Specificity and Reversibility of Chemotactic Deactivation of Human Monocytes

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IMMUNITY, May 1981, 464-468 Vol.32,No.

0019-9567/81/050464-05$02.00/0

Specificity and Reversibility of Chemotactic Deactivation of Human Monocytes

WERNERFALK* ANDEDWARD J. LEONARD

Laboratory of Immunobiology, National Cancer Institute,NationalInstitutes of Health, Bethesda, Maryland 20205

The chemotactic deactivation of human monocytes was studied to provide insight into the mechanism of chemotaxis. Deactivation was dependenton the dose of chemoattractant and time of incubation. A concentration in the cell suspension of

10'

MN-formylmethionylleucyl phenylalanine(FMLP) for 45min at370C ledto60%suppression of the subsequent specific chemotactic response.

Higher concentrations of FMLP ledtoalmost100%specific suppression.Deacti- vation was specific under all conditions used. The response to a nonrelated chemoattractant, human serum-derived C5a, was unaffected by incubation in FMLP.Deactivation was also transient. If cellswere deactivated at370Cwith FMLP, they recovered within 6 hat370C from this deactivation. Both phenomena,deactivation and recoveryfromdeactivation, weretemperature dependent.

Monocytes could not be deactivated at 00C, and they did not recover from deactivation when kept at00C. Thus, specific deactivation appears to require cellular metabolism, involving loss of receptors or blocking of a step between receptor occupancyandresponse.

Chemotactic deactivation refersto attractant- induced loss of migratory function, which is testedbyreexposureof cellstochemotacticfac- tor (19). Deactivation has been described for rabbitperitonealpolymorphonuclear leukocytes (20), human peripheral bloodneutrophils (4,9, 11,12-14), andeosinophils (21). Human peripheral bloodmonocytes arealsosubjecttodeacti- vation(6, 8). Deactivation of humanperipheral neutrophils has been dissociatedintotwo differ- entcomponents. Lowconcentrationsof chemotactic factor lead to inhibition of chemotactic response that is specific for the attractant, whereashigh concentrations resultin inhibition ofthemigratorysystem in anonspecific manner.

Several possible mechanisms have been pro- posed to explain these phenomena. Loss of an esterase activity (20) or damage to the cell by production of oxidative intermediates (13) or release of lysosomal enzymes (2) hasbeen sug- gested for the mechanism ofdeactivation. Re- cently, specific deactivation of human peripheral blood neutrophils has been shown to correlate withlossof specificcellsurfacereceptors for the attractant (16, 17). In this report, we show the dynamics of deactivation of human peripheral bloodmonocytes. Only specific deactivation was observedeven when high concentrations of attractant orrelatively long incubationtimes were used.Wealso show that, in contrastto human neutrophils, human monocytes are able to re- coverfrom deactivation.

MATERIALS AND METHODS

Cellpreparation.Bloodwasdrawnfromhealthy donors andheparinized. Mononuclearcells wereiso- latedby themethod ofBoyum (3).Blood was diluted with anequalvolume of Dulbeccophosphate-buffered saline without calcium ormagnesium, and 35 ml of diluted blood was underlaid with 15 ml ofFicoll-Paque (Pharmacia Fine Chemicals, Inc., Piscataway, NJ.).

Aftercentrifugationat 400 xgfor40minat20°C, the interface cell layerwas removed and washed twice withGey balancedsaltsolutioncontaining 2%bovine serumalbumin(Cohn fraction V) (Gey-BSA; National Institutes of Health MediaUnit, Bethesda, Md.). The cellsuspensioncontained 15to 35%monocytes, 65to 85%lymphocytes, and less than 1% granulocytes. Vi- abilitywasbetter than 99%, as measured by trypan bluedye exclusion. Total and differential counts were made for the finalwashed preparations. Total leuko- cyte recovery was 1 x 106 to 3 x 10'cells per ml of whole blood. Differential counts were made after stain- ingcell suspensions with euchrysin 3RX (Roboz Sur- gical Instruments Co., Washington, D.C.) and observ- ing them with a fluorescence microscope. These num- bers were used to standardize thecell number added per well.

Chemotactic factors. N-Formylmethionylleucyl phenylalanine (FMLP) was purchased from Sigma Chemical Co., St. Louis, Mo. Stock solutions (10-3 M) wereprepared ineither ethanol or dimethyl sulfoxide andstored at-20°C. Human serum-derived comple- ment component C5a was prepared by a modified procedure ofFernandez and Hugli (7) as described previously (6). Briefly, fresh serum was incubated with zymosan(MannResearchLaboratory) in thepresence ofE-aminocaproic acid (Sigma Chemical Co.). C5a was 464

at REGENSBURG on July 15, 2009 iai.asm.orgDownloaded from

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thenpurified byconsecutive chromatography on CM Sepharose Cl 6B (Pharmacia Fine Chemicals Inc.) and SephadexG-100. The final preparation was stored as asolution inphosphate-buffered saline at-20°C.

