Granulocytes, macrophages, and dendritic cells arise from a common major histocompatibility complex class II-negative progenitor in mouse bone marrow

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1 Proc. Ntl. Acd. Sci. USA Vol. 90, pp , April 1993 Immunology Grnulocytes, mcrophges, nd dendritic cells rise from common mjor histocomptibility complex clss II-negtive progenitor in mouse bone mrrow KAYO INABA*t, MUNEO INABA*, MASASHI DEGUCHI*, KATSUHIKO HAGI*, RYoJi YASUMIZUf, SUSUMU IKEHARAt, SHIGERU MURAMATSU*, AND RALPH M. STEINMAN *Deprtment of Zoology, Fculty of Science, Kyoto University, Skyo, Kyoto 606, Jpn; tfirst Deprtment of Pthology, Knsi Medicl University, Moriguchi, Osk 570, Jpn; nd Lbortory of Cellulr Physiology nd Immunology, The Rockefeller University, New York, NY Communicted by Znvil A. Cohn, December 21, 1992 ABSTRACT The developmentl origin of dendritic cells, specilized system of mjor histocomptibility complex (MHC) clss 11-rich ntigen-presenting cells for T-celi immunity nd tolernce, is not well chrcterized. Grnulocyte-mcrophge colony-stimulting fctor (GM-CSF) is known to stimulte dendritic cells, including growth nd development from MHC clss 11-negtive precursors in suspension cultures of mouse bone mrrow. Here we studied colony formtion in semi-solid methylcellulose cultures, clssicl biossy system in which GM-CSF induces the formtion of mixed grnulocytemcrophge colonies. When colonies were induced from MHC clss II-negtive precursors, smll subset (1-2%) of typicl dendritic cells developed longside mcrophges nd grnulocytes. The dendritic cells were distinguished by their cytologic fetures, high levels of MHC clss II products, nd distinct intrcellulr grnule ntigens. By using differentil dherence to plstic, enriched popultions of the vrious myeloid cell types were isolted from colonies. Only the dendritic cells stimulted primry T-celi immune response, the mixed leukocyte rection, nd the potency ws comprble to typicl dendritic cells isolted from spleen. Mcrophges from mixed or pure colonies were inctive s stimultor cells. Therefore, three distinct pthwys of myeloid development-grnulocytes, mcrophges, nd dendritic cells-cn develop from common MHC clss fl-negtive progenitor under the egis of GM-CSF. Dendritic cells constitute system of ntigen-presenting cells in the T-cell-dependent res of lymphoid tissues nd t the portls of ntigen entry into the body such s skin, irwy epithelium, nd fferent lymph. These cells express high levels of ntigen-presenting mjor histocomptibiity complex (MHC) clss II products, cell surfce dhesion nd costimultory molecules (1), nd certin cytoplsmic ntigens loclized primrily within intrcellulr grnules (2, 3). Dendritic cells ct s specilized ccessories to induce immunity (4) nd tolernce (5-7). In contrst to other hemtopoietic cells, the developmentl linege of dendritic cells is not worked out. A reltionship of dendritic cells to phgocytes is suggested by the shred responsiveness of these cells to grnulocytemcrophge colony-stimulting fctor (GM-CSF). GM-CSF mintins dendritic-cell vibility (8-11) nd medites their development from less-mture but nonproliferting precursors in skin (8, 9). More recently, GM-CSF hs been shown to induce the extensive growth of dendritic-cell precursors in suspension cultures (12-14). In these cultures, the mture nonproliferting progeny express high levels of MHC clss II nd other mrker ntigens tht re chrcteristic of dendritic cells. Interestingly, dendritic cells do not respond to the more The publiction costs of this rticle were defryed in prt by pge chrge pyment. This rticle must therefore be hereby mrked "dvertisement" in ccordnce with 18 U.S.C solely to indicte this fct. linege-restricted mcrophge nd grnulocyte colonystimulting fctors (M-CSF nd G-CSF, respectively) (8, 10, 12) Ṡince GM-CSF cn induce the formtion of mixed popultions of grnulocytes nd mcrophges in semi-solid colony