Immunology 1983 50 139

Cultivation, proliferation and characterization of thymic macrophages

RUTH GALLILY & N. SAVION* The Lautenberg Centerfor General and Tumor Immunology, The HebrewUniversity-Hadassah Medical School, Jerusalem, Israel

Acceptedfor publication 18 April 1983

Summary. Successful long-term culture of murinethymic macrophages was achieved by plating adherentthymic cells, in the presence of L cell-conditionedmedium, on dishes coated with an extracellularmatrix. Adherent thymic cells in normal conditions ofin-vitro culture do not proliferate. Those maintainedon plastic tissue-culture dishes, and exposed to Lcell-conditioned medium, proliferate slowly to alimited degree and form very small colonies. Incontrast, when cultured in dishes coated with anextracellular matrix formed by corneal endothelialcells, in the presence of L cell-conditioned medium,adherent thymic cells proliferate rapidly and after12-21 days in culture form large colonies (about 3-5mm in diameter). The proliferating cells were identi-fied to be mononuclear phagocytes by their morpho-logical appearance, their ability to ingest both bacteriaand antibody-coated erythrocytes and by their non-

* Present address: Maurice and Gabriella GoldschlegerEye Research Institute, Tel Aviv University-Sackler Schoolof Medicine, Sheba Medical Center, Tel Hashomer, Israel.

Abbreviations: DMEM, Dulbecco's modified Eagle'smedium; EA, antibody coated erythrocytes; ECM, extracel-lular matrix; FCS, foetal calfserum; FcR, Fc receptors; FITCR&M, fluorescein isothiocyanate-conjugated rabbit anti-mouse IgG; FITC R&R, fluorescein isothiocyanate-conju-gated rabbit anti-rat IgG; LCM, L cell-conditioned medium;NSE, non-specific esterase activity; SEM, scanning electronmicroscope; TG, thioglycollate.

Correspondence: Prof. Ruth Gallily, The LautenbergCenter for General and Tumor Immunology, The HebrewUniversity-Hadassah Medical School, Jerusalem 91010,Israel.

specific esterase activity. These cells were also shownto exhibit cell surface antigens that are characteristicof differentiated macrophages, e.g. Fc receptors andthe specific macrophage cell surface marker F4/80. Ahigh percentage of these cultured cells were found tobear I-A antigens. The adherent thymic mononuclearphagocytes could be trypsinzied and passaged whilemaintaining both their ability to proliferate and theirspecific macrophage characteristics for a period of 70days. Thus, monocyte-macrophage stem cells ae pre-sent in the thymus, and under appropriate in-vitroconditions, can be made to proliferate and mature toI-A-bearing macrophages.

INTRODUCTION

It has been proposed that thymic non-lymphoid cellsplay a crucial role in several stages ofT-cell differentia-tion, including T cell-receptor diversification andselection of self H-2 restricted T cells. Two cell typeshave been postulated to be of importance in thisrespect, the epithelial cells (Zinkernagel, 1978; Jenkin-son, Owen & Aspinall, 1980) and thymic macrophages(Beller & Unanue, 1978; Lango & Schawartz, 1980).The study of the role of thymic macrophages in

T-lymphocyte differentiation and H-2 restrictionspecificity has been limited both by their low numberin this organ and by absence of an effective method ofin-vitro cultivation (van der Meer, 1980). Recentstudies on the effects of a substratum on the prolifer-

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ation of various cell types in culture demonstrated theadvantages of using a natural substratum such as theextracellular matrix (ECM), produced by culturedbovine corneal endothelial cells (Gospodarowicz, Del-gado & Vlodavsky, 1980). The ECM is a naturalsubstrate upon which cells migrate, proliferate anddifferentiate. It has been suggested, moreover, thatclose contact of cells with the ECM substratumrestores their sensitivity to mitogenic factors present inserum and plasma, whereas adherence to plasticprevents such a response (Gospodarowicz & Ill, 1981).In our study we successfully cultivated the adherentthymic cells on ECM-coated dishes in the presence ofLCM, and characterized the cells as Ia-bearingmononuclear phagocytes.