Deactivation procedure.Cell preparations were diluted to concentrations of 1 x 105 to2x 105 monocytes perml of Gey-BSA in 50-ml polypropylene tubes (no. 25335; Corning Glass Works, Corning, N.Y.).

FMLP stock solution was diluted in Gey-BSA so that it could be diluted 1:100 into the cell suspensions.

Standard incubationtime for deactivation was 45min unless indicated otherwise. After incubation, thecells werewashed twice in medium at room temperature, andtotal and differential counts were made for the final suspensions.

For recovery experiments, the protocol was slightly changed. Cellstobetested for recovery at0°C were putin iceimmediatelyafter deactivation, washed with cold medium, and centrifuged in the cold. Cells for testingat37°C were washed andcentrifugedatroom temperature and thenincubated at 37°C.

Fortimecoursesof deactivation and recovery from deactivation, cells were kept at the concentrations used for deactivation and tubes were frequently shaken. At the indicated times, samples were removed fromthe tubes andcellswerecentrifugedat200 x g.

Pelletswerethen suspended in Gey-BSA in 1/10 of theoriginal sample volume. This suspensionusually contained 1 x 106to 2x 106monocytes per ml and wasused for the chemotaxis assay.

Controlcellsuspensions were subjected to the iden- ticalprotocol,butnoFMLPwasadded for deactivation.

Chemotaxis assay.Chemotaxiswasassayedina multiwellchamberasdescribed previously (5). Bottom wellswerefilled with 25pl of attractant solutions in Gey-BSA.ANucleporefilter sheet(Neuroprobe Inc., Bethesda, Md.; Polyvinylpyrrolidone coated, 10

tim

thick,5-,umholesize)wasplacedoverthewells. Gasket and topplatewereassembled,and50plof cell suspen- sioncontaining 50,000to100,000 monocytes, depending onthe experiment,was addedto each top well.

Chamberswereincubated in moist air containing 5%

carbon dioxide for90min. Afterincubation,chambers were disassembled and filters were removed. Cells remainingonthe topsidewerewiped off;filterswere

immersed in methanol for 15s and then stained in Diff-Quick (Harleco,Gibbstown,N.J.).The numberof cells per millimetersquaredwascounted withanim- ageanalyzer(between 1,500and3,000).Withoptimal concentrations of FMLP or C5a, 20 to 40% of the monocytesmigratedtoward the attractant,depending

onthe donor(6). These numberswereusedtocalcu- late percentage of control migration. Assay points wereperformedintriplicate,and the standarderrorof the mean wasnotgreaterthan 15%.

RESULTS

Dependence of degree of chemotactic deactivationondoseof chemotactic factor.

Monocytes were incubated at 370C in various concentrationsofFMLPtodetermine therange overwhichdeactivationoccurred. The cellswere

thenwashed, checkedbytrypanblue exclusion

for viability (better than 99%), and tested for chemotaxis to FMLP and C5a. In all experiments,C5aservedtwopurposes:as anindicator ofcellviability andunimpairedmigratorymech- anism and, moreimportant, as a discriminator between specific and nonspecific deactivation.

The extentofdeactivation, asmeasured byre- sponses tooptimalconcentrations of FMLP and C5a,dependeduponthe concentration of FMLP usedtodeactivate (Fig.1).AnFMLPconcentra- tion oflo-8 Mresulted in about 60% deactivation.Deactivationwascompleteataconcentra- tion of

10-'

M. The responseto C5a remained unchanged,exceptpossiblyat

10'

M, the high- estconcentrationof FMLPused.Thisindicates thatdeactivationwasspecificand that cell damage, at least as measured by chemotactic response,didnot occur.

Deactivation didnotleadto ashiftinconcen- tration of peptide neededfor maximal chemo- tacticresponse.Doseresponsesfortreatedcells

still capable of responding to the deactivating attractant weremeasured from 10-9to 10-6 M, and the location of the response peak was the same asfor thecontrol.

The cell concentration used in deactivation experiments was between 1 x 105 and 2 x 105 monocytesperml. Deactivation in suspensions withhigher monocyte densitieswasunsuccess- ful; thiswaspresumablydue to loss of chemotactic factor, sincesupernatantsof thesesuspen- sionsshoweddecreased chemotacticactivity.

