Terminal complement complex C5b 9 stimulates mitogenesis in 3T3 cells

Terminal complement complex C5b-9

stimulates mitogenesis in 3T3 cells.

J A Halperin, … , A Taratuska, A Nicholson-Weller

J Clin Invest. 1993;91(5):1974-1978. https://doi.org/10.1172/JCI116418.

The membrane attack complex of complement (MAC) can induce reversible changes in cell membrane permeability resulting in significant but transient intracellular ionic changes in the absence of cell lysis. Because ion fluxes and cytosolic ionic changes are integral steps in the signaling cascade initiated when growth factors bind to their receptors, we

hypothesized that the MAC-induced reversible changes in membrane permeability could stimulate cell proliferation. Using purified terminal complement components we have documented a mitogenic effect of the MAC for quiescent murine 3T3 cells. The MAC

enhances the mitogenic effects of serum and PDGF, and also stimulates cell proliferation in the absence of other exogenous growth factors. MAC-induced mitogenesis represents a novel effect of the terminal complement complex that could contribute to focal tissue repair or pathological cell proliferation locally at sites of complement activation.

Research Article

Find the latest version:

Terminal Complement Complex

C5b-9

Stimulates

Mitogenesis

in

3T3 Cells

JoseA.Halperin,*AlaneTaratuska,*and Anne Nicholson-Wellert

*Department of Medicine, Brigham and Women's Hospital, and Department of CellularandMolecularPhysiology, Harvard Medical School; and tCharlesA. DanaResearchInstitute, Harvard-Thorndike Laboratories and the Department ofMedicine, Beth Israel Hospital, Harvard Medical School, Boston,Massachusetts 02115

Abstract

Themembrane attack complex ofcomplement(MAC) can

in-ducereversible changes incell membranepermeability

result-ing in significantbuttransient intracellularionic changes in the

absence of cell lysis. Because ion fluxes and cytosolic ionic changes are integral steps in the signaling cascade initiated when growthfactors bindtotheirreceptors, we hypothesized that the MAC-induced reversiblechanges in membrane

perme-ability could stimulatecellproliferation. Usingpurified termi-nal complement components we have documented a mitogenic

effectof theMAC forquiescent murine 3T3cells. TheMAC enhances the mitogenic effects ofserum and PDGF, and also

stimulates cellproliferation inthe absenceofother exogenous

growth factors. MAC-induced mitogenesis represents anovel

effectof the terminal complement complex that could

contrib-ute tofocal tissue repairorpathological cell proliferation lo-callyatsites of complement activation. (J. Clin.Invest. 1993.

91:1974-1978.)Keywords: complement*C5b-9*mitogenesis . cellproliferation *growthfactors

Introduction

Thecomplementsystemis composedof12activation proteins

andatleast 13regulatory proteinsthat functionaseitherfluid

phaseormembrane-attached inhibitors. Therearetwomajor

pathwaysofcomplement activation, classicalandalternative,

which converge at the level of the C3 step. Thereafter both

pathwaysshareacommonsequencethroughthelate compo-nentsC5, C6, C7, C8, and C9, leadingtothegeneration of the membrane attack complexofcomplement

(MAC).'

Assembly of the MAC isinitiateduponcleavageofCS andgenerationof

C5b.C5breactswith C6 and C7formingC5b-7 which bindsto

thecellmembranes; then,bindingof C8occursand the result-ingC5-8complex incorporates several molecules ofC9toform

C5b-(9

)n,

theMAC. The numberofC9 moleculespercomplex

(n) is believed to be one to two in homologous cell/serum

systems and three to fourin heterologous systems(1). The

MAC isatransmembraneporewhich isfunctionally expressed

asanincreased membranepermeability (2, 3).Thecation leak

Address correspondence to Jose A. Halperin, M.D., Department of Cellular and MolecularPhysiology,Harvard Medical School, 25 Shat-tuckStreet,Building C1 #607, Boston,MA02115.

Receivedfor publication14October 1992 and in revisedform 7 De-cember 1992.