MATERIALS AND METHODS

ReagentsDulbecco's modified Eagle's medium (DMEM, H- 16)was obtained from Grand Island Biological Co(Grand Island, NY). Foetal calf serum (FCS), calfserum and horse serum were obtained from Bio-Lab(Jerusalem, Israel) and were heat-inactivated at 560 for30 min. Penicillin, streptomycin and saline-trypsin-versin (STV) solution were obtained from Bio-Lab(Jerusalem, Israel) and fungizone from Squibb (Prin-ceton, NJ). Fibroblast growth factor was prepared asdescribed previously (Gospodarowicz, Bialecki &Greenberg, 1978) and was a generous gift from DrGospodarowicz (University of California, San Fran-cisco, CA). Tissue-culture dishes and flasks werepurchased from Nunc (Roskilde, Denmark). Dextran(40,000 molecular weight), hexazotized pararosanilineand a-naphthylbutyrate were obtained from SigmaChemical Co. (St. Louis, MO). [3H]-thymidine wasobtained from New England Nuclear (Boston, MA)and emulsion (type V) from Inford Nuclear Research(England).

AntibodiesA monoclonal anti-I-Ab (Igy 2a, undefined publicspecificity) from the cell line 25-9-17S originating fromthe laboratory of Dr Sachs (NIH, Bethesda; Ozato &Sachs, 1981) was provided to us by I. Zanbar (TheWeizmann Institute, Rehovoth, Israel). Anti-F4/80, amonoclonal antibody Igy 2b subclass which is directedspecifically against mouse macrophages (Austyn &Gordon, 1981), was kindly provided by S. Gordon(Oxford, England). Fluorescein isothiocyanate

(FITC)-conjugated rabbit anti-mouse IgG (R&M)and FICT-conjugated rabbit anti-rat IgG (R&R) wasobtained from Sigma Chemical Co. (St. Louis, MO).

Cell culture conditionsMurine thymuses were carefully removed from12-15-week-old C57BL/6 mice (obtained from ourmouse colony). Care was taken with the aid of adissecting microscope to avoid contamination of thethymus with connective tissue or perithymic lymphnodes. The thymuses were dissociated into cell suspen-sion using two forceps. Fifteen million cells wereplated per 35 mm uncoated or ECM-coated tissueculture dish in 2 ml ofDMEM supplemented with 15%FCS, 5% horse serum, 30% LCM, (see below), 10 mMHEPES, 2 mm glutamine, penicillin (100 units/ml) andstreptomicin (100 pg/ml). The cultures were incubatedin a humidified CO2 (10%) incubator at 370, and themedium was changed weekly. After 21 and 42 days inculture, cells were dissociated by exposure (5-6 min,24°) to a solution containing 0.9% NaCl, 0-01 Msodium phosphate (pH 7 4), 0.05% trypsin, and 0.02%EDTA (STV solution). When cells rounded up theywere collected into a serum-containing medium, cen-trifuged (300g, 10 min) and resuspended in the culturemedium described above, at an initial cell density of105 cells per uncoated or ECM-coated 35-mm dish.

Cultures of bovine corneal endothelial cells wereprepared from steer eyes as previously described(Gospodarowicz, Meschner & Birdwell, 1977). Cul-tures were maintained on tissue culture dishes inDMEM H- 16, supplemented with 10% calf serum, 5%FCS, 2 mm glutamine, penicillin (100 units/ml), strep-tomicin (100 Mg/ml), fungizone (2 5 jig/ml) and 5%(w/v) dextran (40,000 molecular weight). Fibroblastgrowth factor (100 ng/ml) was added every other dayuntil the cells were nearly confluent. Plastic dishescoated with an ECM produced by corneal endothelialcells were prepared by exposing 2-week-old confluentcultures to 0 5% Triton X-100 solution followed byexposure to 0-1 M NH40H solution and washing inphosphate-buffered saline (Gospodarowicz et al.,1980).Mouse L cells (106 cells/264 ml flask) were plated in

20 ml of DMEM H-16 containing 10% FCS, 10 mmHEPES, 2 mm glutamine, penicillin (100 units/ml) andstreptomycin (100 ,g/ml). Cultures were incubated ina humidified CO2 (10%) incubator at 370 for 5 days,after which time the culture medium was collected.LCM was centrifuged at 200 g to remove cells anddebris, passed through a 0 45 Mm filter and stored at

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-20°. Thioglycollate (TG)-elicited peritoneal macro-phages (Gallily & Feldman, 1967) cultured for 1-5days in DMEM and 10% FCS were used for compara-tive purposes in the characterization assays.

