcncration of G?’ indicillors with grCiltly improved fluorescent propcrlics, J. Uiol. Clwn. 260, 3440. I Icnson, P.M.. II.B. Johnson und H.L. Spiclgclhcrg, 1972. The reIcescof grunulc cnzymcs from hummt ncutrophils stimulutetl hy :lggrCgillCll
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G6mez-Garre,
Manuel
Carlos Lahoruforio
de Nefrologia.
Guerra,
Caramelo,
Fwrdacidtr Jimk
Eva Gonzilez,
Jestis Escanero
Diaz. U~rirwsidad
A fttbwrmt
Received 2X July lYY2. accepled
Antonio
Lbpcz-FarrC,
Amparo
Riesco.
and Jestis Egido
dc Madrid, Ar wit/u RQ-CS Cardlims 2 SW0
Madrid, Spcrirr
I September 1902
The mechanisms by which cndolhclin-I (ET-l) acts on polymorphonuclcar Isukocytcs (PMN) arc insufficiently known. In this that ET-l is a PMN-aggregating agent, and that platelet-activating factor (PAF) is the principal mediator of ET-l-induced PMN aggregation. ET-I induced doserclatcd PMN aggregation. which started I min after ET-l exposure. Two diffcrcnt specific PAF rct’cptor antagonists blocked the cffcct of ET-I on PMN aggregation. In addition, study. WC asscsscd the hypothcscs
FT.1
ifl&cpd
I! qjyifirant
inrresr
in !hc prrr&~!+
nf Ph.!:
+
PMN
rclcasc from PMN rather than accumulation. This PAF prodiction
nftrr
2 !n 5 mip
was dcpcndcnt
-f
FT-!
on intra-
inr!~h+ir>n
FT-1
and cxtraccllular
;-A.--A .::ciii Li ?4F
Ca’*.
In this regard, the PAF rcccptor xntsgonists significantly blunted the ET-I-induced peak in cytosolic free Ca’ ’ ([Cn” Ii). Our results. thcrcforc, indicate that ET-I is cffcctivc in causing aggregation of human PMN and that its action appears tcr hc mediated by PAF production via a Ca”+=dcpcndcnt mechanism. Endothclin; Aggregation: Polymorphonuclcar leukocytes; PAF (platclct-acaivalirig factor, PAF-accthcr)
1. Introduction Inflammation is characterized by the local accumulation of polymorphonuclear leukocytes CPMN), plasma pro!eins and fluid at the site of injury or infection (Pobcr and Cotran, 1990). The aggregation and adhesion of PMN to cndothclial cells are early steps in inflammation. Furthermore. cndothelial cells exposed to inflammatory mediators, such as thrombin, tumor necrosis factor (TNF) and interleukin-I, reacts by synthesizing platelet-activating factor (PAF) and cndothclin-t (ET-11 CKon and Bade, 1991; Zimmerman et al., 1990; Braquet et al., 19871. ET-I is a potent vasoconstrictor peptide which was first purified from the culture supernatant of porcine aortic cndothelial cells (Yanagisawa et al., 19881, although several other cell types can synthesize it after appropriate stimuli (Ken and Badr, 1991). As mentioned above, the concentration of ET-l increases significantly at sites of inflammatory injury (Ken and Badr, 1991). However, the putative role of ET-1 as a coactivator of inflammatory cells, e.g. PMN, has not been sufficiently explored. Recent experiments have
shown that ET-I is capable of eliciting a transient increase in cytosolic free Ca”+ ([Ca’+]liI in PMN (L6pcz-FarrC ct al., 1991b,c) and cnhanccs supcroxidc anion production (lshida et al., 1990). Howcvcr, the functional consequences of the cffr;ct of ET-I on PMN were not asscsscd in cithcr of thcsc studies (LBpezFarr6 et al., 199lb,c; Ishida et al., 1990). The first aim of the present study was therefore to examine the ability of ET-l to activate PMN, asscsscd by PMN aggregation. In a second part, the possible role of PAF as a mediator of ET-l-induced PMN aggregation was inv&Wigatrd by studying P.AF production and the effect of two types of PAF receptor antagonists. PAF is a phosphoiipid with a wide range of biolugical activities and plays a pivotal role in inflammation. It is also a key molecule in PMN activation, causing chcmotaxis, aggregation, supcroxidc rcleast and dcgranulation (Braquct ct al., I987).