systems (15), we sked whether dendritic cells could lso rise from colony-forming precursor tht is common to phgocytes. Cells with some of the fetures of dendritic cells hve been detected in humn cell colonies tht were induced with lectin-conditioned medium (16) nd more recently with mixture of tumor necrosis fctor nd GM-CSF (17). Here we isolte cells from colonies tht re induced by recombinnt GM-CSF from MHC clss II-negtive precursors nd show tht the colonies contin significnt numbers of dendritic cells by severl criteri. The dendritic cells re redily distinguished from phgocytes prticulrly in their high levels of MHC clss II expression, content ofintrcellulr ntigens, nd ntigen-presenting-cell function. Dendritic cells, therefore, represent distinct immunostimultory differentition pthwy tht is induced by GM-CSF but shres common MHC clss II-negtive precursor with phgocytes. MATERIALS AND METHODS Mrrow Cells nd Cultures. Bone mrrow suspensions were prepred from 6- to 8-week-old (BALB/c x DBA/2)F1 mice s described, including tretment with monoclonl ntibodies nd complement to deplete cells bering MHC clss II, CD4, CD8, nd B220 mrkers (13). Approximtely 6 x 104 cells from BALB/c x DBA/2 dult mle mice were suspended in 1 ml of culture medium [RPMI 1640 medium/10% (vol/vol) fetl clf serum/50 um 2-mercptoethnol, gentmicin (20,ug/ml)] in the presence of 1% methylcellulose (Nkri Tesque, Kyoto) nd recombinnt mouse GM-CSF ( units/ml, 9.2 x 107 units/mg; Kirin Brewery, Tkski, Gunm) nd pplied to 35-mm Petri dishes (Flcon, 3001). Phenotype of Mrrow Cells. After 5 or 6 dys ofculture, ""50 mixed colonies were plucked with Psteur pipette from the methylcellulose, resuspended, wshed, nd cytocentrifuged onto glss slides (Shndon, Sewickley, PA). The cytocentrifuged cells were stined with Giems (Wko Pure Chemicl, Osk) or were fixed with bsolute cetone nd stined with monoclonl ntibodies (see Results) followed by peroxidseconjugted nti-immunoglobulin s described (13). To observe the distinctive morphology of dendritic cells in live preprtions, the colonies were exmined t finl mgnifiction of x40 to x 100. To seprte the vrious cell types tht re found in colonies, colonies were pooled nd seprted into three frctions on the bsis of differentil dherence to plstic. Abbrevitions: MHC, mjor histocomptibility complex; GM-CSF, grnulocyte-mcrophge colony-stimulting fctor; MLR, mixed leukocyte rection. tto whom reprint requests should be ddressed. 3038

2 The cells were pplied in culture medium to 35-mm tissue culture dishes nd fter 2 hr were seprted into nondherent (grnulocytes nd some dendritic cells), wekly dherent (dislodged by gentle pipetting), nd strongly dherent [dislodged fter 20 min of culture t 37 C in phosphte-buffered sline (PBS) with 10 mm EDTA]. Stimultion of the Mixed Leukocyte Rection (MLR). Enriched popultions of dendritic cells nd mcrophges were prepred from pools of bone mrrow colonies s bove, treted with mitomycin C (Kyow Hkko, Tokyo), nd pplied in grded doses to 3 x 105 purified T cells from llogeneic C3H/He mice. DNA synthesis in the responding T cells ws mesured with pulse of 1 ACi of [3H]thymidine (1 Ci = 37 GBq) dministered t h of culture. Dt with syngeneic T cells s responders were <10% of the corresponding response with llogeneic T cells. RESULTS A slightly modified colony-forming ssy ws used to evlute the effect of GM-CSF on mouse bone mrrow progenitors. Since hllmrk of the mture dendritic cell is the bundnt expression of MHC clss II products, we first treted the mrrow suspensions with ntibodies nd complement to deplete cells tht express MHC clss II nd the lymphocyte ntigens B220, CD4, nd CD8. Tretment with ntibody nd complement removed detectble B nd T lymphocytes from suspension cultures (12, 13). Smll numbers of the treted mrrow cells (or unmnipulted bone mrrow tht gve colonies similr to those described below) Immunology: Inb et l. DAY 5 i & s b 4j 