Time course of deactivation. Monocytes wereincubated with10-7MFMLP or in medium alone at37°C. At intervals indicated in Fig. 2,

-i

0

8

0 U.0

UI- E

us z

2cn uies

501

PMLP

0 10' 104 10-7 10 10-' MOLARCONCENTRATIONOF FMLP USED

FOR DEACTIVATION

FIG. 1. Dose-dependent specific deactivation of

humanmonocytes.Mononuclearcellsat adensityof

2x I05monocytes permlwere incubatedin FMLP solutionsormedium at37°Cfor45min. After two

washes inGey-BSA,monocytechemotacticresponse to 10-8MFMLP anda 1:800dilutionofC5a stock solutionwas measured. Error bars representstan-

darderrorsofthemeanfortriplicateassaywells.

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466 FALK AND LEONARD

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100l \FLA

z

ZU.

00

0 FMLP

uiz3560

F5G. 2.e dtim dreprendenced dactivatn cyespowere clincubatedat3Cinmedithe a0

indicated times.

equal portions,were removed, washel sayed for theirchemotactic response t FMLP at two different concentratic optimal range of the dose-responsecu]

tivation occurred very rapidly. Afte deactivation was complete when therl 10-8MFMLP was tested and was65%

higher concentration was used. Aftei no response was detected at eitherc

tion. The response to 05a remained throughout theexpermnent.

Temperature dependence of tion. Toestablish whether deactivati ply blocking of receptors at the cell anenergy-requiring process, deactiva and 000 was compared. Cells were with10-7MFMLP or with medium as Equal portions wereremoved atvani

washed, andtested for chemotacticr~

05a and threedifferent concentrations One other set of cells was deactivate with lo-7M FMLP to show deactival normalconditions. The chemotactic r cells deactivated at 3700 for 50 mi 10%oof thecontrol (Fig. 3). Nodeacti~

detectedin cellsincubatedat000. The to C5a and optimal concentrations wereunchangedafter3.5 h. Only at a concentration of FMLP (10-9 M) cc deactivation be observed. This marg could be explained by the amount

bound at 000 which might have cans

deactivation when the cells were u

3700 for the chemotaxis assay. The

ments show thatdeactivation requirerf olizing cell; occupation ofreceptors a

face, whichoccurs at00C (16, 17),isinsufficient fordeactivation.

Recovery of monocytes from deactivation. If deactivation is induced by receptor depletion, deactivated cells should be able to re- coverwith timeat370C.Monocytesweredeac- tivated at370C by incubation in lo-7MFMLP for1h. Controlcells without FMLP were iden- tically treated. The cellswerethenwashed, and each tubewasdivided intotwoequalportions.

One setwaskeptat 370C inanincubator, the 12D other was kept onice, andatvarious times cells weretested for chemotaxis.Figure4B shows the results with cells incubatedat370C after deac-

on. Mono- tivation. Deactivated monocytes gradually re- 7M

FMLP

covered from deactivation andreached control ,e

removtaXd

^levelsbetween 5 and 7 h.

Recovery

of the cells dilution Of

kept

^at^{OC reached}^{50% of the} controls.Inter-

ttage

of the pretationof thisresult is

complicated by

the fact one forthe that recovery canproceed during the assay in the chemotaxis well at 370C. Thus, the 50%

recovery of these cells represents the sum of possiblerecoveryat00C andat370Cduring90 d, and as- min of incubation for the assay.Cell concentra-

;oC5a and tion during recovery did not affect the result.

ns in the Wecannotsaywith absolutecertaintythat there rve.Deac- was norecovery at00C,but itmusthave been ,r 20 min, considerably slower thanat370C. The control esponseto response to C5a shows again that monocyte z when the chemotaxis function was unimpaired and that

r 100 min, theeventsinvolving deactivation and recovery concentra- werespecificfor the attractant used for deacti- I constant vation.

dleactiva- DISCUSSION

ion issim- Wereport in this paper on the dynamicsof surface or chemotactic deactivation of human peripheral itionat 37

incubated

acontrol. 1

Ous times, esponseto

ofFMLP. 0i '

,dat370C U

tionunder _z _0MWVW_PA_X3r

esponseof ^0,To U ,

was only 1 2 3 4

vationwas DEACTVAO4TIMEMHOURS

responses FIG. 3. Deactivation of human monocytes at0°C.

of FMLP Cells were incubated in either 10' M FMLP or uboptimal mediumat0°C.Anothersetofcellswasincubatedat

)uld some 37°Cin either10-F7MFMLPormedium. Deactivation inal effect at 370C was terminated after 45 min; cells were

of FMLP washed twiceandassayed forchemotaxis with10-7

ofd artalP

^MFMLP^asattractant.Cells deactivated at0°Cwere wdpartial washed twice at the indicated times, and the chemo-

varmed

to tactic responsetoFMLP(10-7, 10-8,and10-9M) and

,se

expern-

to a1:500dilution of C5a was measured.The control sametab- for each data point is theresponse of cells incubated itthe sur- in medium for the indicated deactivation time.

