Each Human

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0019-9567/80/09-0953/07$02.00/0 Vol. 29,No. 3

Human Monocyte Chemotaxis: Migrating Cells Are a Subpopulation with Multiple Chemotaxin Specificities on

Each Cell

WERNER FALK* ANDEDWARD J. LEONARD

Immunopathology Section, Laboratory of Immunobiology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205

Only 20 to 40% of human blood monocytes were capable of responding to chemotaxins invitro.This limit is not due to restrictions of the in vitro

system,

but isdue tothe existence of a migrating subpopulation. Over a wide range, the number of cells migrating toward a given concentration of chemotaxin was directly proportional to the number added to the chemotaxis chamber. These monocytes respondedto all of the three stimuli used: human serum-derived C5a, human lymphocyte-derived chemotactic factor, and a synthetic peptide. It was possible to deactivate cells to one attractant, leaving the response to other attractants intact. This suggested that these attractants were recognized by different receptors. Several lines of evidence showed that most migrating cells hadreceptors for all three chemotaxins tested. Thus, if cells were assayed for migrationto oneattractant, no additional migration occurred when the remaining cellswereassayed formigrationto a differentattractant.Furthermore,thesame cells that had migrated toward one attractant were able to respond to other chemotaxins. Wealsofound that a single attractant attracted as many cells as a combination oftwo or three attractants. Calculations from these data showed thatatleast 75% of the migratingmonocyteshave different receptors for all three attractants.

Macrophages play an important role in the

immunological

response to neoplasms and are found at the site ofprimary and transplanted tumors. Since peripheral blood monocytes are precursors of these effector macrophages, the ability ofmonocytes tomigratemaybeessential foraccumulationattumorsites.Monocytes from cancer patients have abnormal chemotaxis in vitro. Decreased monocyte chemotaxis was as- sociated withpoorprognosis (14); the abnormal response wasreversedby surgical removal of the tumor (13). Boetcher and Leonard showed differences in monocyte response totwodifferent chemotactic agents,

lymphocyte

derived-chem- otacticfactor (LDCF) and activated serum (6).

Only

50% ofcancer

patients

with

impaired

responses toLDCF also had reducedresponsesto activated serum. These

findings

suggest that either there are specific subpopulations of mi- gratingmonocytes withdifferentchemotaxin receptors or thatspecific receptors ina

homoge-

neous population with

multiple

receptors on each cell were blocked. Toresolve these alter- natives,weexamined

subpopulations

of

migrat-

ingmonocytes in normal

subjects.

Weshow

by

various chemotaxis

experiments using

10-,um- thickpolycarbonate filtersthatthere is

only

one

subpopulation

among allmonocytesthatis able

tomigrate invitro, and thatmostofthesecells respond to all three chemotactic stimuli tested.

MATERIALS AND METHODS Cellpreparation.Bloodwasdrawnfromhealthy donors andheparinized. Mononuclearcellswereiso- latedby the method ofBoyum(7). Bloodwasdiluted with anequal volume of Dulbeccophosphate-buffered saline withoutcalciumormagnesium(PBS)and35ml of diluted bloodwasunderlaid with15ml oflympho- cyte separation medium (Bionetics, Rockville, Md.).

Aftercentrifugationat400xgfor40minat20°C,the interface cell layer was removed and washed twice withGey balanced salt solutioncontaining2% bovine serum albumin (Cohn Fraction V) (Gey-BSA, Na- tional Institutes of Health Media Unit, Bethesda, Md.). Thecellsuspensioncontained 15to35%mono-

cytes,65to85%lymphocytes,and less than 1% gran- ulocytes.Viability wasbetterthan99%, asmeasured by trypan blue dye exclusion. Total and differential countsweremade for the final washedpreparations.

Totalleukocyterecoverywas10' to3 X 10' cells per mlof whole blood.Differentialcountsweremade after staining cellsuspensionswitheuchrysin3RX(Roboz SurgicalInstrumentsCo.,Washington, D.C.)and ob- servationwithafluorescencemicroscope.Thesenum- berswereusedtostandardize the cell number added perchemotactic chamber.

