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INFECTIONAND IMMUNITY, Aug.1981, p.629-631 Vol. 33, No. 2 0019-9567/81/080629-03$02.00/0

Chemotactic Activity of Bone Marrow-Derived Macrophages Changes with Time in Culture

WERNER FALK,* SHOGO TOMISAWA, VOLKER KLIMETZEK, ANDMONTE S. MELTZER LaboratoryofImmunobiology,ImmunopathologySection,National CancerInstitute,

Bethesda, Maryland 205

Received13February 1981/Accepted15May1981

The number of bone marrow-derivedmacrophagesabletorespondtochemo- tactic stimulivaries with time in culture. Chemotacticresponsewasoptimalat2 weeks and maydependoncellmaturationordifferentiationorboth.

Morphological, biochemical, and functional activities of mononuclear phagocytes change with maturationordifferentiationorboth(2,4, 5). It isgenerally believedthat theinflammatory macrophagesthat accumulateatsitesof inflammation originate in the bone marrow and are trnsportedviathe bloodasmonocytes(13, 14);

monocytes arrive at or arerecruitedto sites of inflammation and mature or differentiate into effectormacrophages.The itaostprobablemech- anism for this monocyte recruitment is chemotaxis along a concentration gradient. On their way from bone marrow through the blood to tissue,these immature mononuclearphagocytes could change characteristics and develop into mature macrophages. On the otherhand, Bur- sukerandGoldman proposedthatresidentand inflammatory macrophages may in fact derive fromdifferentprecursorstemcelLs (3). Culture of bonemarrow-derived celLs hasbeen usedto follow thedevelopmentofmacrophagefunction (3, 12). In this note, we report on changes in chemotactic and phagocytic properties of bone marrow-derived cells at different times in culture.

Bone marrowcellswereprepared from femura ofadultC3H/HeN male mice (10), 20 to 25 g, obtained from stockat the National Institutes ofHealth,and culturedaspreviously described (12). One modification was culture in 250-ml polypropylenecentrifugetubes (no. 25350,Corn- ing Glassworks, Coming, N.Y.). Cells were seededat adensityof2 x107per 10 ml per tube.

Tubeswereincubatedontheir sides in moist air with5%carbondioxide. Cellswere refedevery other day from day 5 on with 5 ml ofculture medium. Cultured cellswere harvested by cen- trifugationof tubesat250 xg for10 min. Ad- herentcellsweredetached with a rubber police- man beforecentrifugation or by rinsingwith a pipette. More than 95% ofcellsrecovered were viable asassessedby trypanblue exclusion. Re- sultsobtained with these cellswerevery

smilar

to results obtained with bone marrow macrophages recovered from Teflon-coated dishes (10). Chemotaxis was performed in a 48-well chemotaxis chamber (6). Bottom wells were filled with 25

p1

ofendotoxin-activated mouse serum(11)diluted in RPMI1640 (GIBCOLab- oratories, Grand Island, N.Y.). A Nucleopore filter sheet (Neuroprobe Inc., Bethesda, Md;

polyvinylpyrrolidone-coated, 10pmthick,5-pm holesize) was placed over thewells. Gasket and top plate were assembled, and 20,000 macrophages in 50

pl

ofculture medium were added to eachtop well. Chambers wereincubated in moist air containing 5% carbon dioxide for 4 h. After incubation,chambers were disassembled and fil- terswereremoved.Cells remaining onthe top sidewere wipedoffand thefilterwasair dried andstained inDiff-Quick(Harleco,Gibbstown, N.J.).The number ofcells per squaremillimeter was counted with an image analyzer, and the percentage ofinput macrophagesmigratedwas calculatedintriplicate samples (total filter area available for chemotaxis was 8mm2).Whenneu- trophilswerepresent onthe bottom of thefilter, differentialcountsweremadefor each triplicate sample, and the number of migrated macro- phageswascalculated.