Characterization ofmacrophages(i) Non-specific esterase activity (NSE) was assayed

according to the method of Koski, Poplack & Blaese(1976).

(ii) The presence ofFc receptors (FcR) was detectedby rosette formation at 40, using sheep red blood cellsopsonized with IgG rabbit anti-sheep erythrocyteantibodies (EA) according to a method describedpreviously (Schroit, Kedar & Gallily, 1976).

(iii) Detection of Ia-bearing cells was assayed byindirect immunofluorescence staining of the culturedcells, using a 25-9-1 7S monoclonal anti-I-Ab antibody.Following fixation of the adherent thymus-derivedcells with 1% paraformaldehyde for 15 min at roomtemperature, the plates were washed and a definedarea was covered with 10 4l of anti-I-Ab (diluted 1:5)for 30 min at 4°. Unbound antibody was removed byrinsing, 10 p1 of absorbed FITC R&M (diluted 1:50)was added and incubation proceeded for 30 min at 4°.The FITC R&M was preabsorbed extensively in thecold on peritoneal macrophage monolayers cultivatedfor 5 days in serum-free medium (0-2 ml antibodypassed over 20 16-mm wells containing 1 x 106 cul-tured macrophages each). The treated areas were thencovered with phosphate-buffered saline-glycine (1:1)and the cells (100-500/plate) were examined with aZeiss fluorescence microscope.

(iv) The expression of F4/80, a surface antigenspecific for murine macrophages (Austyn & Gordon,1981), was detected by indirect immunofluorescencestaining. The cells in the plates were fixed with 1%paraformaldehyde and 10 p1 anti-F4/80 antibody(diluted 1:50) was then layered, on a defined area, for45 min at 4°. After rinsing, FITC R&R, extensivelypreabsorbed with C57BL/6 spleen cells, was added for40 min at 4°. The rinsed cells were examined andscored as described above.

(v) Phagocytic activity of the cultured cells wasevaluated by adding either 2 x 107 Staphylococcusalbus (Gallily, Douchan & Weiss, 1977) or 2% EA(prepared as described above) suspended in DMEM,and 10% FCS. After incubation for 45 min at 370 the

cells were washed, fixed with methanol, stained withGiemsa and 100-500 cells/plate were examined. Cellswhich phagocytozed more than three particles werescored as positive.

Scanning electron microscopy (SEM)Cultures of thymic macrophages (21 days old) werefixed (60 min, 37°) with a 2-5% solution of glutaralde-hyde in phosphate-buffered saline and further incu-bated at room temperature for overnight. Incubationwith 2% osmium tetraoxide, dehydration and critical-point drying were performed as described (Murakami,Yamamoto & Itoshima, 1977). Dried specimens weresputter coated with a thin layer ofgold-palladium andexamined with a JEOL/35 SEM at an acceleratingvoltage of 25-35 kV.

Experimental designEach experiment was performed at least twice induplicate.