Chemotactic factors. N-Formyl-methionyl-leu-

953

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

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cyl-phenylalanine-methylester (referredto aspeptide) was a generousgift of K. Ho(EliLilly&Co.,Indian- apolis, Ind.). The optimal concentration range for chemotaxiswas10-7to10' M,asdescribed(9). Hu- man serum-derivedcomplementcomponent C5awas prepared as described by Fernandez and Hugh (8) withminor modifications.Briefly,100ml ofserumwas incubated at 370C with 13 g ofepsilon-aminocaproic acid(SigmaChemicalCo.,St.Louis, Mo.)and100mg of zymosan(Mann ResearchLaboratories)which was finely suspended in 2 ml of PBS; after 45 min of incubation, itwasprocessedasdescribed(8).The total preparation, about260ml afterdialysis againststart- ing buffer, was addedto acolumn ofCM-Sepharose C1 6B (Pharmacia FineChemicals, Inc., Piscataway, N.J., 2.6-cm indiameter,100ml ofgel)andeluted with 0.1 M ammonium formate buffer(pH 5),containing 0.5 mMethylenediaminetetraacetic acid. Littleorno activity was found in the eluate. The columnwasthen washed with250mlof0.3M ammoniumformate(pH 5), and theactivitywaseluted with0.5Mbuffer. The pooled fractions were dialyzed against water, mem- brane filtered(Millipore Corp., Bedford,Mass.),and lyophilized.This material was reconstituted with 10 ml ofPBS, and 5 ml was applied to a column of Sephadex G-100 (2.5by80cm) and eluted with0.1M ammonium formatebuffer, pH5.Chemotacticactivity wasdetected in the 12,000-dalton region. The active fractions were pooled, dialyzed against water, and lyophilized. The material wassolubilized in 10 ml of PBS and stored insamplesat-20'C.Thispreparation is referredtoasC5a. Adose-responseisshown inFig.

1. In some experiments, yeast-activated serum was used. Forthis, baker's yeastwasboiled in hotwater and washed withwater four times. Itwasaddedat 1 mg/ml ofserumand incubatedat37°Cfor50min in the presence ofepsilon-aminocaproic acid. The Fer- nandez and Hugh procedure for activation was fol-

ch 200

w

0Z C/)

0a OLLLL_J 0 -

=)w

Za- z

_

10-4

10-4 10-2

lowed, but theacidified samplewasdialyzedagainst PBS and usedforchemotaxis without fractionation.

Lymphocyte-derivedchemotactic factorswerepre- pared bystimulation ofleukocytecultures made from Ficoll-Hypaqueseparatedcellsasdescribedpreviously (2).Concanavalin A(MilesLaboratories,Inc.,Elkhart, Ind.)^wasusedat aconcentration in thecultureme- dium of1 or5,ug/ml. Homologousserumwasadded to0.5% concentration.Incubation timewas 24h.The supernatants were put through

0.45-,tm

filters, di- alyzedagainstwater, andlyophilized.The residuewas solubilized inPBS, appliedto aSephadex G 100 col- umnandeluted with PBS. Peakchemotactic activity wasfound in the15,000-dalton region.Fractionswere

pooled,storedat4°C,andusedasthestandard preparation ofLDCF. Thepreparation showedsomeactiv- ityat adilution of1:80and thedose-responsecurve reachedaplateauat 1:10.A 1:5dilution was usedto give optimalchemotaxis.

Chemotaxis protocol.Monocytechemotaxiswas

assayed in blind well chambers (Neuroprobe Corp., Bethesda, Md.)withpolycarbonatefilters (5-Mumpores;

Nuclepore Corp., Pleasanton, Calif.). Chemotactic agentin theindicated concentrations in Gey-BSA(200

,ul,

totalvolume)wasaddedtothelower chamber. The upperchamberwasfilled with 0.3 ml of cellsuspension inGey-BSA containing 8x 104 monocytes. The cell dose-responsewaslinearover a100-fold range(104to 10" cells per chamber, Fig. 2). We chose a low cell numbertoreduce the effort of counting and thenumber of cells needed. Thechamberswereincubated for 2h inhumidified air with 5% CO2 at 37°C. The cells in the topchamberwereremoved, and the filters were washed for10 sin absolute methanol before staining asdescribed (10). Counting was done as previously described(6). Each pointwasassayed intriplicate and results wereexpressed as the mean ± the standard errorofthe mean of the number ofmigrated cells per 20 oil fields. The percentage of migrating cells was calculatedasfollows: the size ofahigh power oilfield

0 8W

' 2000/

R /

100 /

00

104 5

0 0

z~~~~~~~~~~50

z06 a

DILUTIONSOF C5a PREP.