0..,, %* # x ^. *,. * 0 c *' Proc. Ntl. Acd. Sci. USA 90 (1993) 3039 Tble 1. Yields of colony-forming units from bone mrrow depleted of T, B, nd MHC clss II-rich cells Colony-forming units, no. per Type of colony 105 bone mrrow cells Grnulocyte 79 ± 12 Mixed-type 108 ± 15 Mcrophge 57 ± 14 Eosinophil 10 ± 2 Burst 1.6 ± 0.9 Vlues re the mens ± SEM of qudruplicte ssys in four experiments. were then suspended in medium tht ws mde semi-solid by dding methylcellulose nd ws supplemented with fetl clf serum nd vrious cytokines. We found it helpful to omit the horse serum, stndrd supplement in this culture system. At 5-8 dys, colonies ppered, mny of which were of the "mixed" type contining more compct round cells nd mny dispersed cells. The yield of different types of colonies is shown in Tble 1. By severl criteri, we were ble to show tht some of the dispersed cells were typicl dendritic cells s long s GM-CSF (but not interleukin 3, M-CSF, or G-CSF) hd been used to generte the colonies. Identifiction of Cells with the Phenotype of Dendritic Cells. Under phse-contrst microscopy, the presumptive dendritic cells in individul colonies extended distinct processes in mny directions from the cell body. These lrge lmellipodi or "veils" re chrcteristic of dendritic cells from mny tissues nd species (18-20). Colonies were picked nd pooled *I: d - W: 0 * w. :. #I, 4 L.. 9r e 9. f 9 or DAY 6 ql_ *n 0~~~~~~~~~~~~~~~~~~~~~~~~~~~s 'Ak0 * ^ *.. " s~~~~. h..'!,...-, 0 f:. 4 0 *.. %, 4 "., Xb FIG. 1. Identifiction of dendritic cells in mixed colonies of mcrophges nd grnulocytes t dy 5 nd 6 of culture in GM-CSF. Presumptive dendritic cells in these cytocentrifuged preprtions of pooled colonies re indicted by rrows, except in d nd i where grnulocytes re identified. (-f nd h-j, x 100; g, x250.) ( nd f) Giems stin. Mcrophges re round nd hevily vcuolted; dendritic cells (rrows) re lrger, less-vcuolted, nd hve mny spinous processes. (b nd g) MHC clss II ntigens (monoclonl B21-2, ATCC TIB229) re strongly expressed on subset of lrger spiny cells. (c nd h) 2A1 intrcellulr ntigen, found in the perinucler region of dendritic cells but not phgocytes (unpublished dt), stins subset of the colony cells. (d nd i) RB6-8C2 grnulocyte ntigen. (e nd j) FA1l intrcellulr grnule ntigen of mcrophges (21). A few mononucler cells, presumbly the dendritic cells, re FAll-negtive..v 'o... i 11 V-1 ".r. '' A....," v w. I.r

3 3040 Immunology: Inb et l. Proc. Ntl. Acd. Sci. USA 90 (1993) --..:-.:;.;.;, * V....1,,.i *1 4,I... ;,t.:.1 "I I...:. 41 MU b FIG. 2. Higher-power views of intrcellulr ntigens within the endocytic vcuolr system. Cytocentrifuged cells from 6-dy colonies were stined by 2A1 nti-dendritic cell (), FAll nti-mcrophge (b), nd MAC-3 nti-lysosome (c) ntibodies. The less-vcuolted dendritic cells (rrows) stin strongly for 2A1 but more wekly for FAll nd MAC-3 ntigens. The reciprocl pplies to hevily vcuolted mcrophges. for exmintion with the stndrd Giems hemtologic stin (Fig. 1 nd f). Three cell types were evident. There were typicl mcrophges (i.e., lrge round mononucler cells with mny cytoplsmic vcuoles). There lso were grnulocytes (cells with ple cytoplsm but chrcteristic irregulrly shped condensed nuclei often ssuming "doughnut" shpe). Then, there were mononucler profiles (Fig. 1 nd f, rrows; usully lrger thn most of the phgocytes, with n irregulr "frilly" shpe nd few cytoplsmic vcuoles). After stining with ntibodies to MHC clss II ntigens, the lrge cells were stined very strongly nd selectively, nd the dendritic processes were clerly evident (Fig. 1 b nd g, rrows). When we pplied the M342 (3) or 2A1 (unpublished dt) monoclonl ntibodies tht recognize cytoplsmic vcuoles within dendritic cells nd some B cells (but not phgocytes), subset of cells gin stined selectively (Fig. 1 c nd h, rrows; Fig. 2). Grnulocytes were further identified with the RB6 monoclonl ntibody (Fig. 1 d nd i, rrows). The FAll