1.Abbreviations used in this paper:

[Ca2+]i,

intracellular free calcium; MAC, membraneattackcomplex of complement.

through the MACcanleadtocolloidosmoticswellingandlysis ofthetarget cell or, at sublytic concentrations, to large but reversible changes in theinternalcomposition ofthe cell(4).

Ca2"

ions that leakthroughthe MACtransiently increasethe internal

Ca2"

concentration with different functional conse-quences tothetargetcell(5-8).Inerythrocytes, recruitmentof

aCa2 -activated K+ channel balances theinternal cation con-tentpreventingcellswelling (9),andCa2-dependent vesicu-lationcontributestoterminatethecomplement lesion(10).

It is well established that the response ofgrowth-arrested cellsto avariety ofgrowth factors andpharmacological

mito-gensinvolvesaseriesof ionic changes suchas arapidincrease incytosolic free

Ca2+

[Ca2+

_]i,

_{activation of}Ca2 -sensitiveK+

channels with membranehyperpolarization, and stimulation ofNa+/H+exchange which results in alkalinizationofthe

cy-toplasm (1 1-13). These changes that occur within minutes

aftermitogenic stimulationarebelievedtoplayacriticalrole in theinitiationofDNAsynthesisthatbeginsmanyhours later.

Weinvestigated whether thetransient changes in membrane

permeabilityinduced by the MAC could alsoinducecell prolif-eration.We reporthere that in Swiss 3T3cells,the MAC

en-hancesthemitogeniceffect ofserumandPDGF, and also stim-ulates cell proliferation in the absence ofexogenousgrowth

factors.The phenomenon ofMAC-inducedmitogenesis may represent a novel role for the complement system: directed

regulationof cellproliferation.

Methods

Cell culture.MouseSwiss 3T3 cells(kindly providedby Dr. H.Green,

Department ofPhysiology, HarvardMedical School), were used

be-tweenpassages 17 and 32. The cellsweregrown in DMEsupplemented

with 10% heat-inactivated calf serum, 100 U/ml penicillin,and 100

qg/mlstreptomycin,at370Cin95%air/5%CO2. Formitogenic

as-says, the cellswereseeded in 48-wellplatesat I04cells per well. When

theyreachedconfluence, usuallybetween3 and4d,the mediumwas

replaced withDME 0.5%serumtomake themquiescent,and mitogen-esisassayswereperformed24 h later.

MeasurementofintracellularCa2` activity.Swiss 3T3 cellswere

platedat adensity of6 X 103cells/cm2on 12-mmglasscoverslips,

which had been placed in the wells of 24-well sterile culture plates. The cellswere grown toconfluency, asdescribed above. Theday of the

experiment,the cellswereloadedwith5AMfura-2for 30 minat37°C,

washed, and placed in serum-freeDMEmediumcontaining 0.1% gela-tin. Thecoverslipswereplacedinawater-jacketedchamberona light-shielded, heated stage of an inverted fluorescence microscope (37±0.5°C). FluorescencemeasurementswereperformedinaCM2 dual excitationspectrofluorimeter (SPEXIndustries Inc.,Edison,NJ) (14). The excitation wavelengths were 340 and 380±1.8 nm. A

dichroic mirror deflected the excitedlight (400nmcut-off)tothe per-fusion chamberonthe stage ofaninvertedepifluorescencemicroscope (Nikon). TheFura-2 emissionsignalswerecollected for each

wave-lengthat500±5nm.Cellswereindividuallycalibrated in situby

expo-sure toCa2+-freebufferscontaining LaCl3.Intracellular free calcium

([Ca2+]i)

was determined by the equation:

[Ca2+]J

= Kd X Q(R

J.Clin.Invest.

© The AmericanSocietyforClinicalInvestigation,Inc.

0021-9738/93/05/1974/05 $2.00

-Rmin)/(Rin,,,-R) (15),where thecorrection factor (Q)accountsfor thedifferentspectralpropertiesofFura-2 whencomplexedwithLa"

instead of Ca2+.

