E. J. Pettit and M. B. Hallett* SUMMARY

Transcription

1 Journl of Cell Science 109, (1996) Printed in Gret Britin The Compny of Biologists Limited 1996 JCS Loclised nd glol cytosolic C 2+ chnges in neutrophils during enggement of Cd11/CD18 integrin visulised using confocl lser scnning reconstruction E. J. Pettit nd M. B. Hllett* Moleculr Signlling Group, University Deprtment of Surgery, University of Wles College of Medicine, Heth Prk, Crdiff CF4 4XN, UK *Author for correspondence SUMMARY A confocl lser scnning technique ws used to provide opticl sections in the verticl plne through living neutrophils during enggement of integrin nd shpe chnge. This hs permitted the visulistion of cytosolic free C 2+ rises loclised to the sites of integrin immoilistion. These loclised C 2+ chnges resulted from the relese of C 2+ from intrcellulr stores, nd were not inhiited y removl of extrcellulr C 2+ or y the C 2+ chnnel locking nickel ions. After integrin enggement nd initil cell shpe chnges, second rpid phse of cell spreding occurred, which ws ccompnied y glol C 2+ signlling ursts tht continued spordiclly fter mximum spreding. This glol signlling ws driven minly y C 2+ influx from the extrcellulr medium. We propose tht the loclised nd glol C 2+ signlling triggered y integrin enggement results from common underlying mechnism nd these signls re importnt for neutrophil shpe chnge nd extrvstion. Key words: Confocl imging, Cytosolic C 2+, Integrin, Neutrophil, Signlling INTRODUCTION Adhesion molecules on neutrophils re importnt for the process of extrvstion to occur during the inflmmtory response (Springer, 1990; Hynes, 1992). Although selectins re involved in slowing neutrophils from the circultion nd inducing rolling long the endothelil cell lyer (Bevilcqu, 1993), it is only fter integrin enggement t sites of ICAM-1 expression tht neutrophils re signlled to chnge shpe, ecome motile nd extrvste (Springer, 1990; Hynes, 1992). C 2+ signlling is triggered y cross-linking ntiodies ound to integrin CD11/CD18 on the neutrophil surfce (Richter et l., 1990; Ng- Sikorski et l., 1991; Petersen et l., 1993) nd results from loclised increse in C 2+ t the periphery of the cell (Petersen et l., 1993). The mjor prolem with the interprettion in previous studies hs een the inility to determine the time nd position t which integrin enggement occurred. Furthermore, it hs not een possile to correlte integrin enggement induced C 2+ signlling with cell shpe chnge nd dherence. Here, we hve used confocl lser scnning microscope to reconstruct verticl (z plne) view through fluo3-loded neutrophils. This provides n opticl slice in which the contct points etween the cell nd the underlying sustrte cn e clerly defined nd their reltionship to cytosolic free C 2+ concentrtion chnges determined. We show here tht loclised C 2+ signlling occurs t specific contct sites, where integrin enggement occurred. MATERIALS AND METHODS Neutrophil preprtion Neutrophils were isolted from heprinized lood of helthy volunteers s descried previously (Hllett et l., 1990). Following dextrn sedimenttion, centrifugtion through Ficoll-Pque (Phrmci) nd hypotonic lysis of red cells, neutrophils were wshed nd resuspended in Kres uffer (120 mm NCl, 4.8 mm KCl, 1.2 mm KH 2PO 4, 1.2 mm MgSO 4, 1.3 mm CCl 2, 25 mm Hepes nd 0.1% ovine serum lumin, djusted to ph 7.4 with NOH). Integrin inding surfce Antiodies (Dko, High Wycome, UK) to the integrin suunits α (CD11) or β (CD18) were dhered to glss surfces in sufficient density to ensure tht integrin enggement on neutrophils occurred (Pettit nd Hllett, 1994). The mount of dherent ntiody, quntified using n ECL second ntiody technique, ws estimted t pprox molecules/µm 2. This ws sufficient to cuse crosslinking nd oxidse ctivtion (Pettit nd Hllett, 1994). Confocl imging nd mesurement Neutrophils were loded with fluo3 y incution with cetoxymethyl ester (Moleculr Proes, Oregon) s previously descried (Mint et l., 1989), to give n estimted cytosolic concentrtion of 100 µm, resulting in n increse in C 2+ uffering cpcity of the cytosol of pproximtely 5-10% (Al-Mohnn nd Hllett, 1988). Confocl z- plne imges, which permitted the timing of neutrophil touch-down onto the integrin engging surfce to e determined were otined using CLSM-Fluovert confocl lser scnning microscope (Leic,