RESULTS

Proliferation of munine thymic adherent cellsMurine thymic cells were plated on uncoated plastic orECM-coated 35-mm dishes (1-5 x 107 cells/dish) andexposed to medium supplemented with 15% FCS, 5%horse serum and 30% LCM. After 24-hr incubation,cells plated on ECM-coated dishes adhered to thematrix. Most cells remained small, rounded and diedafter 3 days, while a few larger cells well attached onthe matrix displayed a rounded morphology (Fig. I A).When uncoated plastic dishes were used, most of thecells remained unattached and only a small fraction,consisting of large cells, attached and spread on thedish; these cells had a flattened morphology (Fig. I B).The fraction of small and rounded cells which diedafter 3 days were probably thymocytes. The attachedlive cells were counted and evaluated on day 3 andrepresented about 0 03% of the total thymic cellsplated on ECM-coated and plastic dishes, respect-ively, began to divide, and on day 7 colonies largerthan four cells each appeared. On ECM-coated dishes,40% of the colonies were larger than 10 cells percolony, while on plastic dishes only 5% were largerthan 10 cells per colony (Table 1). During the next 2weeks, most of the colonies ceased proliferation andon day 21, only 29 and 40 colonies larger than 0 5 mmin diameter per plastic and ECM-coated dish, respect-ively, could be observed. On plastic dishes, however,

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Figure 1. Morphological features and [3H]-thymidine autoradiography of thymic mononuclear phagocyte cells cultured in vitrofor 21 days. (A) SEM, single thymic macrophage cultured on ECM-coated dish (magnification x 3600). (B) SEM, singlemacrophages plated on plastic dish (magnification x 4800). (C) Light microscopy, colony of mononuclear phagocytic cells.Arrow indicates dendritic-like cell; arrowhead indicates firmly attached rounded cells (magnification x 100). (D) Lightmicroscopy, central area of colony, note the piled-up cells (arrow) in center (magnification x 100). (E) SEM, central area ofcolony (magnification x 3000). (F) Autoradiograph of 21-day-old cultures which were labelled for 72 hr with [3H]-thymidine(2 5 pCi/ml), washed with PBS and fixed with methanol, coated with emulsion and exposed for 5 days (magnification x 500).Note black stained nuclei in contrast to unstained ones.

142

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Table 1. Proliferation of thymic mononuclear phagocytic cells in vitro

No. No.per 35-mm % Attached per 35-mm % Attached

Culture ECM dish cells plastic dish cells

Attached cells 4400+ 200 4600+ 175on day 3 (13) (13)Colonies of 4-10 385+50 87 190+25 41cells on day 7 (12) (13)Colonies of 10 260±40 5-9 10+5or more cells (12) (12) 0 2on day 7Colonies of 35+4 0-8 29+2 0-605 1-0 mm (6) (3)diameter on day 21Colonies of I-0 mm 5-3+2 3 0-12 0 0or more diameter (6) (3)on day 21

Murine thymic cells (1 5 x 107 cells) were plated either on a plastic orECM-coated 35 mm tissue culture dish as described under 'Materials andMethods'. On days 3, 7 and 21 the plates were examined by invertedmicroscope and single attached cells and/or colonies were counted. Valuesrepresent mean+ SE. Numbers in parenthesis indicate the number ofdishesscored.

none of the colonies exceeded the size of 1 mm indiameter, while on ECM-coated dishes about fivecolonies per dish reached 3-5 mm in diameter, Cul-tures grown for 3 weeks on ECM-coated dishes weretrypsinized and counted. Each 35-mm dish yieldedabout 2 5 x 105 cells which were replated on ECM-coated dishes (PI) at an initial density of 105 cells per35-mm dish. The plated cells adhered to the ECM, afew of them began to proliferate and after 3 weeksapproximately 30 colonies larger than 1 mm indiameter appeared per 35-mm dish. At 3 weeks (PI)culture were trypsinized and replated once more (P2).These cells were maintained and proliferated asbefore, but began to deteriorate at about 70 days. Onthe other hand, cells plated on ECM-coated dishes, inthe absence ofLCM scarcely proliferate. After 3 weeksin culture, only very small colonies were seen, eachcolony consisting of less than 50 cells with most of thecells having rounded morphology (data not shown).Similar characteristics were observed when cells wereplated on plastic dishes but exposed to LCM-contain-ing medium.

Morphological characterization of the cultured thymic-adherent cellsThe morphological features of thymic-adherent cellsproliferating on ECM-coated dishes are shown in Fig.