Phatocytosiswasquantified by uptakeof anti- Forssman immunoglobulin G-coated sheep erythrocytes,labeled with51Crasdescribedear- lier (11). The numbers represent the totaluptake of2 x

10'

cells ineach group. In bonemarrow cultures grown withL-cell conditioned medium, the number ofmorphologicallydefined macro- phagesincreasedlinearlyuntildays8 to 10and then remained constant. From day 9 on, cells weremorethan 90%macrophages.Thepercent- age of peroxidase-positive macrophages in- creased untilday4 (Fig. 1), remainedconstant for5days,and thendecreased;fromday14on, no peroxidase-positive celLs were found. From days 4 through 10, the only other cells found were polymorphonuclear granulocytes. From

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630 NOTES

about day 10 on, 100% of thecellsweremacro- phages.

Wetested phagocytic andchemotactic properties aftervarioustimesinculture. Phagocytic activity ofbone marrowcellsincreased withtime inculture (Fig.2).Therateofincrease was rapid duringthe first days of culture and then slowed so that by day 26, these cells were about six times as active as peritoneal exudate macrophages. This is consistent with previously re- ported observations that the phagocytic capacity of macrophages in culture gradually increases with time (la).

12

\Tta Cellb 10

x 8

E TotalMacrophages

z-6_

4

2 Macrophages

0 4 8 12 16 20 26

DaysinCulture

FIG. 1. Growth ofmouse bone marrow cells in vitro.

The chemotactic activity of bone marrow macrophages alsoincreaseddramaticallyduring thefirst days of culture. Peak activity occurred around day 12 and then decreased to that of residentperitonealmacrophages by day 22 (Fig.

3). No contaminatingcells werepresent atthis time. In numerous experiments, we found this peak activity between days11and 14, with 70 to 100%oftheinputmacrophagesmigrating. That chemotactic responses reached a plateau with increasing concentration ofattractant suggests thatonlya subpopulation of macrophageswas abletorespond (Fig. 4). Changesinchemotaxis by bonemarrowmacrophages withtime in culture, then, must represent changes in this re- sponsivesubpopulation. Theusualmigration of macrophages or granulocytes fromavariety of sources has not been more than 20 to 40% of inputnumber (2,7,9). Thefinding that 100%of the bonemarrowmacrophagescanmigrate is of greatinterest.Itshowsthat limitedmigration is not caused by physical restrictions within the

U,w (3 I CD0.

z0

1001-

50 -

4 8 12 16 20 22 24 26

DAY IN CULTURE

FIG. 3. Chemotactic responsesofbonemarrow-de- rivedmacrophages. Resultsareexpressedastheper- cent totalinputmacrophagesthatmigrated^to1:200 endotoxin-activatedmouse serum.

aU,

Coo

^-

0

c /

4 I

cc day 1

E ~~~d22 day26 C.

DaysinCulture

FIG. 2. Phagocyticresponses of bone marrow-derived macrophages. cpm, Counts per minute; sem, standarderrorofthemean.PEC,Peritonealexudate cells; 5"Cr-EAIgG, sheep erythrocytes labeled with 5"CrandimmunoglobulinGantibody.

DILUTION OF EAMS

FIG. 4. Chemotacticdoseresponsesofmousebone marrow-derived macrophages aftervarious daysin culture.Standarderrorsofthemeansdidnotexceed 15%.EAMS, Endotoxin-activatedmouseserum.

INFECT. IMMUN.

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VOL. 33, 1981

assay system but that specific requirements must be met by the macrophages before they are ableto expresschemotaxis. These require- mentscould bemetby changes instageofmat- uration or differentiation or by production of required accessory factors by the cells them- selves. Thepercentmigration ofmouseresident peritoneal macrophages, for example, can be manipulated by the number of lymphocytes in the cell mixtureorsimply by cell density(E. J.

Leonard and A. H. Skeel,J. Reticuloendothel.