2 1690 E. J. Pettit nd M. B. Hllett Heidelerg, Germny), y stepping the microscope stge through the confocl lser line scnning in the x direction to produce trnsverse imges t totl rte of 0.08/second (i.e. the intervl etween successive imges ws 12.5 seconds). The time to cquire single imge ws 4 seconds. In order to prevent photoleching of fluo3 during the experiment, the lser power ws ttenuted. The loss of signl ws offset y incresing the voltge of the photomultiplier detectors with susequent increse in the noise on the imges. This is pprent in unprocessed imges (see Fig. 1), ut ws reduced y nerest neighour smoothing lgorithm. A similr procedure ws used to cquire imges of integrin distriution in neutrophils, pre-coted with fluorescein-conjugted ntiody to the β-suunit (CD18, Dko High Wycome, UK) efore sedimenttion on to nti-α-suunit coted glss. The z-plne imging rte ws sufficiently fst to cpture events during touch-down of fluo3-loded nd integrin lelled neutrophils onto surfces coted with nti-cd11 ntiody. Fster time resolution C 2+ mesurements were mde y positioning the lser scnning line in the x direction pproximtely 1 µm ove the ntiody-coted surfce, prllel to the plne of the surfce. Neutrophils were then permitted to sediment onto the surfce. Cells which fell into the scnning line were consequently imged with x direction sptil informtion nd with temporl resolution of 12.5 milliseconds. By ngling the ntiody-coted surfce reltive to the lser scnning direction, rpid scnning, with the sme resolution ws lso cheived through plne which sectioned oth the cell nd its underlying sustrte. All mesurements were performed with cells mintined t 37 C, using specilly constructed heting chmer. The cytosolic free C 2+ concentrtion ws clculted using the stndrd eqution with K d for fluo3 of 864 nm (Merritt et l., 1991). cells in which shpe chnge ws prevented y cytochlsin B (Fig. 3). This demonstrted tht C 2+ signlling ws not consequence of cell shpe chnge. Buffering the loclised C 2+ chnges with intrcellulr BAPTA (concentrtion estimted to e pprox. 5 mm) prevented oth firm dherence nd the susequent cell shpe chnge (Fig. 3). These results were, therefore, consistent with the loclised C 2+ signlling resulting from integrin enggement t the surfce nd eing responsile for mediting n erly stge of neutrophil dherence. 24 s 6 min RESULTS Loclised C 2+ signlling during integrin enggement As neutrophils contct the nti-cd11-coted surfce, they rpidly deform nd spred out (Fig. 1). The cytosolic free C 2+ concentrtion closest to the deformed regions of memrne, t the integrin-engging contct points, ws trnsiently elevted to out 1 µm (Fig. 1). As cell spreding continued, nd new contct points were formed, further loclised nd trnsient elevtions in cytosolic free C 2+ concentrtion were triggered (Fig. 1). Blocking the neutrophil surfce integrins y pre-tretment with nti-integrin ntiody prevented oth cell shpe chnge nd C 2+ signlling (Fig. 1), demonstrting their dependency on integrin enggement. At higher time resolution, y fst lser scnning in single line pproximtely 1 µm ove the ntiody-coted surfce nd prllel to the plne of the surfce, cells which fell into the scnning line were imged with x direction sptil informtion with temporl resolution of 12.5 milliseconds (Pettit nd Hllett, 1995). This technique demonstrted tht the loclised C 2+ trnsients hd n up-phse of out 3 seconds nd recovery of out 4-6 seconds (Fig. 2c). The elevted C 2+ ws loclised to pproximtely 4-5 µm 3 of cytosol over this time scle. Integrin clustering t the contct surfce ws visulised y fluorescently stining the β chin (CD18) on neutrophils efore contcting n nti-α-chin (CD11). This reveled the ppernce of symmetricl ptterns t the contct surfce (Fig. 2,) with similr time course s initil C 2+ signlling. This ws consistent with the hypothesis tht loclised C 2+ signlling resulted from loclised integrin cross-linking. The loclised C 2+ signlling t the contct site continued in 36 s 48 s 1000 nm 400 nm 200 nm 75 nm Fig. 1. Trnsverse confocl imges of cytosolic free C 2+ in neutrophils during integrin enggement. Imges show the cytosolic free C 2+ concentrtion, pseudo-coloured ccording to the colour scle, in trnsverse sections through neutrophil contcting n nti- CD11 ntiody coted surfce: () n untreted neutrophil (0-48 seconds); () neutrophil pre-coted with nti-cd11 to lock cell surfce integrin nd prevent CD11 enggement (0 nd 6 minutes). Both sets of imges re rw dt, showing the photon noise nd light sctter from the contcting surfce. The photon noise originted from the low lser power nd high voltge on the photomultipier detectors. In the first imge there ws distortion during sedimenttion s result of the movement of the cell reltive to the scnning line. The dt shown re typicl of similr experiments using either nti- CD11 or nti-cd18 coted glss (n=52).