FIG. 1. Dose-response curveofhumanperipheral monocytestopurifiedC5a.C5ainzymosan-activated human serumwaspurifiedbyCM-SepharoseCl 6B chromatographyandSephadexG-

IX00

gelfiltration.

NUMBEROFMONOCYTESINUPPERCHAMBER

FIG. 2. Celldose-responsetopeptide. Thenumber ofmonocytes addedtothechemotaxis chamber in 300

tol

ofmedium is shown on the abscissa. The two insertsrepresentdifferentexperiments. The concentration ofpeptidewas10"M.

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wascalibrated; 20 countedfieldsrepresented 1/142 of the whole filter area sothat the counted number x 142 X 100 divided by the input monocyte number equals the percent of migrating cells. The migrated cells were more than 99% monocytes. Only very few monocytesfound on cover slips placed in the lower chamberduring incubation showed that the migrated cells remained attached to the filter.

Deactivation procedure. Deactivation was carried out by preincubation of the cells at a concentration of 2.6x 105 monocytes per ml. The deactivating concentration for the peptide was l07 M and for activated serum was a dilution of 1:5. Incubation time was 40 to 50 min at room temperaturein Gey-BSA.

Controlpopulationswerepreincubatedunder identical conditions in Gey-BSA alone. After incubation, the cells werecentrifuged at 200 x g for 10 min and washed 3 times withGey-BSA.After thelast centrifugation, thecells were suspended in 1 ml of medium for counting. Differential counts and viable counts (by trypan blue dyeexclusion) were done, and the suspension was thendiluted to provide 8 x 104 monocytes percham- ber. The cell recoverywasusually70% withaviability of over95%.

Filtertransfer. The filter transferprocedurewas asfollows. When the cell layer on the top of thefilter wasneeded, the supernatant was carefully removed with a pipette, the cell layer was washed once with medium, and the filterwastransferred to a new chamber. When the top cell layerwasnot needed,itwas removed witha cotton swab. The new chamber was filled with195 p ofmedium or attractantdilution, and thefilter wascarefullyplaced in it upside down or up so as toavoid air bubbles. A300-ELIportion of medium wasaddedtothe top chamber.All manipulations were carried out very quickly to preventthe filters from dryingout.Notmorethan three chambersweredis- assembled for filter transferat atime.

Sephadex G-10 separation of mononuclear cells.SephadexG-10wasswollen inGey-BSAfor3h.

Five ml of gel was added to a 35-ml syringe. All manipulationswerecarriedout at4VC.Fivetoseven ml ofa cellsuspension obtained by Ficoll-Hypaque separation andcontaining107to 2x 107mononuclear cells per mlwasplacedontopof thesyringecolumns.

Thelymphocyte-rich eluantwascollected ina50-ml polypropylene centrifuge tube. The column was washed three times with 5ml ofGey-BSA, and the washing fluidswere combined with the first eluant.

The gelwasthen transferred intoa50-mlcentrifuge tube andsuspended in 40 ml of medium. After the tubewasgently shaken,itwascentrifugedfor15s at 100x g. The supernatantwasfilteredthroughloosely packed siliconized glass wool and centrifuged. The cells were suspended in 1 or 2 ml of medium and counted.

RESULTS

Influenceof

lymphocyte-monocyte

ratio.

Thecelldose-responsecurve

(Fig. 2)

shows that thenumberof

migrated

cellsis

directly

proportional to the number ofmonocytes in the top chamber. Sincedonor variationwith

respect

^to the

monocyte/lymphocyte

ratio is in therange

HUMAN MONOCYTE CHEMOTAXIS 955

of 15 to35% monocytes to 65 to 85% lymphocytes in the Ficoll-Hypaque separated fraction, we determined whether this ratio had any influence onthe migration. A cell suspension containing 24% monocytes and 76%lymphocytes was frac- tionated on Sephadex G-10. The cell suspension which was separated from the beads by shaking wascomposed of 64% monocytes and 36% lymphocytes with a viability of over 99%. Monocyte recovery wasabout 40%. Thedose-responses of this and the original cell suspension to chemo- tacticpeptidewerecompared (Fig.3);therewas nosignificant difference in the response.