monoclonl ntibody (22), which identifies "mcrosilin" tht is bundnt in cytoplsmic grnules of mcrophges (21), stined most of the mononucler cells in the colonies. However, some FAll-wek presumptive dendritic cells were lso noted (Fig. 1 e nd j, rrows; Fig. 2b). A monoclonl ntibody (MAC-3) to lysosoml membrne glycoprotein stined the phgocytes strongly reltive to the wek stin on presumptive dendritic cells (Fig. 2c). The yield of the subset of MHC clss II-rich nd 2A1 cytoplsmic ntigen-positive cells ws determined in colonies t different times. At the pek, dy 5-6, the presumptive dendritic cells represented % of the progeny (Tble 2). Tble 2. Frequency of dendritic cells rising in mixed colonies generted from MHC clss II-negtive bone mrrow precursors Dy of MHC clss II- 2A1-positive colony ssy rich cells, % cells, % ND ± ± ± ± ± ± 0.08 Vlues re the mens ± SEM of three experiments in which >400 cells were counted in cytocentrifuged cells hrvested from mixed colonies. Prior work hs shown tht the 2A1 nd M342 cytoplsmic ntigens pper reltively lte in dendritic-cell development, fter the cell hs stopped proliferting, wheres MHC clss II is expressed during nd fter growth (12, 13). We conclude tht GM-CSF induces MHC clss IT-negtive precursors to form mixed colonies tht contin typicl phgocytes (mcrophges nd grnulocytes) plus some dendritic cells, s ssessed by cytologic fetures nd surfce nd intrcellulr ntigens. Seprtion of Colony-Derived Mcrophges nd Dendritic Cells. To verify tht ll the distinctive mrkers of dendritic cells (Figs. 1 nd 2) were being expressed by the sme popultion, we pooled the cells from mny colonies nd prepred mcrophge- nd dendritic-cell-enriched popultions by using dherence criteri. It is known tht dendritic cells, unlike mcrophges, do not stick firmly to plstic surfces (19, 20, 23). When colony-derived cells were pplied to plstic, the bulk of the grnulocytes nd some of the dendritic cells were nondherent nd could be removed by swirling the pltes. When we gently pipetted buffer over the remining dherent cells, most of the dendritic cells were dislodged s verified by double lbeling for MHC clss II nd the 2A1 intrcellulr dendritic cell ntigen (Fig. 3 -c; e.g., cells t rrows), but not the FAll mcrophge grnule ntigen (dt not shown). The vcuolted phgocytes remined firmly ttched nd could only be dislodged fter wrming the cultures in PBS supplemented with 10 mm EDTA. Almost ll the cells in the firmly ttched popultion (Fig. 3d) hd low or bsent MHC clss TI (Fig. 3e), nd expressed the FAll ntigen tht is bundnt in mcrophge grnules (Fig. 3f), but lcked 2A1 (dt not shown). In ddition, mcrophges expressed much higher levels of the lysosoml membrne glycoprotein detected by the MAC-3 monoclonl ntibody thn did dendritic cells (dt not shown). With dditionl GM-CSF, we could not convert the phgocytes to dendritic cells or vice vers. Function of Colony-Derived Dendritic Cells nd Mcrophges. The enriched popultions (nondherent, wekly dherent, nd firmly dherent) were then evluted for immunostimultory ctivity by using the MLR s test system. In the MLR, leukocytes from one strin of mice, prticulrly dendritic cells (25, 26), initite strong prolifertive response in T cells from other strins tht differ in MHC. Stimultion of the MLR requires presenttion of the foreign MHC prod-

4 Immunology: Inb et l. Proc. Ntl. Acd. Sci. USA 90 (1993) 3041 FIG. 3. Two-color immunofluorescence to test for coexpression of MHC clss II nd other ntigens in enriched bone-mrrow-colony-derived dendritic cells nd mcrophges. Cells were double-lbeled for MHC clss II nd either the 2A1 ntigen of dendritic-cell intrcellulr grnules (unpublished dt) or the FAll ntigen of mcrophge intrcellulr grnules (21). ( nd d) Phse-contrst imges of the cytocentrifuged cells fixed in cetone. (b nd e) MHC clss II monoclonl ntibody N22 (24) (ATCC HB225). (c nd f) Double-lbeled cells with rt monoclonl ntibodies to intrcellulr