INTRODUCTION

The modulation of the expression and localization of MHC-IIhas been extensively studied in DC. The reason is that DCdisplay unique Ag presentation ability as compared to otherprofessional APC (MΦ and B lymphocytes). DC efficientlypresent antigens (Ag) to sensitized T lymphocytes and canprime virgin T and B lymphocytes. It is well established thatupon in vitro maturation by inflammatory molecules (TNF-α,IL-1β, LPS), DC up-regulate the expression of MHC-II and thepresentation of Ag at their plasma membrane.

MHC-II are highly polymorphic glycoproteins which aremainly expressed at the surface of professional Ag presentingcells (APC). MHC-II bind exogenously derived antigenicpeptides and present them to T lymphocytes (Watts, 1997).During their biosynthesis, the α and β chains of MHC-II aretranslocated into the endoplasmic reticulum and associate witha non polymorphic third glycoprotein, the invariant chain (Ii),to form a nonameric complex (Cresswell, 1996). Ii facilitatescorrect folding of the αβ subunits and guides the complexthrough the Golgi apparatus and the trans-Golgi network.These αβIi complexes are then either targeted away from thebiosynthetic pathway to the cell surface (Saudrais et al., 1998),and internalized to endosomes and lysosomes (Benaroch etal., 1995; Roche et al., 1993, Warmerdam et al., 1996), or

transferred from the trans-Golgi network to the endocyticpathway (Benaroch et al., 1995; Peters et al., 1995). Ii is thenprocessed by resident lysosomal proteases and removed fromthe complex by human leukocyte antigen-DM (HLA-DM)which is found enriched in MHC-II containing lysosomes(Busch and Mellins, 1996).

It is now established that lysosomal MHC-II-containingcompartments (MIIC) play a critical role in the process of Agpresentation mediated by MHC-II. In B cells and DC, thesecompartments appear to be heterogenous and have beenclassified into at least four types according to their morphology,protein content and accessibility to endocytic tracers. They canbe MIIC of irregular shapes with only a few internal vesicles,multivesicular MIIC, multilaminar MIIC and intermediateMIIC with internal vesicles and concentrically arrangedmembrane sheets (Kleijmeer et al., 1997; Nijman et al., 1995).In MΦ, tubular electron dense prelysosomes were shown tocontain MHC-II (Harding and Geuze, 1992). All MIIC aremidly acidic and contain lysosomal components such as β-hexosaminidase, cathepsin D and lysosomal associatedmembrane proteins (Lamp). The distinct MIIC types arevariably accessible to endocytic tracers, reflecting theirdifferent positions in the endocytic pathway and possiblydifferent functions in MHC-II trafficking and Ag processing(Kleijmeer et al., 1997).

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The macrophage-colony stimulating factor (M-CSF) hasbeen already shown to affect the function of dendritic cells(DC). Therefore, the differentiation of dendritic cells intomacrophages (MΦ) might represent a pathway which couldinhibit the immune response initiated by DC. BecauseMajor Histocompatibility Complex class II molecules(MHC-II) are crucial for DC function, we asked whetherM-CSF may influence the intracellular transport of MHC-II in monocyte derived DC. We found that, at early stages,M-CSF induced first a rapid redistribution of MHC-IIfrom the MHC-II containing compartments (MIIC) to theplasma membrane and second an increase in MHC-IIsynthesis as observed with LPS or TNF-α. These processes

were associated with the sorting of MHC-II from lysosomalmembranes which underwent a drastic structuralreorganization. However, in contrast to tumor necrosisfactor (TNF)-α or lipopolysaccharide (LPS), M-CSFneither potentiated the allostimulatory function of DC norallowed the stabilization of MHC-II at the cell surface, butrather increased MHC-II turnover. We conclude that therapid modulation of MHC-II transport and distributionmay participate in the inhibitory effect of M-CSF on DCfunction and differentiation.