Complement components/assays. C5b6 was made from purified C5 and C6,using factorB,cobravenomfactor(all purchased from

Quidel,SanDiego, CA),andrecombinantfactorD(giftofT.White, scios,MountainView, CA) ( 16). Ni+ (1.0mM)wasused tostabilize

the cobravenomfactor Bbconvertase(17). TheC5b6wasfractionated by HPLCon aDEAEcolumn(AP-l ProteinPak8HR;Waters Asso-ciates,Milford, MA).Thereaction mixturewasappliedinbufferA(60 mM NaCl, 10 mM sodiumphosphate <pH7.6) and elutedwitha

linear60 mingradientofbufferAandbufferB(500 mM NaCl, 10 mM sodiumphosphate,pH7.6).TheC5b6elutedas adistinctpeakwitha

characteristic absorptionspectraat220,250,and280nm(DiodeArray

Spectrophotomer 1040A; Hewlett-Packard Co.,Avondale,PA)anda

reproducible retentiontimeof 39-40 min.

TheC5b6 poolwastitratedthe same dayoftheexperiment using

human red blood cells. 1 U ofC5b6wasdefinedasthe amountofC5b6

required to produce 50%lysisof 5 x 10'human red blood cells when

incubatedinatotalvolumeof300 ,ulwithC7(0.1 ,ug), C8(0.5 ,ug),

andC9(0.5,g)(Quidel).

Uptake of[3H] thymidine.3 hafterexposure ofquiescentSwiss 3T3 cellsto amitogenicstimulus, I,uCi/mlof[3H]thymidinewasaddedto

thewells,and the cellsmaintainedat37°C/5% CO2 foranadditional

21 h. Then the cells were washed threetimeswith DMEmediumand theacid-precipitable radioactivitywasextractedwith cold 10% TCA.

Afterneutralization with 0.3 N NaOH, aliquotswere counted in a

Tri-Carb Scintillation counter (Packard Instrument Co., Downers

Grove, IL). Fornegative controls,the cellswerekeptin 0.5% serum, andforpositivecontrolstheywerestimulated with 10%serum.Inall

experiments, each control andexperimental pointwasperformedin

quadruplicate(four wells).Theproteincontentofeachwell,measured

bytheCommassie-PlusProteinAssayReagentaspermanufacturer's instructions (Pierce Chemical Co., Rockford,IL),wasusedtoassess the numberofcells/well.When thedispersion oftheprotein readingsis

<5%,incorporationdataareexpressedas countsperminuteper well.

Results

The MAC transiently increases intracellular

Ca2".

Cultured

Swiss 3T3 cells can be made to enterreversiblythe

nonprolifer-ative

Go

phaseof the cell cycleby thewithdrawal ofserum.

When suppliedwith the appropriate growth factors,the cells reenterG. and progress into the cell cycle. Because the early ionic changes that occur in responsetogrowthfactorsare very

similar tothose inducedbytheMAC,wehypothesizedthat the MAC couldprovidea stimulus for cell proliferation. To test

thishypothesis, confluent serum-deprived quiescent Swiss3T3

cells wereexposedtothe MAC assembled frompurifiedhuman

C5b6plusC7(3 minat37°C),andsubsequentaddition of C8 andC9. To correlate thehemolytic titerof the MACwith re-versible nonlethal ionic changes in the 3T3 cells, we measured

the effect oftheMAContheintracellular

Ca2"

concentration

of thesecells. WhenFura-2-loaded3T3 cells were exposed to1 U ofC5b6 and 9MAgofC7 (3 minat37°C), additionC8 and C9

(9Mgeach)resulted inatransient increase in intracellular Ca2+ (Fig. 1). Similar to the effect of growth factors such as PDGF

andEGF (12),the MACatthe concentrationsusedpromoted

arapidrise in

_[Ca2+]i

that reachedapeakbefore decliningto a newlevel,slightly higherthan theunstimulated value. The ef-fect oftheMACon

[Ca2+]i

shown inFig. 1 wasexpected from the workofothers ( 18); however, this isthefirst demonstration ofatransient risein

_[Ca2+]i

induced bythe MAC assembled frompurifiedcomponents andatthesinglecelllevel. The ad-vantageofusing purifiedcomponentsis that it avoids the early,

nM

500-[Ca++]

100-0 150 300

Time,s

Figure1.Effect oftheMAConthe free intracellular calcium of Swiss 3T3 cells. Fura 2-loaded 3T3 cellswerepreexposedtoIUof C5b6 and9 MgofC7 inavolumeof 400,lfor 3min. Thearrowindicates thetime when C8/C9(9ggeach,inavolume of 200Ml)wereadded.