Inthe remainder of this paper, wediscuss the results which show that humanmonocytes can be selectively deactivated to different attractants;thisimpliesdifferentchemotaxinreceptors.

Then, after examining a series ofdifferent ex- perimental approaches, we conclude that most ofthe responding monocytes have receptors for allthreeofthe chemotaxinstested.

Selective deactivation by preincubation.

Mononuclear cellswereincubated in dilutedac- tivated serum, in a chemotacticpeptide solution, or in medium as a control.Afterextensive washing, the cells were counted, adjusted to equal monocyte numbers, and assayed for their responses to activatedserumandpeptide (Table 1).Theresponse ofthecellstothedeactivating chemotaxin was suppressed, whereas the response tothe new chemotaxin was notaltered.

This was true for bothdeactivatingagents. The deactivation was therefore chemotaxinspecific and migration to other chemotaxins was not inhibited.

Selective

migration

in the presence of

deactivating

concentrations of chemotax-

- Ficoll-Hypaquecells ---MonocytesafterG10 90

cr0 ,,

\\

zoI

10-10 ¹⁰8 io4

MOLAR CONCENTRATION OF PEPTIDE

FIG. 3. Comparison of thedose-responsecurves of Ficoll-Hypaque cells and a SephadexG-1Oenriched monocyte suspension. The twocellsuspensionswere adjusted to the same monocyte numberper ml. The chemoattractant was peptide.

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ins. An experiment similar to the above was donewith a three-attractantsystem. Cellswere suspended in medium containing different chemotaxins in deactivating concentrations.

Thesesuspensions were placed in thetopchambers. The bottom chambers were filled with chemotaxinsolutionsasshown in Table

2; again,

the chemotaxin response was specific. The attractants in the mixture of all three had the same concentrationsaswhen used alone. Little or noresponsewasobservedtoattractantin the lowerchamber if thesameattractant waspres- entin the upperone

(Table 2,

italicized

data).

However, iftheattractantsin the topand bottom chambers were different, the monocytes respondedtothestimulus

by migration.

Witha mixture of all threeattractantsin thetop chamber, there was little or no migration to any stimulus in the bottom (Table 2, bottom line).

Thisshows thatthese threeattractants arespe- cifically recognized

by

the cells and that it is

TABLE 1. Specific deactivation of monocytes Mean no.

Cellspreincubated of mono-

for40min at220C Attractant cytes per

in"I 20oil fields

±SEMh Medium Activated serum' 90±10

Peptide 115 ± 15

Peptide

(10-7

M) Activatedserum 110 ± 15

Peptide 35 ± 5

Activatedserum Activatedserum 30 + 5 (1:5dilution) Peptide 150± 10

" After preincubation, thecells were washed three times with medium and thenassayed.

bSEM,

Standarderrorof themean.

'Yeast-activatedhumanserum;dilution1:40.

possibletodesensitizespecifically tooneattract- ant,leaving responsiveness to other attractants intact. At the desensitizing concentrations shown, capacity of the cells formovementwas

notaffected.

Evidence for different chemotaxinrecep- tors on the ^same monocyte. The previous experiments ledtothequestions ofwerediffer- entpopulations respondingtodifferent attractants,or was onepopulation capable of respond- ingtoall of the three stimuli used? To evaluate thesequestions,wesetupanexperimenttotest formigration of additional cells afterastandard chemotaxis incubation. Cells in the first run

migrated to medium, peptide, or C5a (Fig. 4).

After the 2-h incubation period, the superna-

tants in the top chambers were carefully re-

moved and the filter top surfaces were gently rinsed so asnot to removeadherent cells. The filters were then placed in new chamberscon-

taining medium or different attractants in the bottom wells. Medium was added to the top well. The chambers were incubated for 2 h to determine if additionalcellsmigratedduringthis period. Results forthesequences medium-peptide or medium-C5awerethe same asforpep-

tide-mediumor C5a-medium, showingthat the cells remained viable during the transfer and

were capable of responding to a chemotactic stimulus after afirst incubationperiod (Fig. 4).

No increase in numbers ofmigrated cells was

observed whenthecellsmigratedtowardspep-

tideorC5ainthe firstrunandwerethen tested forresponsetoeither stimulus in the secondrun.