IC-E. Cultures on ECM-coated dishes which wereexposed to medium containing LCM actively prolifer-ated and formed impressive multicellular colonies(Fig. ID, E). In 2-week-old cultures, the colonies wererather homogenous containing mainly large and flat-tened cells, while each of the 3-week-old coloniesconsisted of at least three distinct kinds of cells bymorphologic criteria. The first type of cells which haddendritic characteristics were well adhered and flat-tened cells, with short or long projections (Fig. IC,arrow). The second type of cell consisted of welladhered but rounded cells, (Fig. IC, headarrow), whilethe third group of cells appeared mainly at the centerof the colony and consisted of smaller, slightlyadhered but rounded cells, (Fig. IC, arrowhead), whileup on top of each other and to be shed from the cellmound into the incubation medium (Fig. ID, E). Inorder to identify the proliferating cells in the colonies,the cultures were exposed to [3H]-thymidine (2-5uCi/ml) for a period of 72 hr, followed by autoradio-graphy. In each colony many labelled nuclei wereidentified as shown in Fig. IF.

Differentiated properties of proliferating thymic cellsNon-specific esterase (NSE). One of the discrimi-

nating cytochemical indicators for mononuclear

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Ruth Gallily & N. Savion

phagocytes is the NSE. In our study, all the cellsderived from the thymic-adherent cells and cultivatedfor 3-70 days on ECM, demonstrated a very strongNSE activity (see Table 2 and Fig. 2A). Ninety-eightpercent of the control TG macrophages, as well as100% of 9-day-old cultured bone-marrow macro-phages were positive, whereas murine thymocyteswere all negative (data not shown).

FcR. The EA-rosetting test indicated that 88-98%of the thymic-adherent cells cultured for 3-70 days onECM possessed FcR (Table 2, Fig. 2B).

Phagocytic activity. The capacity of the thymic-der-ived cells to ingest bacteria and opsonized erythrocyteswas evaluated following incubation of the cells withStaph. albus and EA. As seen in Table 2, 85-100% ofthe cells cultured for up to 60 days ingest the bacteria,whereas somewhat smaller fractions, 46-47%,engulfed EA (see also Fig. 2C). A decrease in phago-cytosis was observed in cells cultured for more than 60days.

Detection of surface antigens(i) The detection of F4/80, an antigen specific formacrophages, was evaluated by the indirect immuno-fluorescence technique; 77-97% of the thymic adher-ent cells culture for 7-50 days demonstrated the F4/80antigen while neither thymic lymphocytes nor peri-toneal granulocytes were stained (see Table 2). Furth-ermore, thymic-adherent cells did not stain at all withFITC R&R preabsorbed with C57BL/6 splenocytesthus indicating both the absence of surface IgG onthese cells and the non-cytophylic property of theantibody.

(ii) Surface expression of Ia-antigens on culturedthymic-adherent cells was detected by the indirectimmunfluorescence technique using anti-I-Ab monoc-lonal antibody. The results are shown in Table 2 andFig. 2D. Sixty-five to 97% of thymic-adherent cellscultivated for up to 60 days demonstrated the presenceof I-Ab antigen. Cultures cultivated for more than 60days had a much lower ratio of I-A"-positive cells(22%). It should be noted that the less adherent,rapidly proliferating cells were removed by rinsing theplates during the fluorescence assay and that thoseincluded proportionately few Ia-bearing cells (preli-minary experiments, data not shown). The specificityof the anti-I-A" FITC R&M reaction was shown bythe following findings: (a) no positive staining was

obtained when 7- and 10-day-old cultures of thymic-adherent cells of unrelated haplotype, C3H (H-2k),were treated with both anti-I-Ab and FITC R&M; (b)no positive staining was obtained when anti-I-Ab ofIgM class was used with FITC R&M IgG; (c) nopositive staining was obtained when C57BL/6 thymiccells were incubated with FITC R&M in the absence ofanti-I-A".