MAC-independent, oscillationson

[Ca2+]i

observedwhen the

complexwas activated from C8-deficientsera( 19). The

con-centrations of C5b6 used for subsequent experiments was

correlated with its

hemolytic

activityinourstandardizedassay.

TheMACenhances the mitogenic

effect of

serum.

_Synthesis

ofDNA

by quiescent

fibroblastswasstimulated

by

serumina

dose-dependentmanner

(Fig. 2,

lower curve). When terminal

complementcomponentswerepresentin themediumtoform

theMAC

simultaneously

with the addition ofserum,the

mito-genic

effectwas_{significantly enhanced,} asdetectedbythe

in-creased

incorporation

of[3H]thymidine

(Fig.

2, upper

curve).

Thestimulatoryeffectwasnotproduced byC5b6alone,nor

by

2000

IC~b-9

C5b-7 Control

.1000-c#-l

.

0.0 2.5 5.O

Serum Concentration (%)

Figure 2. Effect of the MAC on the mitogenic effect of serum. Cells (4 x 103)wereplated in 48 multiwell plastic culture plates. 24 h after reaching confluency, the cells were made quiescent by incubation for

12-18 h in DME-0.5% calf serum. Then the cells were stimulated with different concentrations of serum in the presence and absence of the MAC. Complement components and different concentrations of serum diluted in DME were added sequentially as follows: 200 Ml of C5b6 ( 1 U), 200 Ml ofC7 (9 Mg), and 3 mm later 200 Ml of the appropriate serum dilution to obtain final concentrations of 0, 0.1, 0.25, 0.5,1,E2,and 5% with andwithout C8 + C9 (9 Mg each). DNA synthesis wasdeterminedby measuring the uptake of

_[p3H

thymidine.

Each point represents the mean±SEM of quadruplicate values

ob-tamned in one representative experiment which was performed four times with comparable results. (Inset) Lack of an effect of single or combinedcomplementcomponentsin mixtures that do not form the MAC. Theserumconcentration was 2.5%, and maximum [3Hothy-midine uptake was 4,200 cpm/well.

theindividual complementcomponents alone orbyany combi-nation thatdidnotinclude theserial addition of C5b6, C7, and C8 + C9 (inset, Fig. 2). Thus, the stimulatory effect of the terminalcomplementcomponentsfor themitogeniceffect of

serumrequired the formation of the MAC.

TheMAC enhancesthe mitogenic effects ofPDGF. Because

theprincipal mitogenpresentin whole bloodserumis PDGF,

wedetermined the effectofthe MAC onthemitogenic effect of purified PDGF. These experimentswereconducted in

serum-free medium containing 0.1% gelatin, 30ng/ml IGF, and 10

,gg/ml

transferrin, the empirically determined minimum

re-quirementsfor PDGF-stimulated cellsto enterinto the growth cycle in the absence ofserum. The MAC significantly

en-hanced themitogenic effect of PDGF (Fig. 3). Similartothe

effect on serum-stimulated mitogenesis, the effect ofPDGF

was notpotentiated byeither individualcomplement compo-nents or anycombination ofthese componentsthat does not

form the MAC. These results indicate that cells targeted by the MACaresensitizedtothemitogenic effect of PDGF.

The MAC is mitogenic in the absence ofexogenous growth factors. In someexperimentsthe MACstimulatedtheuptake of

[3H

] thymidinein the absenceofany serum orPDGF,

sug-gestingapossible mitogenic effect oftheMAC by itself. Indeed,

exposure ofquiescent fibroblasts to the MAC in serum and PDGF-freemedia, stimulatedDNAsynthesisina dose-depen-dentmanner(Fig. 4).