2. Materials and methods
Blood was drawn from healthy human donors and PMN wcrc isolated using density-gradient ccntrifugation, scdimcntation and hypotonic lysis of rrythrocytcs (Hcnson ct al., 1072). After separation, the PMN wcrc
suspcndcd in incubated medium. The PMN prcparation was more than Y5% pure (with a contamination of platclcts Iowcr than 3’i 1 and more than YS’ii viahlc hilSl3.l on tepitfl hluc exclusion.
USA) or IO-H M fMLP was added. The incubation was continued for different times at 37°C. Incubations were stopped by the ;rddition of 3 ml I N HCI in methanol.
PMN wcrc resuspcndcd in Krcbs-Hcnsclcit buffer containing, in mM: 11X.4 NaCI, 4.7 KCI. 2.5 CaCI,, 1.2 5 glucose and M&O,, 1.2 KH ZPO,. 25 NaHCO,, 0.2%; bovine strum albumin. pH 7.4. A suspension of PMN (10’ cells/ml) was incuhatcd at 37°C for 2 min in ;I Payton dual-channel aggrcgomctcr with 1000 rpm continuous stirring at then stimulated with ET-I (human. porcine, Bachcm Chcm. Bubcndorf. Switzcrlandl, PAF f l-O-alkyl-2-O-acctyl-sn-~lyccryl-3-ph~~sph~9rylcholine. Sigma Chcm. St. Lc9uis, MO, USA) of fMLP 1N-formyl-mcthionyl-Icucyl-phcnylalaninc, Sigma chcm. St. k9uis, MO. USA). Results arc cxprcsscd as a pcrccntago ci.angc in light transmission with rcspcct to . &;;&;: ;j&i;\bii di’* ;{;i;,; !. iii xmiC ii@Ii iiilfthiitkiiiil;.
matography on precoatcd plates of silica gel 60 in propionic : propanol : water : chloroform (2 : 2 : 1: 1I. The silica was scrapped off in narrow bands hascd on their comi~rati~~nwith PAF standard. The distribution of PAF in the incubation buffer and in the cells was mcasurcd by incubating PMN as dcscribcd and centrifuging the cell suspension tl20t_lO rpm for 1 min) at the end of the agonist incubation period. The cell-free supcrnatant was rcmovcd, the pellet was rcsuspcndcd, and parallel extractions wcrc complctcd on the two fractions.
cxpcriments, PMN wcrc incuba~cd in a Caz+-free medium containing 2.5 mM ctl~ylcnc~lyc~9l~bis-~ pamino-ethyl-cthcr)-N.N ‘-tctraacctic acid (EGTA, Sigma Chcm.. St. Louis. MO, USA)) or prcincubatcd ItI min with IO ’ M YI-!N.N-dictytamino)-octyi-3,4.5tr~mcth~9xybenz~?atc hyd-ochloridc fTMB8, Sigma Chcm., St. Lewis, MO, USA) or the PAF rcccptor antagonists. 5 * iOF’ M BNS202l (Institute Hcnri Beaufour. LC Plcssis-Robinson. France) and !i - 10-“ M WEBZOt(h (Bochringcr Ingclhcim. Germany).
PMN wcrc loaded with fura- as dcscribcd prcviously (Hcnson ct al., lY72). Cells pcllctcd by low-speed ccntrifugation wcrc washed twice and rcsuspcndcd t I.5 x ltl”/mlJ in PSS buffer containing, in mM: 140 NaCl, 4.6 KCI, 1 MgCl,, 2 CaCl?, IO glucose, It) HEPES, pH 7.4 and incubated for 30 min at 37°C. Tr~lnsmissi~)nwas measured at 37°C in a spcctrofluorometer (LS 50 Perkin Elmer) with a stirring cuvettc containing 8tOOr;ll of PSS buffer and 200 ~1 of PMN suspcncion. [Ca”‘li was calculated by using standard equations (Grynkicwicz ct al., IYXS; Okada ct al., IYYO~. assuming a K,r of furafor [Ca” li of 224 nm (L&cz-Farrc c\ al., lYYlb,c; Grynkicwicz ct al., 1985; Okada ct al.. IYYO). F,,,,,, and F,,,,, wcrc dctcrmincd in each oxpcrimcnt by the addition of Triton X-100 (tl.lp/i.) arid 10 mM EGTA. rcspcctivcly.