Key words: M-CSF, MHC class II molecule, Dendritic cell

SUMMARY

Modulation of MHC class II transport and lysosomedistribution by macrophage-colony stimulating factorin human dendritic cells derived from monocytesCarole L. Baron 1, Graça Raposo 2, Suzy M. Scholl 3, Huguette Bausinger 4, Danielle Tenza 2, Alain Bohbot 5,Pierre Pouillart 3, Bruno Goud 1, Daniel Hanau 4 and Jean Salamero 1,*1UMR 144 CNRS-Institut Curie, Laboratoire des Mécanismes Moléculaires du Transport Intracellulaire, 26, rue d’Ulm, Paris, France2UMR 144 CNRS-Institut Curie, Laboratoire de Microscopie Electronique, 26, rue d’Ulm, Paris, France3Institut Curie, Service de Medecine Oncologique, 26, rue d’Ulm, Paris, France4INSERM E 99-08, Laboratoire d’histocompatibilité, ETS Strasbourg, France5Service d’Onco-Hématologie, Hôpital de Hautepierre, Strasbourg, France*Author for correspondence (e-mail: salamero@curie.fr)

Accepted 19 December 2000Journal of Cell Science 114, 999-1010 © The Company of Biologists Ltd

RESEARCH ARTICLE

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The modulation of the expression of MHC-II in DC byinflammatory molecules, such as TNF-α and LPS, isaccompanied by the reorganization of intracellular membranescontaining these molecules. A study performed in humanmonocyte-derived DC has implicated an inhibition of αβ dimerrecycling at the cell surface, an increase in their half-life anda transient increase in their rate of synthesis during maturation(Cella et al., 1997). Up-regulation of surface MHC-IIexpression has also been observed during the in vitrodifferentiation of mouse bone marrow-derived DC from an‘early’ to a ‘late’ phenotype. In this model, both themodification of the transport pathway of newly synthesizedMHC-II induced by the regulation of Ii proteolysis and thegeneration of new class II containing vesicles (CIIV) drivingpreexistant MHC-II from lysosomes to the plasma membraneare responsible for the induction of MHC-II/peptide complexesat the cell surface within 2 days of activation (Pierre et al.,1997). In both human and mouse DC, intracellular MHC-IIdisappear during DC maturation. Consistently, mature DC losethe capacity to present new Ag.

DC bear receptors for non-inflammatory cytokines like M-CSF (Szabolcs et al., 1996; Zhou and Tedder, 1996), whichhave been shown to affect the differentiation of DC at differentstages. M-CSF specifically binds to a high affinity membranereceptor, the proto-oncogene c-fms(Sherr et al., 1985). C-fmsis mainly expressed by cells of the mononuclear lineage(monocytes, MΦ, immature myeloid DC) and their precursors(Stanley et al., 1997) and in DC, the expression of c-fms isdevelopmentally regulated since it is inhibited with theirterminal differentiation (Akagawa et al., 1996; Caux et al.,1996; Zhou and Tedder, 1995). Immature DC, which expressc-fms, can differentiate into MΦ in the presence of M-CSF(Hausser et al., 1997; Szabolcs et al., 1996). While theinhibitory activity of M-CSF on DC differentiation andfunction has been well documented, the early events of this‘M Φ pathway’ of differentiation as well as the effect of M-CSFon MHC-II transport and distribution have never beendescribed. Since the modulation of MHC-II transport andthe reorganization of MIIC are of crucial importance forAg presentation and immune response induced by DC, weinvestigated the effect of M-CSF in MHC-II and MIICdistributions in immature DC obtained from blood monocytesin the presence of GM-CSF and IL-4.

We found that M-CSF induced a rapid translocation ofMHC-II to the plasma membrane and a long term increase inMHC-II synthesis. This translocation was associated with thesorting of MHC-II from one of the markers of MIIC andlysosomes, CD63/Lamp-3 (Metzelaar et al., 1991). In contrastto what we found in LPS treated DC, CD63 was notupregulated at the plasma membrane and remained mostly inlysosomes which became tubular. At the ultrastructural level,MHC-II containing compartments were also affected and oftenbehave as hybrid structures of mutilaminar and elongatedlysosomes. Although M-CSF up-regulated MHC-II at theplasma membrane as do LPS or TNF-α, it did not stabilizeMHC-II αβ dimers but rather increased their internalizationand their subsequent degradation. Moreover, treatment with M-CSF for 24 hours did not lead to an increase in the expressionof CD86 molecules or to an up-regulation of theallostimulatory activity of DC. Our results suggest that thedepletion of the internal pool of MHC-II present in immature

DC combined with an increase of their turnover rate and thereorganization of lysosomal membranes may participate inthe known inhibitory effect of M-CSF on DC function(Gabrilovich et al., 1996; Hausser et al., 1997; Menetrier-Cauxet al., 1998).