This lack of additional migrationsuggeststhat there is predominantly a single population of cellscapable ofbeing attracted by either peptide

TABLE 2. Selective migration of human monocytes

Mean no. of migrated monocytes±SEMW with the followingattractant in lower chamber:

Attractantinupper cham-

ber" LDCF+C5a+

Medium LDCF' C5a" Peptide" Peptide'

Medium 65±7 266±57 184 ±20 247±47 252±52

LDCF1:55 85±10 75±5 160±15 170±35 270±30

C5a 20±10 190±20 40± 15 300±20 340±20

Peptide 10±5 75±25" 240±10 35±5 340±15

LDCF+C5a + Peptide 10±5 45±20 10±5 35±15 40±5

"The cellswere mixed with the indicated attractants and placed into the upper chamber.

SEM, Standard error of the mean. Foreach row in the table, the response with different attractants in upper and lower chamber was significantly greater than the response with the same attractants in both chambers.

'A1:5dilutionof stock solution in both chambers.

"A1:50dilutionin upperchamber; 1:500dilution in lower chamber.

"A10-7Mconcentration in upper chamber;10'8Mconcentration in lower chamber.

fTheconcentration of single attractants in the mixture was the same as when used alone.

-'Seetextforpreparation of stock solution dilutions.

hThe lowresponse to LDCF when peptide is on top was observed intwoother experiments and has not been explained.

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FCh.a Fdor

1-

Attractants Meannumber of migrated monocytes per First run Secondrun 20oilfields ± SEM

Medium Medium 20± 5

Peptide 70± 5

C5a 85± 10

Peptide Medium 75±10

Peptide 85± 10

C5a 90± 5

C5a Medium 90± 10

Peptide 85±10

C5a 85± 30

C5aprep. 1:500 Peptide10-'M

FIG. 4. Absence ofcell populations with specificity for onlyonechemotaxin. A normal chemotaxis experiment was performed during the first run with the indicatedattractants.Thefilterswith cells remaining onbothsurfaces were then transferred to new chambers withtheindicated chemotaxins, incubated for 2 h, stained, and counted to determine if the presence of different chemotaxins in the second run increased the numberof migrated cells. SEM, Standard error of the mean.

orC5a and thus havingreceptors for both.

Toobtaindirect evidence for thepresenceof multiple receptors onthe same cell, we set up the

following

experiment. During the first 2-h incubation period the cells were tested for response toactivatedserumor

peptide

inthe usual way (Table 3).

Supernatant

andcellswerethen removed from the top chamber. Control medium, activated serum, or peptide was then placed in thetopchambers in thesameconcen- trations as used for the first period. The cells which

migrated

to the bottom of the filter in response tothe firststimulusnowrespondedto the second stimulus

by upward migration

tothe top of the filter. After the second incubation

period,

the cells

remaining

on thefilter bottom were counted. These numbersare presented in Table 3; reduction in number

compared

tothe mediumresponse means back

migration.

Cells selected

by

responseto one attractant were ca-

pable

of back

migration

to adifferentattractant (see italicized data in Table3). In the firstrow of the

table,

cells selected

by

response toacti- vated serum migrated backto

peptide

and vice versa for the second row. Similar results were obtained for athree attractantsystem

(LDCF,

peptide, and

C5a).

Reduction in cell number on the bottom of the filter is, of course, an indirect measure of back

migration,

and it is

possible

that cellswere

simply falling

offthefilterinto the bottom well.

However, as noted

above, migrated

monocytes did not fall off filtersonto cover

slips placed

in

HUMAN MONOCYTE CHEMOTAXIS 957

the bottom wells. A second migratory response was also shown directly byremoving thefilter after the first incubation and placing it upside down ina newchamberwith a newattractant.

Themigrated cells, now on the top of the filter, migratedasecondtimeandwere counted on the bottom of thefilter afterthesecondincubation.

Quantitation. The previous experiments demonstrate in a qualitative waythat there is onlyonepopulationofmigrating monocytes that responds to the stimuli used. The results are qualitative because they may be affected by manyfactors such asdiffusionofchemotaxins, aging ofcells,or exhaustion of themotility ap- paratus. To get quantitative information, we arranged an experiment with allpossible combinations of3attractantsin a normalchemotaxis assay. Seven different attractant combinations are possible withrespect to 3attractantsandthe corresponding receptors onthe cells (Table4).