DISCUSSION

Semi-solid liquid culture systems provide a convenientmethod for studying the presence and characteristicsof haematopoietic stem cells (CFU-S) in bone marrowand other organs (Pluznick & Sachs, 1965; Bradley &Metcalf, 1966). When bone marrow-derived cells werecultivated under defined conditions in the presence ofcolony-stimulating factors (provided by feeder layersor added as conditioned medium) primarily two typesof colonies develop, mononuclear phagocytic coloniesand granulocytic colonies. In the past few years,committed stem cells for mononuclear phagocyteshave also been shown to be present in tissues otherthan bone marrow, including the peritoneal andpleural cavities, spleen, lymph nodes and peripheralblood (reviewed by van der Meer, 1980). It is generallyaccepted that these originate in the bone marrow.However, the conditions for obtaining optimal growthof colonies from these stem cells have been quiterestricted and often required inflammatory stimuli.Even primary bone-marrow cultures begin deteriora-ting after 3-4 weeks unless specific conditions areemployed (van der Meer, 1980).

Recent studies in vitro using a natural substratumsuch as the ECM produced by cultured bovine cornealendothelial cells demonstrated the importance of theECM in supporting the proliferation of various celltypes, such as bovine granulosa, adrenal cortex(Gospodarowicz et al., 1980), vascular endothelial(Gospodarowicz & Ill, 1980a), vascular smoothmuscle (Gospodarowicz & Ill, 1981) and cornealendothelial cells (Fujii et al., 1982). As macrophagesare among the most adherent cells in vitro, their highaffinity to the substratum might reflect their ability tointeract with the surrounding substratum in vivo, andmight indicate a role of the extracellular matrix inmacrophage proliferation and differentiation. Indeed,the ECM allowed long-term proliferation of thymicmacrophages at high clonal density. In contrast, whenplated on plastic dishes and exposed to LCM, thymicmacrophages proliferated slowly and to a very limited

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Thymic macrophages

Table 2. Characterization of thymic adherent cells during cultivation in vitro

% Positive cells*

Fc lat F4/80t Phagocytosis PhagocytosisPassage Days in culture NSE receptor surface antigen surface marker of S. albus of EA

PO 3 100 98 88 42 100 467-10 100 88 75 79 89 NT:11-20 99 99 65 77 90 7621-30 98 98 68 83 NT 64

Pi 31-40 100 97 NT NT NT 5441-50 100 96 89 97 NT NT51-60 100 100 96 NT 85 NT

P2 61-72 100 97 22 NT 45 48

TG-elicited 1-5 98 99 6§ 89 93 72peritonealmacrophages

Primary cultures (Po), first (PI) and second (P2) passages of murine thymic macrophages were prepared fromthymic cell suspension, maintained and passaged on ECM coated dishes as described under 'Materials andMethods'. On the days indicated in the Table, cultured cells from duplicate dishes were assayed formacrophage-specific activities as described in 'Materials and Methods'. Due to the extensive washing involved inthe assays, most of the rounded and less adherent cells were removed from the dishes. Thus, mainly well adheredand flattened cells were scored. TG-elicited peritoneal macrophages were assayed for comparison.

* The numbers represent averages of at least two experiments. A rough estimate ofthe spread in the percentage ofpositive cells within each experiment was 1, 1, 10,4,5 and 18% for NSE, Fc, Ia, F4/80 engulfment ofS. albus and EArespectively. The spread across experiments was about twice as great.

t Background has been subtracted ( < 4%).$ NT = not tested.§ Normal peritoneal macrophages.

extent. Moreover, because macrophages maintainedat a clonal density on plastic dishes respond poorly tofactors present in LCM, but do respond when main-tained on ECM, it is likely that the close contact of thecells with the ECM restores their sensitivity to agentspresent in the LCM.The thymus of an adult C57BL/6 mouse contains

about 2 x 104 macrophages. Most of these cells have avery limited ability to proliferate in culture, even underoptimal conditions such as those described in thisstudy. Less than 1% of these cells demonstrated theability to proliferate in culture and to produce largecolonies (about 3-5 mm diameter) when culturedunder optimal conditions. This small fraction ofadherent colony-forming cells which proliferate exten-sively in culture probably represents the small popula-tion ofmonocyte-macrophage stem cells present in thethymus. The cells in each colony were not homo-genous in morphology and included three types ofpopulations. Well adhered cells without cytoplasmicprojections formed one population. A second popula-