Soc., in press). It is of interest thatsomebutnot all mouse macrophage cell line cells exhibit a 100% migration(1). The highchemotactic activity of bonemarrowcellsatcertain developmen- talstagesshown in thisreportprovides a possi- ble mechanism by which macrophages needed at inflammation sites are recruited from the bone marrow. This recruitment may be medi- ated through complement. The complement split product C3e facilitates releaseof cells from thebonemarrow(8), andthe complement frag- ment C5a serves as a strong chemoattractant.

Botharegenerated during inflammation.

LITERATURE CITED

1.Asaamit, R.R., W. Falk, and E. J. Leonard. 1981.

Chemotaxisofmacrophage celllines.J.Immunol. 126:

2194-2199.

la.Bianco, C.,F. M.Griffin,Jr.,and S. C.Silverstein.

1975.Studies of the macrophage complement receptor.

Alteration of receptorfunctionupon macrophage acti- vation. J.Exp.Med.141:1278-1290.

2. Boumsell,L,and ML S.Meltzer. 1975. Mouse mono- nuclearcell chemotaxis.I.Differentialresponseon mon- ocytesandmacrophages. J. Immunol.115:1746-1748.

NOTES 631

3. Bursuker, I., and R. Goldman. 1979. Derivation of resident and inflammatorymacrophages from precursor cellsdiffering in 5'-nucleotidaseactivity.J. Reticuloen- dothel.Soc.26:205-208.

4. Daema, W. T., H. K. Koerten, and M. R. Soranzo.

1976. Differences betweenmonocyte-derivedand tissue macrophages,p.27.InS. M.Reichard,^M.E.Escobar, and H.Friedman, (ed.), The reticuloendothelial system in health and disease: functions and characteristics.

PlenumPress, NewYork.

5. Edelson,P.J.,and Z. A. Cohn. 1976. 5'-nucleotidase activity of mouseperitoneal macrophages.J.Exp.Med.

144:1581-1595.

6.Falk, W.,R H.Goodwin, Jr.,and E. J. Leonard. 1980.

A48-well microchemotaxisassemblyforrapidandac- curate measurement ofleucocytemigration.J. Immu- nol.Methods 33:239-247.

7.Falk, W., and E. J. Leonard. 1980. Human monocyte chemotaxis: migrating cells are asubpopulation with multiple chemotaxin specificities on each cell. Infect.

Immun. 29:953-959.

8.Ghebrehiwet, B.,and H. J.Miiler-Eberhardt. 1979.

C3e: anacidicfragment of humanC3 withleucocytosis- inducing activity. J. Immunol. 123:616-621.

9.Harvath, L.,W.Fa4l,and E. J.Leonard.1980.Rapid quantitationofneutrophilchemotaxis:useofapolyvi- nylpyrrolidone-free polycarbonatemembrane inamul- tiwellassembly.J.Immunol.Methods 37:39-45.

10.Klimetzek, V., and H. G. Remold. 1980. The munne bone marrowmacrophage, asensitive indicator cell for murinemigration inhibitory factoraindanewmethod for their harvest.Cell.Immunol.53:257-266.

11. Stevenson,M.M.,J.C.Rees, and M. S. Meltzer. 1980.

Macrophage function in tumorbearingmice:evidence for lactic dehydrogenase-elevating virus-assOciated changes. J. Immunol. 124:2892-2899.

12.Stewart,C.C.,and H Lin. 1978.Macrophagegrowth factorand itsrelationship tocolonystimulating factor.

J.Reticuloendothel. Soc.23:269-285.

13. vanFurth,R. 1976.Origin and kinetics of mononuclear phagocytes. Ann. N.Y. Acad. Sci. 278:161-175.

14. vanFurth,R.,and Z. A.Cohn 1968.Theorigin and kinetics of mononuclearphagocytes. J. Exp. Med. 128:

415-435.