MATERIALS AND METHODS

Medium, cytokines and reagentsDC were cultured in a complete medium consisting of RPMI 1640supplemented with 2 mM L-glutamine, 1% (v/v) sodium pyruvate, 1%non-essential amino acids and 10% (v/v) heat inactivated FCS (allfrom Life Technologies, Paisley, UK). Human (h) rM-CSF was agenerous gift of the Genetic Institute Inc. (Cambridge, MA) and wasused at 1000 U/ml. HrGM-CSF (50 ng/ml) and hrIL-4 (1000 U /ml)were kindly provided by Novartis (Rueil Malmaison, France) andSchering Plough (Dardilly, France), respectively. HrTNF-α (30ng/ml) was provided by Genzyme (Cambridge, MA). LPS (E. colisérotype 0128:B12) used at 1 µg/ml was obtained from Sigma (StLouis, MO), as were all chemicals used in this study, except whenspecified.

Cell cultureMonocytes, obtained from normal, healthy volunteers with theirinformed consent, were isolated from human blood by continuousflow centrifugation leukapheresis and counterflow centrifugation(Faradji et al., 1994). Immature DC were derived from thesemonocytes by culture in complete medium containing hr GM-CSFand hr IL-4, as previously described (Sallusto and Lanzavecchia,1994). The differentiation of monocytes into immature DC wassystematically followed by the analysis of cell surface and internalmarkers. Immature DC were obtained after 7 days culture andtreatment with TNF-α or LPS treatment for 24 hours induced theiractivation or maturation (Sallusto and Lanzavecchia, 1994).

AntibodiesThe antibodies used in this study were L243 (mouse IgG2a, anti-DRαβ dimers, Becton Dickinson, Mountain View, CA), DA6.147(mouse IgG2a, anti-HLA-DRα) previously described (Guy et al.,1982), BL6 (mouse IgG1κ, anti-CD1a, Immunotech, Marseille,France), 3-4A4 (rat IgG2b, anti-c-fms, kindly provided by Dr M.Roussel, St Jude Children’s Research Hospital, Memphis, TN), M5E2(mouse IgG2a, anti-CD14, PharMingen, Le Pont De Claix, France),H5C6 (mouse IgG1, anti-CD63, kindly provided by F. Lanza, ETS,Strasbourg, France), anti-EEA1 (rabbit antiserum, kindly provided byHarald Steinmark, Heidelberg, Germany), HB15A (mouse IgG2b,κ,anti-CD83, Immunotech, Margency, France) and BU63 (mouse IgG1,anti-CD86, Serotec Ltd, Oxford, UK). L243 was FITC-labelled withthe FLUOS reagent from Boehringer-Mannheim (Mannheim,Germany) and H5C6 was Cyanin 3-labelled with a kit fromAmersham France (Les Ullis, France) according to the manufacturer’sinstructions. FITC-labelled secondary antibodies were obtained fromJackson Immunoresearch Laboratories, Inc. (West Grove, PA).Isotype controls (rat IgG2b and mouse IgG1, 2a and 2b) werepurchased from Caltag Laboratories (Burlingame, CA) and used at1/50 dilution.

FACS analysesCells were washed once and resuspended in PBS containing 3% FCS(v/v) and 0.1% NaN3 (PBS/FCS/NaN3 solution) in 96-well plates. Thecells were incubated for 30 minutes at 4°C with different primaryantibodies, washed and further incubated with species-specific FITC-labelled secondary antibodies for 30 minutes at 4°C. After washing,the cells were analysed on a FACScan spectrofluorimeter (BectonDickinson). Staining with isotype controls was performed in parallel.