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1 3 5 7 1 3 5 7

RECOVERY TIME IN HOURS

FIG. 4. Recoveryof human monocytes from deactivation. Cellswere incubatedat37°C with 10'- M FMLP or medium. After incubation, cells were washed twiceto removeFMLP and divided into two portions each. Onesetofcells, incubated in FMLP or medium, was kept at0°C (A), and one was kept at 37°C (B). At various times monocytesweretestedfor chemotactic responsetoFMLP(10-7and10-8M)and toa 1:500dilutionof C5a. The figure combines two experiments. Oneexperiment, in which thecell density during recoverywas 2 x106/ml,representstimes of20min and5h10min.Theremainingtimepoints arefor the otherexperiment, in which cell density was 2 x105/ml.

blood monocytes with FMLP as chemoattractant. Deactivation, or loss of chemotactic re- sponsiveness after prior exposure to chemoat- tractants, didnot occur at00C evenafter rela- tively long incubationperiods.Itproceededvery rapidlyat 370C, with almosttotal deactivation after 20 to 30 min of incubation. Deactivation andchemotaxiswerealso evidentat room temperature (data not presented). The degree of deactivation was dependent upon the dose of attractantused,withapeptideconcentration of 108 Mleadingto about 60% deactivation. Not only an optimal concentration of peptide but also a minimal absolute amount per cell was necessary. With monocyte concentrations of about 2 x 106 per ml, deactivation under the standard conditions (10-7 M FMLP) did not occur(datanotshown).Alikely explanationfor thisphenomenon isdigestionof thepeptide on orin themonocyte, sincesupernatantsofthese deactivations showed decreasedchemotacticac-

tivity,indicating loss of chemotactic factor. Al- thoughrelease of FMLP splitproductshasnot yet been reported for monocytes, it has been shownforothercells (1,16).

The deactivation of monocytes was always specific.Inallexperiments,weusedthe response to anunrelated attractant,C5a,as acontrol for specificity and functional capacity of the cell.

Evenatverylongincubationtimesoratconcen-

trations ofattractantthatwere 100-fold higher

than theconcentration required for90% deactivation, nonspecific deactivation was not observed. We haveshown in a previousreport (6) thatthis is also true for other attractants used for deactivation.This is in sharpcontrast to the behaviorof polymorphonuclear cells from rab- bits and humans (12-14). Polymorphonuclear cell deactivation has been reported to have a specific and a nonspecific component, depending onthe concentration of chemotatic factor used for deactivation. Only 40 to 50% deactivation could be achieved, and specific andnonspecific componentscouldnot be clearlyseparated. The nonspecificevent leading todecreasedcell mo- tilitywasascribedto several factors,such as (i) depletion ofanenzyme or proenzymepool or (ii) damage of the cellcaused byoxidative intermediates. These mechanismsdo notseem to oper- ateinmonocytes; if they do, thecapacity of the cell formovement is not affected.Production of oxidative intermediatesin monocytes has been shown to be poor compared with neutrophils (10, 15), andmeasured oxygenuptake by monocytes uponstimulationwith FMLP is also very small (data notshown). Deactivation of mono- cytesforaspecific attractantwith retention of responsivenesstoother attractants suggests that deactivation is at the level of the attractant receptor or at somepointin the path leading to directionalmovementbefore thecommon path- way activated by all attractants. Since deactivation did not occur at 00C, but required a metabolizing cell, binding to receptors by at- tractantappears tobeinsufficient for deactivation.Binding studies with labeled FMLP showed thatthe peptidewas specifically boundat0°C.

Theamountbound within30minof incubation was70% of theamountboundat220C(W.Falk, L. Harvath, and E. J. Leonard, manuscript in preparation).

Ithas beenreported forneutrophils and humanmonocytes(17; J. J. Muscato,J. E.Niedel, and J. B. Weinberg, Blood 54:89a, 1979) that incubation with chemotactic factor is followed byaggregation and internalization ofthe receptor-attractant complex. This is an energy-dependent phenomenon and is inhibited by low temperatures. Thisfindingprovidesaplausible mechanism for specific deactivation in that it leads to depletionof the cells of receptors for thatspecificattractant.Intothat schemefitsour observation that monocytes recovered from specific deactivationwithin about6h. The recovery was inhibited by low temperature. This time period correlateswith the timemonocytes need to recoverfromdepletionofimmunoglobulinG receptors (18), which has been shown to be approximately 6h also. It does not meanthat theremustbealink betweenimmunoglobulin^G

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receptorand FMLP receptor, but this timespan seemsto be sufficient torebuild the surfaceof the cell and elaborate new receptors. Experi- ments areinprogressto correlate deactivation and chemotaxis with disappearance and reap- pearanceof receptorsonthe surface.

LITERATURE CITED

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