Lipids wcrc cxtractcd from the methanol phase as dcscribcd by Biigh and Dyer ( 1YiYj. Samplss were dried under N,, dissolved in 200 ~1 of chloroform: methanol (1 :!I) and separated by thin-layer chro-
PAF activity was dctcrmincd by the rclcesc of [ “H~scrstonIr; (Nzw England Nuclear, Boston, MA, Us&j l&in prcioaocd i&&it piareicis in rhc prebcncc of IO’” M indomcthacin, as d~scribcd previously tSunchcz-Crcspo et al.. lY83l. In parallel cxpcrimcnts, PAF production by stimulatcd PMN was mcasurcd as [jH]acctatc (New Eng land Nuclear, Boston, MA, USA) incorporation into PAF. The i~cu~iti~)n prot~~c~)lwas the same as dcscribed above cxccpt that 25 FGi of [‘Hfacctatc was added to 1 ml of PMN suspension. After the silica WBS scrapped off, the radioactivity was dctcrmincd by liquid scintillation spcctromciry (Sisson ct al., 1987). The results arc cxpresscd as the pcrccntage of [*‘H]acctatc inc~)rp~~~tcd by PMN under basal coi~dit~ons{t~kcn as itlt~~~) for caeh cxpcrimcnt. In order to bc ccrtnin that the matcriai cxtractcd and mcasurcd was really PAF, selcctcd samples wcrc analyzed by HPLC using a dual pump Kontron Model 420 (Konrron lnstrumcnt, Zurich, Switzerland) with 2W x 4.t9 mm Sphcri-5 silica c~~lurnn, as dcscrihed prcviousiy (Blank and Snyder, lYK3t. The rctcntion time of PMN [‘I-!lPAF was idcntical to a synthctir labclcd PAF (Cl&CIX mixture, Amcrsham, 5uckinghamshirc, UK).
All values arc mcnns f S&M, C~jrnpar~s~~~sbctwccn means of multiple groups wcrc analyzed by one-way analysis of variaracc and Schcffd’s multiple comparison test. 3, Results
Isolated PNN wcrc rcsuspcndod in Krcbs-Hcnselcit buffer (5.10” PMN/mli and after IO min IO”’ M ET-l, 10 @M A23187 (Sigma Chcm. St. Louis, MO,
TABLE
1
Changes in agpr~gatiun in EGTA- or TMUK-incuhatrd humian PMN dmulatcd by ET-I. Pk4N WCI’Cstimufated for 111min with III-’ M ET-I in a f’;t’ ‘-fret medium cnntaining 2.5 mM EGTA (ad&d at the same rime as ET-I) (n = 5) or pr~~n~u~~~l~~for if1 min with 10 ’ M TMBK in = 5). hggregt;ticm is cxprcssed us fi change in light !rrmsmission.
it P < ft.lF~with respect to PMN stimulated hy ET-I.
aggregated 1 min after ET-1 exposure ifig. II3 Umcline, 2 + I; 10-’ M ET-l, 23 + 5% light t~~~sm~~s~on, P < 0.051, with only a small further increase from 1 to IQ min. The aggregation obtained with ltYx M ET-I was equivslcnt to that produced by lO’-’ M PAF and was significantly elevated compared to that elicited by the ~hemota~ti~ peptide fMLP (IO”” Mf (fig. 18). in an at&S?pt 16 anaiyze the raie of ionized calcium as the intra~e~~u~ar mediator of PMN aggregation induced by ET-l, additional ertpcriments were pcrformed. PMN aggregation induced by ET-l in Ca’+-free mcd~um suntanning 2.5 mM ECTA was signif~~ntly decreased c@mpiarcd to that induced by ET-I in the prrrscncc of Ca”+ (table 1I, Furthermore, ET- I-induced PMN aggregation wats lower in PMN prcincubated with !.O-.e M TME38, an inhibitor of C$.” rclcasc from int~a~~llul~~rstores (table 11. h a second set of experiments, we i~vest~g~lted the role of PAF as a possibtc mediator of ET-l-induced PNN aggr~~ti~9n. As shown in fig. 2, PMN ~~~~re~tiun induced by ET-t was significantly inhibited af!cr 10 min of prcirrcubation with two different specific PAF rl3ccptar ~l?~go~~sts, 5 * fC1m’5M BN52021 or 5 9
IO‘- M WEB2086. Nevertheless, no significant effect of 5 * lQ_” M BN52021 was noted on the PMN aggregation induced by III-^’ M fMII_+P (lUek M WLp, 22 -t 5; ICl-x M tMLP + BNX%21,26 & 5% light transmission). E3NS2tI21(5 * ii)-’ M) by itself did net a&g PMrJ aggr~~tion*
ET-1 enhanced [Ca”+& of PMN in a dose-dcpendent manner, and both Ca”-free medium and TMB8 significantly inhibited the ET-I-induced transient increase in [@a”]i, as recently described by our group (LtSpez-FarrG et al., f99lb,cI. The increase in fCa’+]i induced by IO-’ M ET-l was s~~n~fi~~ntlydiminished by preir?cubating the PMN with 5 1 10e5 M BN52021 for 10 min (fig. 3). As expected, the same results were obtained when cells were stimulated with lo-” M PAF after preincubtition with BN52021. However, no effect of BNS2021 was noted on the increase in fCa’+& irmU3.i @jriii I;.M tfviii-, IIol‘was any efiect oj 3 i i$j .: 111BNS2021 on PMN aggregation.