Growth-arrested cellsreinitiateDNAsynthesis - 12 hafter

mitogenic stimulation.Thus,kineticdifferencesin the

progres-sion of the cells through the cell cycle could explain the in-creased uptake of

[3H]thymidine

observed inthepresenceof

25000 r

20000

15000

z1

cq)

10000

[

CZ

1%

._

5000

n

0 1 2

I

C

/6789

C5b67

PDGF3

(ng/ml)

4 5

Figure 3. Effect of theMAConthemitogeniceffect ofPDGF.Uptake of[3H]thymidinebyquiescent3T3cells exposedtothe same con-centrations of the complement componentsasinFig. 2, but toPDGF

instead ofserum(experimental procedures as described for Fig. 2). DMEused for this experimentwasserumfree and contained0.1% gelatin, 30 ng/ml IGF, and 10Ag/mltransferrin (DMS++). Final concentrationsofPDGFwere0, 0.1,0.5, 1,2.5, and 5 ng/ml. Each

pointrepresents the mean±SEM ofquadruplicatevaluesobtained fromonerepresentative experiment of four experiments performed.

zW

fr~ 0

1.00

-0.75

-0.50

-0.25.

0.00 .

DME C5b6(1U) C5b6(1U)

+ C7,c8,cs

+

.

C5b6 (3U)

+ c7,c8,cs

+

DME

+

10 %serum Figure 4.Mitogeniceffect of the MAC in the absence of other exoge-nousmitogens. QuiescentSwiss 3T3 cells placed in DME++were

exposedsequentiallytocomplementcomponentstoform the MAC. Theconcentrations of C5b6were 1 and3U,andthoseof C7, C8,

andC9were asdescribed forFig.2. Resultsareexpressedasrelative valuesof the[3H]thymidineuptakeinto cellsstimulatedwith 10% calfserum.Each bardepictsthe mean±SEMoftherelative thymidine incorporationcalculated fromtwo setsof quadruplicatevalues ob-tained intwoexperiments.

the MAC. This possibilitywasexplored byfollowingthetime courseofthymidine uptakeinthe presence of either PDGFor

the MAC. The results showninFig. 5 indicate that increased DNA synthesis and not different kinetics of

[3H]thymidine

incorporation explain theeffect of the MAC observed inour

experiments.

To assess whether the transient increase in intracellular

Ca2+ inducedbytheMAC wouldsufficetoinitiate the

mito-genicresponse,quiescentcellswereexposedto aconcentration

of theCa2+ ionophoreA21378 that promotesanincreased

in-12000

- _10000.

Q 8 8000.

2 6000

E

g 4000

6 x 2000

6 _e.~ 2000.

'_{PDGF (2.5ng/ml)}

C5b-9

C5b67 Control

0.0 4 8 12

Time, h

16 20 24

Figure5.Comparison of the kinetics of[3H]thymidineuptake

in-ducedby the MAC and PDGF.Uptakeof[3H]thymidine bySwiss 3T3 cellswasmeasuredatdifferent time intervals aftermitogenic

stimulation ofquiescent3T3 inserum-free medium.(o) DME++;

(A)C56(1 U); (A)PDGF(2.5 ng/ml); (v) C5b-9(1 U C5b6+C7

+C8+C9,15_tg/mleach).

ternal

Ca2"

comparabletothatinFig. 1.To mimicthe tran-sient increase observed with the MACtheionophorewasleftin themedium for a timeinterval of 3 min and then aspirated and

replaced by exactly the same medium withoutA12378. The

transientCa2+influxproduced with the ionophore didnot in-crease DNA synthesis orenhance the mitogenic response to

PDGF (2.5ng/ml).

Discussion

Theresults presented inthispaper areconsistent witha mito-genic effect of the MAC: It sensitized 3T3 cellstothemitogenic stimulus ofserumandPDGF, and alsostimulatedcell

prolifera-tionwhen assembled in the cell membrane in the absence of

anyexogenously added growth factors. Theseexperiments sug-gest that controlof cellproliferationmaybeapreviously

unrec-ognized function of the complementsystem.