3 Integrin triggered C 2+ signls in neutrophils 1691 Loclised C 2+ signlling s result of store relese The loclised C 2+ signlling spikes were oserved oth in the sence of extrcellulr C 2+ (Fig. 4) nd in the presence of the trnsmemrne locking ion Ni 2+ (Fig. 4). These dt demonstrted tht loclised C 2+ signlling ws the relese from C 2+ stores nd tht there ws no requirement for trnsmemrne influx of extrcellulr C 2+. Furthermore, no quenching of fluo3 occurred in the presence of extrcellulr Mn 2+ (Fig. 4c), suggesting there ws no contriution from locl chnnel opening. Glol C 2+ ursts nd cell spreding After contct with the surfce, second rpid stge of cell shpe chnge ws seen s contct with the surfce ecme more intimte (Fig. 5). In pproximtely 60% of cells (n=200), this ws preceded y urst of high C 2+ throughout the whole cell (Fig. 5). This proly represents n underestimte of the correltion etween glol C 2+ signlling nd rpid spreding, s the reltively long inter-imge intervl (12.5 seconds) would not hve permitted detection in ll cses. Fst time resolution single line scnning enled more ccurte determintion of the reltionship etween the initil C 2+ urst nd spreding (Fig. 5). Once firmly ttched, ursts of elevted C 2+ continued spordiclly (Fig. 6,,c). The intervl etween these ursts ws vrile up to 14econds (104±3econds, n=15). There ws no evidence tht these ursts were the result of solule fctor diffusing from neighouring cells, s C 2+ ursts in neighouring cells occurred synchronously (Fig. 6), suggesting tht there ws no communiction etween djcent cells. The C 2+ ursts were medited y dditionl integrin enggement, s they were inhiited y locking cellulr integrin y dding nti-cd11 ntiody to dherent neutrophils (Fig. 7d). Furthermore, loclised ut not glol C 2+ signlling ws triggered in cytochlsin B-pretreted cells (Fig. 3). However, fter dherence, susequent cytochlsin B tretment did not prevent glol C 2+ ursts. These dt suggested tht the genertion of the C 2+ urst were insensitive to cytochlsin B, ut required lrge contct re. Glol C 2+ urst s result of C 2+ influx Unlike the loclised C 2+ signl seen during initil contct, glol C 2+ urst responses were inhiited y lck of extrcellulr C 2+ (Fig. 7) nd y Ni 2+ (Fig. 7c), suggesting trnsmemrne C 2+ influx. Furthermore, lthough smll elevtion in cytosolic free C 2+ concentrtion ws oserved in the presence of extrcellulr Mn 2+, quenching of fluo3 rpidly ccompnied these elevtions ursts. Since the glol C 2+ signl ws dependent on integrin inding nd the site of integrin enggement ws only t the contct surfce, the possiility existed tht C 2+ influx occurred only through this contct surfce. However, no evidence ws found, y high time resolution (12.5 milliseconds) scnning, tht C 2+ ner the contct surfce, i.e. the site of integrin enggement, rose efore C 2+ t the memrne distnt from the contct site (Fig. 6d). It ws therefore concluded tht the locl enggement of integrin t the contct surfce could signl C 2+ chnnel opening t other sites. c Fig. 2. Integrin distriution nd the time course nd sptil spred of two initil loclised C 2+ events. Imges of fluorescein conjugted nti-cd18, s mrker of the distriution of cell surfce integrin, re shown () efore nd () fter contct with n nti- CD11 coted surfce. (c) The 3-D grph shows the time course of two loclised C 2+ events immeditely fter touch-down onto n nti- CD11 coted surfce. The dt were cptured y rpid lser scnning t plne 1 µm ove the contcting surfce. The x xis shows the cell dimension, the y xis time nd the z xis, cytosolic free C 2+ concentrtion. 400 mm C 2+ 5 µm 15 s