ET-1 and fMLP i~~du~~da si~~~if~~antincrease in total PAF production by human PMN after 2 min of in~~l~~at~on and the effect was maintained up to 15 rnin (fig. 4). The: amount of PAF produced by ALP-stimulated PMN ~3s lower than produced by ET-l activated PMN* Several ~eport~ indicwtc that the PAF syntllesi~ed by PMN is retained by the cells. WC malyzcd this issue in ~T-~-stim~~l~~tedPMN by n~ca~ur~ng PAF in both the incubation buffm and the ceils. As shown in fig. 5, the ET- {-jljduced release of PAF into the incubatiuln buffer accounted for 72 & 8% of the total amount of PAF
oyI_;___L_u---___u_L_~
15
6
lime
0 _-lO‘* Y El-l
ET-l*BNSWZl
ET-l’WEB2066
Fig. 2. Effect of two specific PAF receptor antagonists on PMN aggregation ind, ted by IO-& M ET-I. PMN were preincuhated for 10 min with S.lOY” M BNS2021 or S.lO-” M WEBZOX6 hefore stimulation with IO-’ M ET-l. Aggregation is expressed as % change in light transmission. Data are meansfS.E.M., n = 5. P < 0.05 with respect io basal conditions. * P < 0.05 with respect lo PMN stimularion with ET-l.
l
Fig. 5. Percentage of PAF released by human PMN at different times after stimulation with IO-’ M ET-l or IO-’ M fMLP. Data are P < 0.05 with respect to baseline values. means* S.E.M.. n = 8. * P < 0.05 with respect to ET-I values. ( ) IO-” M ET-I. (0) IO-”
M IMLP.
produced in 2 min, reaching 91 f 6% 5 min after ET-1 activation. PAF synthesized in response to fMLP stimulation was detected mainly in PMN and not in the Irrr!!hatin!! rn~dl!!rn ‘fig. 51. To confirm the effect of ET-l on PAF production, additional experiments were carried out in which [“HIacetate incorporation into PAF was measured at a fixed time of 5 min. Thus, lOen M ET-1 stimulated the incorporation of [3H]acetate into PAF (212 f 40% with respect to PMN stimulated under basal conditions, P < 0.05). Pretreatment of human PMN with lO-” M TMB8 or the use of a medium containing 2.5 mM EGTA inhibited PAF production induced by ET-I (table 21.
BN62021
Fig. 3. Effect of thr PAF receptor antagonist, 5~10-s M BN52021. on the [Ca” I, transient induced by IO-’ M ET-I, lO1n M PAF or 10 -” M fMLP. The rise in (Ca’ ’ 1,was calculated as the increase in [Ca’* 1,relative to baseline for each individual experiment. Data arc meansfS.E.M., n = 5. * P < 0.05 with respect to stimuli without 1 I\!-* M ET-l, (a) 10~’ M PhF, (a) 10KH M NLP.
250
(mid
fl
4. Discussion Our result?; demonstrate that ET-I is an effective aggregating agent of human PMN and that this effect is mediated, in a significant proportion, through PAF production. In this regard, BN52021 and WEB2086, two potent specific PAF receptor antagonists (Braquct et al., 1985; Casals-Stenzels ct al., 1983, blocked ETI-induced PMN aggregation. Upcz-FarrC et al. (1991a)
*
TABLE
2
Effect of EGTA or TMBH on PAF production hy PMN stimulated with IO-’ M ET-I. PMN were stimulated 5 min with 10~’ M ET-I in a medium containing 2.5 mM EGTA (added iIt the same time as ET-I) (n = 4) or preincubatcd with IO’.’ M TMBH (n = 4). 2
6
15
Time (min) Fig. 4. PAF production hy human PMN at different times afkr stimulation with IO -” M ET-I, IO yM A23lX7 or IO ” M fMLP. Data are means* S.E.M.. n = X. P < 0.05 with rcspcct to hascline ) Baseline. tfs) IO PM A23187, (I#) IO--” M ET-l, to) IOmH M fMLP.