Twomajortypesofgrowthfactorsreceptorshavenowbeen characterized: receptor tyrosine kinases (PDGF, EGF, and FGF) (20),andGprotein coupledreceptors(a-thrombin, 5-HT) (21 ). Activation of growth factor receptors' transmem-brane signaling molecules stimulates PIP2-phospholipase C, which inturnactivatesthephosphatidylinositolcascadeof sec-ond messengersgenerating IP3 anddiacylglycerol. Diacylglyc-erol is the physiological activator ofproteinkinase C,which

accountsforsomeof the earlymitogenicevents,including acti-vationofNa+/H+exchanger, andphosphorylationofthe ribo-somal S6protein. Associated with these biochemical

phenom-ena, mitogens also inducearapid increase in

[Ca2+]i,

which

may comefrom intracellularstores,and/orfrom influxof ex-tracellular Ca2+ through channels in the plasma

mem-brane (12).

Fragmentsof complementfactorsBandC3caninfluenceB

lymphocyteproliferation (22, 23) through specific membrane

receptors. A novel aspect ofMAC-stimulated mitogenesis is thatit wouldnotberestrictedto aspecificcelltypebecausethe MAC inserts into the lipidbilayer ofthe cell membrane and does notrequire specific membrane receptors. Although the transduction ofintracellular signalsby the MAC isnot

com-pletely understood, it hasbeen established that several

path-waysofintracellular signaling areactivated bytheMAC,

in-cluding protein kinase C (24, 25), the adenylcyclase system

which raises cyclicAMP ( 19), and the

Ca2+-activated

K+ ef-flux (9), which is stimulated bytheMAC-induced increased in

[Ca2+

]i.

Thus, there areseveral pointsofconvergence where the MAC could interact synergistically with the mitogenic

pathwaystopromotecellproliferation.Onecommoncellular effectthatcouldmediate the MAC-inducedmitogenesisis the increased

[Ca2+

]i, particularlysince increased

[Ca2+

]iseems to

beanecessarycondition for the MACtoinduce cell prolifera-tion(Fig. 1).However,MAC-induced mitogenesis isnotonly

related to the changes in

_[Ca2+],

sincean ionophore-induced influx of

Ca2+

comparable inmagnitudeand duration to the oneinduced bythe MACfailedto stimulateDNAsynthesis. This result implies that additional effects oftheMAC

contrib-ute toitsmitogenic effect. Rozengurt showedthatmellitin,an

amphipathic pore-forming polypeptidethat increasesNa

in-fluxalsostimulatesDNAsynthesisinquiescent Swiss3T3 cells

(26). Whether the mitogenic effect ofthe MACis solely the consequence of

Ca2"

and otherMAC-induced ionic

changes,

orwhetherit also

_directly

stimulates chemical

_signaling

path-waysinamannerthat short-circuits the receptor-initiated

reac-tions, isnotknownat present.

We proposethatactivationof complement and formation oftheMACmaytransmitlocalmitogenic signals. The

signifi-canceofthe MACregulatingcellproliferationliesinthe fact thatcomplementisuniquely adapted todeliverafocal

mito-genic stimulus:generation of the MAC istightlycontrolledby

thespecificityandrestriction ofcomplementactivation. MAC

stimulatedmitogenesiscouldnormallycontributetofocal tis-suerepair.Undercircumstances of abnormal complement

ac-tivation, the MACmayprovideadirectstimulus(Fig. 4)and/

orsynergisticaction with other growth factors (Figs.2 and3) for the pathological proliferative response that characterizes

manydiseases inwhichdeposition ofthe MAC has been immu-nohistochemically documented.Theseinclude: the mesangial cells inproliferative glomerulonephritis (27),thesynovialcells

in rheumatoidarthritis (28), and also thesmooth muscle cells

of the atheromatous plaque (29). It is also intriguingto specu-late that the dysregulated hematopoesis characteristic of the

disease paroxysmal nocturnal hemoglobinuriamay result from

mitogenic stimulation bytheMAC. Inparoxysmal nocturnal hemoglobinuria, thedeficiency ofcomplement regulatory

pro-teins normallypresentontheplasmamembrane leadsto

unre-strictedcomplementactivationatthesurface of the PNH cells.