ntigens (2A1 nd FAll, respectively). (-c) Loosely ttched dendritic cell-enriched popultion. (d-j) Firmly ttched mcrophge-enriched popultion. (b nd c) The dendritic-cell frction stins strongly for MHC clss II nd 2A1 intrcellulr ntigens. Two such dendritic cells re indicted by rrows. (e nd f) The mcrophge frction expresses little or no MHC clss II but high levels of the FAll intrcellulr ntigen ( single clss II-rich profile is indicted by rrows). The stining sequence ws N22 hmster nti-mouse clss II, biotin-conjugted rbbit nti-hmster immunoglobulin (Jckson ImmunoReserch), Texs red-streptvidin (Biomedi, Foster City, CA), 2A1 or FAll rt monoclonl ntibody, nd fluorescein-conjugted mouse nti-rt immunoglobulin (Boehringer Mnnheim). (x250.) uct plus other ccessory functions [e.g., the ligtion of severl dhesion or costimultory molecules including CD2, CD11, nd CD28 (1)]. The nondherent cells from the colonies (contining grnulocytes nd dendritic cells) nd the loosely dherent cells (contining primrily dendritic cells; Fig. 3 -c) were very much enriched reltive to the bulk or unfrctionted popultion in MLR stimulting ctivity (Fig. 4, compre dt for wekly dherent nd nondherent GM-colony popultions with tht for the bulk GM-colony popultion). The strongly dherent GM-colony mcrophges were inctive. The wekly dherent dendritic-cell component ws indistinguish- E 105 Q L- o :5.c 104 ) I Stimultor cells, no. per well ble from splenic dendritic cells in MLR stimulting ctivity (Fig. 4, compre wekly dherent GM-colony popultion nd spleen dendritic cells). Some of the methylcellulose colonies were not of the "mixed" type but contined either typicl mcrophges nd grnulocytes s described (15). When cells from these colonies were tested, no MLR stimulting ctivity ws pprent. DISCUSSION Bowers nd Berkowitz (27) first reported tht dendritic cells could be generted from MHC clss II-negtive precursors in.2 0 Spleen dendritic cell V Cells from GM colony * Bulk popultion v Nondherent popultion A Wek dherent popultion * * Strong dherent popultion Cells from M colony O Bulk popultion * Cells from G colony * Bulk popultion FIG. 4. MLR stimulting ctivity of vrious cell types derived from GM-CSF-induced bone mrrow colonies. GM-CSF ws used to induce the formtion of colonies from MHC clss II-negtive precursors s in Fig. 1. Cells from mixed colonies (contining both mcrophges nd grnulocytes, GM colony) or from pure mcrophge (M colony) nd grnulocyte (G colony) colonies were plucked from the methylcellulose semi-solid cultures, wshed, mitomycin C-treted, nd used to stimulte n llogeneic MLR in 3 x 105 purified T cells from C3H H-2k mice. The cells in the mixed GM colonies were seprted into frctions s in Fig. 3. The nondherent frction contins both grnulocytes nd dendritic cells, the wekly dherent frction is primrily dendritic cells, nd the strongly dherent frction is primrily mcrophges. The MLR ws monitored by mesuring DNA synthesis in the T cells ([3H]thymidine uptke) t h of culture.

5 3042 Immunology: Inb et l. serum-free suspension cultures of rt bone mrrow. By dding GM-CSF, much lrger numbers of grnulocytes, mcrophges, nd dendritic cells cn be generted (13). Reid et l. (16) observed tht bulk popultions of humn bone mrrow or blood cells would generte mixed colonies of mcrophges nd dendritic cells upon supplementtion with lectin-stimulted leukocyte-conditioned medium tht likely contined GM-CSF. Recently, Reid et l. (17) hve documented tht GM-CSF nd tumor necrosis fctor work together to generte colonies contining mcrophges nd dendritic cells or dendritic cells only. Here we show tht dendritic cells shre common MHC clss II-negtive colony-forming precursor with phgocytes (grnulocytes nd mcrophges), by using the clssicl GM- CSF semi-solid biossy. We were unble to detect pure dendritic-cell colonies. In mixed colonies, the dendntic cells were enumerted nd distinguished from typicl phgocytes by the following