tion consisted offlattened adherent cells with cytoplas-mic projections displaying the morphology of dendri-tic cells. The third population was composed ofsmaller cells which were rounded and less adherent.The two first well adhered cell types were shown tohave many characteristic properties of differentiatedmacrophages. The population of slightly adheredsmall and rounded cells, which usually are shed intothe incubation medium, are at present under extensivestudy.Our study demonstrates, by the following charac-

teristics, that cells derived from the thymic-adherentcells grown on ECM in the presence of LCM aremononuclar phagocytes. Many of them are dish-adherent with morphological features ofmononuclearphagocytes. Non-specific esterase activity, whichappears dominantly in macrophages (Koski et al.,1976), was detected in all the cells. Similarly, most ofthem expressed both Fc receptors and the F4/80, asurface antigen which is specific for macrophages(Austyn & Gordon, 1981). A high percentage of the

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cells phagocytose both bacteria and opsonized eryth-rocytes. Interestingly, 65-97% of the thymicmononuclear phagocytes bear I-Ab antigen detectedby a monoclonal anti-I-Ab, even after cultivation forup to 60 days and following two passages. The Tasu.rface molecules (class II antigens), which are theimmune-response (Ir) gene products of the majorhistocompatibility complex, were shown to play acentral role in regulation of immune response(Shreffier & David, 1975; Thomas, Yamashita &Shevach, 1977; Benacerraf & Germain, 1978).Furthermore, it has been postulated that the thymicnon-lymphoid Ia-bearing cells, i.e., the epithelial cellsor the macrophages (Zinkernagel, 1978; Jenkinson etal., 1980; Beller & Unanue, 1978; Longo & Schwartz,1980) might determine the T-cell self recognitionrepertoire by a selective process (Zinkernagel et al.,1978; Fink & Bevan, 1978). This hypothesis wassupported by experiments with chimeric animals(Longo & Schwartz, 1980; Sharrow, Mathieson &Singer, 1981).We did not anticipate the expression of Ta on the

surface of a high percentage of cultured thymicmononuclear phagocytes since various studies haveshown that macrophages lose Ta expression uponcultivation. Indeed, thymus macrophages werereported to lose their Ia following cultivation for 3days (Beller & Unanue, 1980). Similarly, the ex-pression of Ia on peritoneal exudate macrophages,although quite low, was shown to disappear followingin-vitro cultivation (Stadecker, Wyler & Wright, 1982)and cultured bone-marrow macrophages were shownnot to express Ia on their surfaces without activationby lymphokines (Calami, Beller & Unanue, 1982).This latter observation, however, is still under discus-sion (Stern, Erb & Gisler, 1979). Furthermore, it wassuggested that the presence of LCM, which waspresent in all our cultures, antogonizes Ta expression(Calami et al., 1982) and that the Ia phenotype ofmacrophages is regulated by immune reactions involv-ing T lymphocytes or their products (Beller, Kiely &Unanue, 1980). Our present data do not support all ofthese findings and assumptions. The cultured thymicmacrophages expressed Ta for a prolonged period oftime, and neither T lymphocytes nor their productswere present during cultivation. Our experimentsfurther negate the claim that Ia on macrophages ispassively acquired from other cells. At present we cannot rule out the possibility that the high percentage ofIa-positive cultured thymic macrophages in our sys-tem is regulated by the specific in vitro conditions we

employed. It is tempting, however, to suggest that thisextensive Ia expression on thymic macrophages is areal physiological phenomenon which might providethese cells with the capacity to regulate T-cell differen-tiation and the generation of T-cell immunocompe-tence in vivo.

ACKNOWLEDGMENTS

We are grateful to Dr N. Haren-Ghera who triggeredour interest in the thymus macrophages, to E. Stein forher important contribution, and to M. Weigensbergfor her help with the manuscript. This work wassupported by the Society of Research Associates of theLautenberg Center, the Concern Foundation of LosAngeles, and Dr 'I' Found.

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