Acknowledgments

We thank Drs. D. C. Tosteson, T. W. Smith, and M. T. Tostesonfor

useful discussions and encouragement during this work. Weare grate-ful to Dr. Hans-Jorg Berger for his help with the[Ca2+]imeasurements,

and toM. Rynkiewiczfor excellent technical assistance.

This work was supported in part by grantHL-33768 from the

Na-tional Institutes of Health, andGrant-in-Aid 13-533-890 (J. A.

Hal-perin) from the American HeartAssociation, Massachusetts Affiliate.

References

1.Stewart, J. L., J. B. Monahan, A.Brickner, and J. M. Sodetz. 1984. Measure-ment of the ratio of the eight and ninth components of compleMeasure-ment on comple-ment-lysed membranes. Biochemistry. 23:4016-4022.

2. Green, H., P. Barrow, and B. Goldberg. 1959. Effect of antibody and com-plement on permeability control in ascitis tumor cells anderythrocytes. J.Exp.

Med. 1 0:699-713.

3. Michaels, D., A. Abramovitz, C. Hammer, and M. Mayer. 1976. Increased ion permeability of planar lipid bilayer membranes after treatment with the C5-9 cytolytic attack mechanism of complement. Proc. Natl. Acad. Sci. USA. 73:2852-2856.

4. Halperin, J. A., A. Nicholson-Weller, C. Brugnara, and D. C. Tosteson. 1988. Complement induces a transient increase in membrane permeability in unlysed erythrocytes. J. Clin. Invest. 82:594-600.

5. Shirazi, Y., F. A. McMorris, and M. L. Shin. 1989. Arachidonic acid mobi-lization and phosphoinositide turnover by the terminal complement complex, C5-9, in rat ologodendrocytes XC6 glioma cell hybrids. J. Immunol. 142:4385-4389.

6. Campbell, A. K., and B. P. Morgan. 1985. Monoclonal antibodies demon-strate protection of polymorphonuclear leukocytes against complement attack. Nature(Lond.). 317:164-166.

7. Hansch, G. M., D. Gensa, and K. Resch. 1985. Induction of prostanoid synthesis in human platelets by the late complement components C5b-9 and channel forming antibiotic nystatin: inhibition of reacylation of liberated arachi-donic acid. J. Immunol. 135:1320-1324.

8. Hattori, R., K. K. Hamilton, R. P. McEver, and P. J. Sims. 1989. Comple-ment proteins C5b-9 induce secretion of high molecular weight multimers of endothelial von Willebrand factor and translocation of granule membrane pro-teinGMP-140 to the cell surface. J. Biol. Chem. 264:9053-9060.

9. Halperin, J. A., C. Brugnara, and A. Nicholson-Weller. 1989. Ca++-acti-vated K efflux limits complement-mediated lysis of human erythrocytes. J. Clin. Invest. 83:1466-1469.

10. Iida, K., M. Whitlow,andV.Nussenzweig. 1990. Membrane vesiculation protects erythrocytes fromdestruction by complement. J.Immunol. 147:2638-2646.

11. Rozengurt, E. 1986. Early signals in the mitogenic response. Science

(Wash. DC).234:161-166.

12.Soltoff,S. P., and L. C. Cantley. 1988.Mitogensandion fluxes.Annu. Rev.

Physiol.50:207-223.

13. Moolenar, W. H., R. Y. Tsien, P. T. van der Saag, and S. W. de Laat. 1983. Na-Hexchange andcytoplasmicpH in theactionof growthfactorsin human

fibroblasts.Nature(Lond.). 304:645-648.

14.Borzak,S.,R. A., B. K.Kelly,B.Kramer, Y. Matoba, J. D. Marsh, and M. Reers.1990. Insitucalibration of fura-2and BCECFfluorescence inadult

ventric-ular myocytes. Am. J.Physiol.259:H973-H98 1.