4 1692 E. J. Pettit nd M. B. Hllett 24 s 24 s 6 DISCUSSION 6 min Fig. 3. C 2+ signlling within neutrophils during integrin enggement. The cytosolic free C 2+ concentrtion chnges occurring during contct with nti-cd18 ntiody coted surfces within neutrophils () pretreted with cytochlsin B (5 µg/ml) to prevent cell spreding nd () BAPTA (intrcellulr concentrtion 5 mm) to prevent cytosolic free C 2+ signlling. The pseudo-colour scle converting colour to cytosolic free C 2+ concentrtion is shown in Fig. 1. The exmples shown re typicl of replic experiments (n>10). The dt shown here demonstrte two spects of integrin medited C 2+ signlling in neutrophils, nmely loclised C 2+ signlling s result of limited integrin enggement during the initil contct, nd the susequent glol C 2+ signlling fter further integrin enggement. The glol C 2+ signlling demonstrted here my correspond to previously reported glol rises in cytosolic free C 2+ concentrtion nd sudden chnge in shpe ccompnying down-touch (Kruskl et l., 1986), nd the spordic glol spiking of cytosolic free C 2+ fter integrin enggement (Jconi et l., 1991), nd which lso occurs during chemotxis, s the result of enggement of integrin molecules (Mrks nd Mxfield, 1990; Jconi et l., 1988; Hendry nd Mxfield, 1993). However, the initil loclised C 2+ signlling reported here (Fig. 1) hs not previously een oserved. The loclised C 2+ signlling, s result of limited integrin enggement, resulted from loclised relese of C 2+ from intrcellulr stores. These C 2+ stores were distinct from the single focl juxt-nucler store relesed y the seven trnsmemrne domin receptors, such s f-met-leu-phe (Hllett et l., 1990; Dvies et l., 1991). The stores which lierte C 2+ in response to integrin immoiliztion my, however, e relted to the C 2+ - ATPse orgnelles tht cluster t sites t the periphery of phgocytotic vesicles (Stendhl et l., 1994). It hs een suggested tht emptying intrcellulr C 2+ stores signls C 2+ chnnel opening in the plsm memrne nd C 2+ influx y relesing diffusile clcium influx fctor (CIF) from the store itself (Rndrimmpit nd Tsein, 1993; Prekh et l., 1993; Dvies nd Hllett, 1995). Loclised C 2+ signlling, in the sence of glol C 2+ rise, my thus result from limited integrin enggement generting insufficient CIF to trigger plsm memrne C 2+ chnnel opening. However, on further cell-surfce contct, dditionl integrin enggement, perhps of newly up-regulted molecules fter the initil touch down (Springer, 1990; Hynes, 1992) would result in n incresed CIF relese nd trigger the glol C 2+ chnge oserved. This would lso provide n explntion for the requirement of incresed integrin inding nd the lrge contct re for glol C 2+ ursts (Figs 5, 6). Although the glol cytosolic free C 2+ increses were driven y influx of extrcellulr clcium (Fig. 7,,c), prt of the requirement for C 2+ influx my e relted to mintining the concentrtion of C 2+ within the stores. However, the liertion of diffusile fctor (CIF) could lso result in the opening of C 2+ chnnels t sites remote from the integrin enggement sites. The differences in the density of integrin cross-linking my thus explin oth loclised nd glol integrin-medited C 2+ signlling y single underlying mechnism. This work lso rises the question of the identity of the intrcellulr messenger responsile for relesing C 2+ from the peripherl C 2+ stores. With f-met-leu-phe, it is thought tht IP 3 medites C 2+ relese (Thelen et l., 1993). Signlling C 2+ relese from remote site ner the nucleus t the centre of the cell (Hllett et l., 1990; Dvies et l., 1991) is theoreticlly fesile s IP 3 diffuses rpidly in the cytosol (Allritton et l., 1992). However, s the peripherl C 2+ store is not triggered y f-met-leu-phe, the messenger responsile for the integrin triggered relese site t the cell periphery, my thus e different. Recently, it hs een shown in crdic cells, tht the C 2+ sprks re generted s the result of the increse in C 2+ ner the C 2+ store (Lopez-Lopez et l., 1995; Cnnell et l., 1995). This c Fig. 4. Loclised C 2+ signlling during touch-down of neutrophils onto nti-cd18 coted surfces: () in the sence of extrcellulr C 2+ (EGTA, 1 mm); () in the presence of the C 2+ chnnel locking ion nickel ions (Ni +, 1 mm); nd (c) in the presence of the fluo3 quenching ion Mn 2+ (0.1 mm). The pseudo-colour scle converting colour to cytosolic free C 2+ concentrtion is shown in Fig. 1. The exmples shown re typicl of replic experiments (EGTA, n=10; Ni, n=12; Mn, n=25).