With theattractantcombinations indicated and counting the numbers of cells attractedbythese combinations, one can set up seven equations for seven unknowns, as outlined in the table (e.g., x + u + v + t= 130). Itisapparentthat the number ofcellsrespondingto amixture of all three attractants is not much greater than the number responding to a single attractant.

Thus, most of the cells have receptorsfor the threeattractantsused;t =75%. The resultsfrom the solutions of the equationsareshown inthe rightmost column of the table. The fact that the total percentage exceeds 100is attributable to the standarderrorsof thecountednumbers.

TABLE 3. Evidenceformorethanonereceptorfor chemoattractantson asinglecell

Meanno.ofmigrated monocytes per 20oil fields±SEM with thefollowing

attractantsin upper chamber for sec- Attractantfor ondrun:"

first run in lower

chamber Activated

serum(1: Peptide

Medium

40 dilu- (10-'M) tion)

Yeast-activated 200± 10 200±50 90±5 serum(1:40

dilution)

Peptide(10' 320+20 90+15 140+20b M)

SEM, Standarderrorof themean.

bTheresponse to the same attractant ispossible because theattractantin the bottom chambermay be dilutedbydiffusion into thetopchamberordestroyed bymigrated cells(4, 12)duringthe firstperiod.Addi- tion ofattractant tothetopchamber thuscreates a

concentration gradient in the direction of the top chamber.

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TABLE 4. Estimationof the fractionofmigrating cells withapeculiar set of receptors

No. of cells Observedno. Calculated

Possible receptorcombinationona withrecep- No. of cellsmigratingtocorrespond- of cellsmi- values for singlecell tor combi- ingattractant combination grating column2'

nation

C5ab x x+u+w+t 130C 10

LDCF y y+u+v+t 150 5

Peptide z z+v+w+t 130 25

LDCF+C5a u x+y+u+v+w+t 130 10

LDCF +peptide v y+z+u+v+w+t 170 0

C5a+peptide w x+z+u+v+w+t 160 0

C5a+LDCF+peptide t x+y+z+u+v+w+t 180 75

"Aspercentageoftotal cellsmigratingtothe combinations of all threeattractants(180). Duetothe standard errors, the percentagescanvaryby±15%.

bOptimalconcentrationswereused:C5a,1:500;LDCF, 1:5;peptide, 10' M.

' Meannumber of monocytes per20oilfields.

DISCUSSION

Only 20 to 40% of human peripheral blood monocytesrespondedby migrationtochemotac- tic stimuli. Thesefindings raised twoquestions that weattempted to answer. First, do there-

sponsive monocytes representadistinctsubpop- ulation among the total blood monocytes in vivo? Second, are there migrating monocytes with different chemotaxin receptors or combinations ofreceptors on their surfaces? An ap-

proach to the first question is to consider the possibility that the limit in the number of mi- gratingmonocytesisnotduetoasubpopulation, but that thepopulation isuniform andthe lim- itation is duetoanaspectof the in vitrosystem, suchasrestriction formovementby smallpore

diameteror lowpore density, cell crowding on

thefilter, inadequate time for migration, disap-

pearanceof chemotaxingradient, orloss of cel- lularcapacity torespond. The in vitro system

wastherefore analyzed in several ways. Itwas

shown that the number of migrated cells was

always proportionaltothe number ofmonocytes in the upper chamber over atleast a 100-fold

range (Fig. 2) and thus, overthisrange, migra- tionwasnotlimited by cell crowding. Further-

more,the resultwasunaffected by the propor-

tionoflymphocytes in the cell suspension (Fig.

3).Thisisinagreementwithtwostudies in the literature (1, 15). The possibilitythat the num-

ber ofmigrating cellswasrestrictedby thepore

density^onthefilterwasruledoutbyourfinding that there was no significant increase in the number ofcells migrating through filters with twice the usual number ofpores per unitarea

(unpublished data). We also showed that the limit in the number of migrated cells was not

due to selection ofa population responsive to

only one chemoattractant (the remaining cells being responsivetoanotherattractant) since in the filtertransfer experiment (Fig. 4), asignifi- cantnumberofcells with the abilitytomigrate

toanotherattractantcouldnotbe detected.This also rulesout duration ofincubation or disap- pearance of chemotaxingradientaslimitingfac- tors. Furthermore, the medium control shows that the cells could still migrate after the first incubationperiodandtherefore thelimit in migration numberswas notdue toloss ofmigratory capacity during the experiment. These studies provide the most compelling evidence to date that the monocytes capable of migrating to chemotaxins represent a subpopulation of the totalbloodmonocytes.