Treatment
pg PAF/S*
Baseline IO-‘” M ET-I ET- I i.2.S mM EGTA ET-I t, IO ’ M TMBH
43* H 17f**40 ” 3Of 3 ” 41)f IO ”
IO” PMN
” P < 0.05 with rcspcct to harclinc values. ” P < 0.05 with rcspcct td PMN stimulalcd with IO ” M ET-I.
171
recently suggested that the effects of ET-I on renal function and on mesangial cell contraction may be related to the incrcasc in PAF synthesis by those cells. Many studies have suggested that Ca’+ influx and the increase in [Ca ‘+j, trigger PMN aggregation induced by several agents (O’Flaherty et al., 1978). The observation that TMBX or Ca2+-fret medium blocked ET-I-induced PMN aggregation further suggests an important role for both extracellular and intracellular Ca2+ in the ET-l effect. ET-I increases the intracellular Ca2+ level in many cells (Ken and Badr, 19911, including human PMN Upez-Farr6 et al., 199lb,c). In the present study, we fo!md that the PAF or ET-l-induced increase in [Ca2+li was inhibited by the PAF receptor antagonists. Neverthelcss, BNS2021 was not able to inhibit the fMLP-induced increase in [Ca’+]i. Thus, the inhibition by BN52021 or WEB2086 of PAP; and ET-l-induced aggregation seems to bc quite specific. To explore further the possibility that PAF production rn?!!sl mrdiqtr !t?P n!=pV+ PffeCtr rf FT-1 nn PMN, we measured PAF synthesis by bioassay and by incorporation of [3H]acetat.e into labeled PAF. Mueller et al. (1984) found previously that human PMN synthesize labeled PAF from t3H]acetate. As shown in this study, PAF production by PMN stimulated with ET-l was demonstrated by both methods. Furthermore, PAF produced as a result of ET-l stimulation was dctectcd in the incubation buffer, whereas, PAF produced as a result of fMLP stimulation was detected mainly in the cell fraction. These data suggcstcd that fMLP stimulates the synthesis but not the rcleasc of PAF from PMN, as reported by other authors (Sisson ct al., 19871, whereas ET-l stimulates both the synthesis and the release of PAF from PMN. This is an important point, since PAF must be released in order to act on the rcccptor localized at the plasma mcmbranc Icvcl. This may explain why PAF antagonists inhibited the PMN aggregation and the intracellular free @a’* increase induced by ET-I but not by fMLP. That this effect was dccrcased by TMB8- or EGTA-containing medium further suggests a pivotal role of both transmembrane Ca”” flux and Ca” rclcasc from intraccllular stores. With regard to the possible mechanism of ET-l-stimulated PAF synthesis, ET-l has been rcported to activate phospholipasc A, in various tissues (DeNucci ct al., 1988) which, if prcscnt in human PMN, may provide substrates for acctyltransferasc and, subsequently, for PAF synthesis. Regarding tl:d possible pathophysiological relcvancc of the action of ET-I on PMN, Ishida ct al. (l990) have recently rcportcd that ET-l primes the enhanccmcnt of supcroxide production by PMN stimulated by the chemotactic pcptide IMLP. In this regard, WC recently rcportcd preliminary results demonstrating that a singlc bolus injection of ET-l into conscious rats induced
a rapid dc*.rcasc in the number of circulating PMN (tipez-FarrC et al., 1991~). In line with this, our present Findings suggest that ET-I may participate in inflamniatory reactions.
Acknowledgements This work was partially supported by grants from Fondo de Investigaciones Sani(arias de la Seguridad Social (F&s) YO/229. 91 /lh2. Y2/972 and Fundacicin Iiiigo Alvarez de Toledo. D.G.C. is ;I fellow of the Fundacicin Conchite Ribago, A.R. is a fellow of FISss. A.L.F. is a postdoctoral fellow of the Fundaci6n JimCnez Diaz and Boehringcr Mannheim. M.G. and J. Escanero are visiting investigators from the University of Zungoza. The authors thank L. Gulliksen for secretarial assistance.
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