15.Grynkiewicz, G.M., M.Poenie, andR. Y.Tsien. 1985.Anewgeneration ofCa2"indicatorswithgreatlyimproved fluorescence properties. J. Biot. Chem. 260:3440-3450.

16.DiScipio,R.,C. Smith, H. J.Muller-Eberhard,and T. Hugh. 1983. The

activation ofhuman complement componentC5 by a fluid phaseC5convertase. J.Biot. Chem. 258:10629-10636.

17.Fishelson,Z., and H. J.Muller-Eberhard.1982. C3convertase of human complement: enhancedformationandstability of the enzyme generated with nickel instead of magnesium. J.Immunot. 129:2603-2607.

18. Morgan, B. P. Complement membrane attack on nucleated cells: resis-tance, recovery and non-lethaleffects.1989.Biochem.J.264:1-14.

19.Carney, D. F., T. J. Lang, and M. L. Shin. 1990. Multiple signal messen-gersgenerated by terminal complement complexes and their role in terminal complexeselimination. J.Immunot.145:623-629.

20. Lammers, R., H.Riedel,and A.Ullrich. 1990. Chimeric receptors reveal

distinctsignalling characteristics ofreceptor tyrosine kinase cytoplasmic

do-mains.In Growth Factors: From Gene to Clinical Application. Sara, V. R., K. Hall, and H. Low, editors. Raven Press, Ltd., New York.

21. Pouyssegur, J.,C.Kahan, and K. Seuwen. 1990. Transmembrane signal-ing pathways initiating cell growth: role of G-protein coupled receptors. In

GrowthFactors: From GenestoClinicalApplication. Sara,V.R.,K.Hall,and H. Low,editors.RavenPress, Ltd., New York.

22.Ambrus, J. L., L. Chesky, T.Chused,K. R.Young,P.McFerland,A. August, and E. J. Brown. 1991. Intracellularsignalingeventsassociatedwith the

induction of proliferation ofnormal human Blymphocytes bytwodifferent antigenicallyrelated human B cellgrowth factors (highmolecularweightBcell

growth factor(HMW-BCGF)andthecomplementfactorBb).J.Biol. Chem. 266:3702-3708.

23. Hivroz,C., E.Fisher,M. D.Kazatchkine,andC.Grillot-Courvalin. 1991.

Differential effects ofthestimulation ofcomplementreceptors CR1(CD35)and

CR2(CD2I)oncellproliferationand intracellular Ca" _{mobilizationofchronic}

lymphocytic leukemiaB cells. J.Immunol.146:1766-1772.

24. Cybulsky, A. V., J. V. Bonventre, R. J.Quigg,W.Liebertal, andD. J. Salant.1990.Cytosolic calciumandprotein kinase C reduce complement

me-diated glomerular epithelial injury.KidneyInt.38:803-811.

25.Wiedmer,T.,S.Ando,andP.J.Sims. 1987. Complement

C5b-9-stimu-latedplatelet secretion is associated withaCa2"-initiatedactivation ofcellular proteinkinases.J.Biol. Chem. 262:13674-13681.

26. Rozengurt, E., T. D.Gelehrter,A.Legg, and P.Pettican.1981.Mellitin stimulatesNa entry, Na-K pumpactivityand DNAsynthesisinquiescent

cul-tures ofmousecells.Cell. 23:781-788.

27.Rauterberg, E. W., H. M.Lieberknecht,A.M.Wingen,and E. Ritz. 1987.

Complement membrane attack(MAC)inidiopathicIgA-glomerulonephritis.

KidneyInt.31:820-829.

28. Auda, G., E. R.Holme,J. E. Davidson,A.Zoma, J.Veitch,and K. Whaley. 1990. Measurementof complement activation productsinpatients with chronic rheumatic diseases. Rheumatol.Int. 10:185-189.

29.Niculescu,F., F. Hugo, H.G.Rus, R.Vlaicu, and S. Bhakdi. 1987. Quan-titative evaluation oftheterminalCOb-9 complement complex by ELISA in

humanatherosclerotic arteries. Clin. Exp. Immunol. 69:477-483.