Theanalysisof monocyte receptorspecificity wasmade with three attractantsthat may have importance inimmune reactions: LDCF, made by stimulation with concanavalin A of human lymphocytes; C5a from human blood, purified by a procedure analogoustothat described by Fernandez andHugli (8); anda syntheticpep- tide, N-formyl-methionyl-leucyl-phenylalanine- methylester. Small peptides are believed tobe the attractantsreleased

by

bacteria (3, 5). The conclusions drawnfrom this part of the study arethatthe threeattractants arerecognized by different receptors andthat the great majority ofresponding cells have all three of theserecep- tors.Evidence forreceptorspecificitywasbased ondeactivation studiessuch as the one showing the preincubation of cells with one attractant selectivelyinhibited thesubsequent response to that attractant,whereas the response to a differ- entattractantremained intact(Table 1). These findings areinagreement withresults for other migratingcells, like rabbit neutrophils (16), hu- manperipheralbloodneutrophils (11), or eosin- ophils (17).

Evidencefordifferent receptor specificities on the same cell was obtained directly from the back-migration experiment shown in Table 3.

The essence of thisexperiment is that a specified cellpopulationwasfirstselected byitsresponse tooneparticularattractant. Thiscell population

958

FALK AND LEONARD INFECT. IMMUN.

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HUMAN MONOCYTE CHEMOTAXIS 959 wasthen used forthe next step, in which it was

determinedwhether the cells could migrate back upwards towardadifferent attractant. Since the cells migrated back,weconcluded that thesame cells that responded to one stimulus could re- spondto asecondorthirddifferent stimulus and therefore musthave atleast three differentre- ceptorspecificities.Thesimplest, albeit indirect, experimenttoshow that therearedifferent re- ceptorspecificitiesonthe samecell is shown in Table4.Withall thepossible combinations of 3 attractants, one should expect an addition be- havior in thecaseofsingle receptorsonone cell and thesame numbers in the caseofmultiple receptors.Table4shows thatatleast 75% of the migrating cells must have receptors for all 3 attractants.

Restriction of chemotactic responsiveness in vitroto a

subpopulation

ofblood monocytes may also occur in vivo. The

ability

to

migrate

may represent oneof several maturationalstagequal- itiesexhibitedby monocytesontheir way from the bone marrow to become effector macrophages in normal tissues and in sitesof inflam- mation.Our

findings

permitus toseparatemon- ocyteson apreparative scale into two popula- tionsonthe basis of their chemotacticresponse and to determine if there are

morphological, biochemical,

or functional differences in these two

populations.

Thedefect in chemotaxis of monocytes from cancer patients can be

explained

eitherby the formation of

subpopulations

of

migrating

cells withmissingorblocked receptors, byachange in dose-response, or by a decrease in the responding cell number. A clinical

study

toanswer these questions willrequire adetermination of (i) the number ofresponding

cells, (ii)

the dis- tributionof receptorsamongthe

cells,

and

(iii)

the

dose-responses

for various attractants.

Thesestudiesareinprogress.

LITERATURE CITED

1. Altman,LC.,C. T.Furukawa,andS. J.Klebanoff.

1977.Depressed mononuclearleukocytechemotaxis in thermallyinjuredpatients.J.Immunol.119:199-205.

2.Altman,LC.,R.Snyderman,J. J.Oppenheim,and S. E.Mergenhagen.1973.Ahuman mononuclear leu-

kocyte chemotactic factor:characterization, specificity and kinetics of production by homologous leukocytes.

J. Immunol. 110:801-810.

3. Asvanikumar, S., E. Schiffman, B. A. Corcoran, C.

B.Pert, J. L Morell, and E. Gross. 1978. Antibiotics and peptides with agonist and antagonist chemotactic activity. Biochem.Biophys. Res. Commun. 80:464-471.

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