5 Integrin triggered C 2+ signls in neutrophils s 6 1 µm C s 72 s 2 5 µm Fig. 5. A glol C 2+ rise fter integrin enggement showing susequent cell spreding () visulised y z sectioning, nd () visulised y rpid lser scnning, shown s 3-D plot where the horizontl xis is the dimension cross the cell, the receding xis is time nd the verticl xis is cytosolic free C 2+ concentrtion. The pseudo-colour scle converting colour to cytosolic free C 2+ concentrtion for the imges in is shown in Fig. 1. The exmples shown re typicl of replic experiments (n= 200). would seem n unlikely mechnism in neutrophils, ecuse no CICR cn e demonstrted nd gents which interct with CICR, such s cffeine nd rynodine hve no effect of C s s 12 signlling. Furthermore, the C 2+ spikes reported here persisted in the sence of trnsmemrne influx. The identity of the messenger for the relese of C 2+ from peripherlly locted C 2+ C 2+ concentrtion (nm) Side B 1 Side A c d 12 C 2+ (300 nm) 144 s ms 168 s 18 Fig. 6. Glol C 2+ chnges in dherent neutrophils: () repeted glol C 2+ elevtions in firmly dherent neutrophil; () synchronous C 2+ ursts in 3 neutrophils in close proximity; (c) the time course of glol C 2+ responses, determined y rpid lser scnning; nd (d) 3-D plot of the initil stge of the C 2+ rise during glol elevtion with the lser scnning line positioned through the neutrophil to intersect the integrin engging surfce (side A) nd the non-engging surfce exposed to the medium (side B). The pseudo-colour scle used in the imges (,) for converting colour to cytosolic free C 2+ concentrtion is shown in Fig. 1.

6 1694 E. J. Pettit nd M. B. Hllett Fig. 7. C 2+ elevtions in neutrophils dherent to nti-cd18 coted surfces efore nd fter replcing the medium with: () incution medium; () C 2+ free medium (1 mm EGTA); (c) nickel contining medium (Ni + ion concentrtion, 1 mm); nd (d) solule nti-cd18 ntiody. The exmples shown re typicl of replic experiments (n>10). stores in neutrophils thus remins unknown. The dt presented here lso provide evidence for role of C 2+ in the regultion of cell shpe chnge. In cells in which the initil C 2+ spike ws suppressed y C 2+ BAPTA, cell shpe nd firm dherence were prevented (Fig. 3). Furthermore, stge of rpid cell deformtion, resulting in firm dherence, which is presumly prelude to trnsendothelil migrtion in vivo, ws preceded y glol cell C 2+ flsh. The possiility exists tht, C 2+ entering the cell vi plsm memrne chnnels cuses depolymeriztion of corticl ctin (Downey et l., 1990; Al-Mohnn nd Hllett, 1990) nd therey permits rpid shpe chnge. The loction of C 2+ storge sites close to the plsm memrne my, therefore, hve strtegic importnce in this process. Although the C 2+ signlling events demonstrted here resulted from engging integrin experimentlly y using immoilised ntiody, we propose tht similr events lso occur during physiologicl enggement y ICAM-1 on endothelil cell surfces. In order to estlish the relevnce of these findings to the physiologicl process, it will e necessry to correlte the C 2+ signlling triggered y CD11/CD18 integrin enggement with trns-endothelil migrtion This my provide the key to understnding neutrophil emigrtion, nd my led to the development of novel therpies for inflmmtory disese. We re grteful to the Wellcome Trust for support. E.J.P. ws Wellcome Prize Student. REFERENCES Al-Mohnn, F. A. nd Hllett, M. B. (1988). The use of fur 2 to determine the reltionship etween intrcellulr free C 2+ nd oxidse ctivtion in rt neutrophils. Cell Clcium 8, Al-Mohnn, F. A. nd Hllett, M. B. (1990). 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