fetures: dendritic or veiled morphology, wek dherence to plstic, nd distinct ntigenic mrkers including high levels of MHC clss II products nd new intrcellulr ntigens. Since this smll subset of dendritic cells could be enriched by dherence methods, it ws possible to study the T-cell stimulting ctivity of different cell types from mixed colonies nd from pure mcrophge nd grnulocyte colonies. The MLR stimulting ctivity of colony-derived dendritic cells ws very strong nd comprble to tht seen in mture lymphoid dendritic cells, wheres the phgocytes lcked detectble ctivity (Fig. 4). Therefore, by the criteri tht we hve studied, the dendritic cells differ mrkedly from the mcrophges nd grnulocytes tht re lso rising from MHC clss II-negtive progenitors within individul colonies. The extent to which this distinct immunostimultory, differentition pthwy occurs in bone mrrow in vivo is uncler. Typicl dendritic cells hve not been identified in fresh bone mrrow suspensions nd re rre in blood, wheres mture grnulocytes nd mcrophges re found in both sites. In vivo, dendritic cell development my not proceed to the sme extent in mrrow s in these semi-solid colonyforming systems. Prolonged exposure to GM-CSF, or other cytokines yet to be identified, my be required to induce the full development of MHC clss II-rich immunostimultory dendritic cells. We thnk Judy Adms for the composite microgrphs. This work ws supported by grnts from the Ministry of Eduction, Science nd Culture of Jpn for Joint Reserch in Interntionl Scientific Reserch ( ), the Mochid Memoril Foundtion of Medicl nd Phrmceuticl Reserch, the Shimizu Foundtion for Immunologicl Reserch, nd Ntionl Institutes of Helth (Grnt A113013). Proc. Ntl. Acd. Sci. USA 90 (1993) 1. Young, J. W., Koulov, L., Soergel, S. A., Clrk, E. A., Steinmn, R. M. & Dupont, B. (1992) J. Clin. Invest. 90, Breel, M., Mebius, R. E. & Krl, G. (1987) Eur. J. Immunol. 17, Agger, R., Witmer-Pck, M., Romni, N., Stossel, H., Swiggrd, W. J., Metly, J. P., Storozynsky, E., Freimuth, P. & Steinmn, R. M. (1992) J. Leuk. Biol. 52, Steinmn, R. M. (1991) Annu. Rev. Immunol. 9, Mtzinger, P. & Guerder, S. (1989) Nture (London) 338, Mzd, O., Wtnbe, Y., Gyotoku, J.-I. & Ktsur, Y. (1991) J. Exp. Med. 173, Inb, M., Inb, K., Hosono, M., Kummoto, T., Ishid, T., Murmtsu, S., Msud, T. & Ikehr, S. (1991) J. Exp. Med. 173, Witmer-Pck, M. D., Olivier, W., Vlinsky, J., Schuler, G. & Steinmn, R. M. (1987) J. Exp. Med. 166, Heufler, C., Koch, F. & Schuler, G. (1987) J. Exp. Med. 167, Nito, K., Inb, K., Hirym, Y., Inb-Miym, M., Sudo, T. & Murmtsu, S. (1989) J. Immunol. 142, McPherson, G. G. (1989) Immunology 68, Inb, K., Steinmn, R. M., Witmer-Pck, M., Ay, K., Inb, M., Sudo, T., Wolpe, S. & Schuler, G. (1992) J. Exp. Med. 175, Inb, K., Inb, M., Romni, N., Ay, H., Deguchi, M., Ikehr, S., Murmtsu, S. & Steinmn, R. M. (1992) J. Exp. Med. 176, Scheicher, C., Mehlig, M., Zecher, R. & Reske, K. (1992) J. Immunol. Methods 154, Metclf, D. (1988) The Moleculr Control of Blood Cells (Hrvrd Univ. Press, Cmbridge, MA). 16. Reid, C. D. L., Fryer, P. R., Clifford, C., Kirk, A., Tikerpe, J. & Knight, S. C. (1990) Blood 76, Reid, C. D. L., Stckpoole, A., Meger, A. & Tikerpe, J. (1992) J. Immunol. 149, Drexhge, H. A., Mullink, H., de Groot, J., Clrke, J. & Blfour, B. M. (1979) Cell Tissue Res. 202, Freudenthl, P. S. & Steinmn, R. M. (1990) Proc. Ntl. Acd. Sci. USA 87, Schuler, G. & Steinmn, R. M. (1985) J. Exp. Med. 161, Rbinowitz, S. S. & Gordon, S. (1991) J. Exp. Med. 174, Smith, M. J. & Koch, G. L. E. (1987) J. Cell Sci. 87, Steinmn, R. M., Kpln, G., Witmer, M. D. & Cohn, Z. A. (1979) J. Exp. Med. 149, Metly, J. P., Witmer-Pck, M. D., Agger, R., Crowley, M. T., Lwless, D. & Steinmn, R. M. (1990)J. Exp. Med. 171, Steinmn, R. M. & Witmer, M. D. (1978) Proc. Ntl. Acd. Sci. USA 75, Inb, K. & Steinmn, R. M. (1984) J. Exp. Med. 160, Bowers, W. E. & Berkowitz, M. R. (1986) J. Exp. Med. 163,