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1001M-CSF and MHC class II molecules in DC

For the blocking assay, unstimulated DC and DC stimulated for 24hours with M-CSF or LPS were incubated at 37°C for 4 hours in thepresence of medium, 50 µg/ml 9E10 (anti-c-myc) or H5C6 (anti-CD63). The cells were then washed and MHC-II plasma membraneexpression was labelled with L243 or an isotype control and asecondary antibody coupled with FITC. The fluorescence was alsoanalysed by FACScan.

Allogeneic mixed leukocyte reaction105 PBL were co-cultured in 96-well microplates (Corning CostarFrance, Brumath, France) with different numbers of irradiatedstimulator APC (3,000 rad from a 137Cs source), pre-incubated ornot for 24 hours with M-CSF, TNF-α or LPS. [3H]thymidineincorporation was measured after 5 days culture following an 18 hourspulse with [3H]thymidine (1 µCi/well). All cultures were set up intriplicate. The negative controls included APC alone or PBL alone andthe positive control was PBL incubated with 100 IU/ml IL-2.

Immunoelectron microscopyImmature DC and DC treated for 24 hours with either M-CSF or LPSwere fixed with a mixture of 2% paraformaldehyde and 0.125%glutaraldehyde in 0.2M phosphate buffer pH 7.4 (PB) for 2 hours atroom temperature. The fixed cells were processed for ultrathincryosection cutting and immunogold labeling as described previously(Raposo et al., 1997). Briefly, after washing in PB and PB-50 mMglycine, the cells were embedded in 10% gelatin. Small blocks wereinfiltrated with 2.3 M sucrose for 2 hours at 4°C and frozen in liquidnitrogen. Ultrathin cryosections prepared with a Leica ultracut FCS(Vienna, Austria) were retrieved in 2% methylcellulose/2.3 M sucrose(v/v) and put onto Formvar-carbon coated grids. In a first step, thesections were incubated with H5C6 (anti-CD63) followed by abridging rabbit anti-mouse IgG antibody (Dako, Denmark) andProtein A coupled to 15 nm gold particles (PAG 15). In a second step,the sections were incubated with rabbit polyclonal anti-HLA-DR andProtein A coupled to 10 nm gold particles (PAG 10). Protein A-goldconjugates were purchased from Dr J. W. Slot (Department of CellBiology, Utrecht University Medical School, the Netherlands).Finally, the cryosections were contrasted and embedded in a mixtureof methylcellulose and uranyl acetate and viewed under a CM120Twin Philips electron microscope (Eindhoven, The Netherlands).

Immunofluorescence staining Cells were allowed to adhere on 12 mm diameter glass coverslips(Polylabo, Strasbourg, France), precoated with 50 mg/ml poly-L-lysine (Polylabo), before fixation for 10 minutes in 3%paraformaldehyde in PBS. The fixed cells were permeabilized for 30minutes in 0.05% saponin in PBS containing 0.2% BSA and incubatedfor 30 minutes with FITC-labeled L243 and Cyanin-3-labeled H5C6.The coverslips were then washed, mounted in Mowiol (Merck,Darmstadt, Germany) solution and examined under a confocalmicroscope (TCS4D, Leica Lasertechnic, Heidelberg, Germany). Inthe anti-MHC-II mAb internalization assay, immature DC wereincubated for 24 hours in complete medium containing M-CSF or LPSand then incubated for 1 hour at 4°C in the presence of 5µg/ml FITC-coupled L243. The cells were washed and some samples were allowedto adhere for 10 minutes to poly-L-lysine coated coverslips. Othersamples were incubated for 20 minutes at 37°C in medium containingcytokines and activators. During the last 10 minutes of this incubation,the cells were placed on poly-L-lysine coated coverslips beforefixation in 3% paraformaldehyde. After permeabilization, the cellswere labelled with a 1/2000 dilution of rabbit anti-EEA1 and stainedwith Texas Red-coupled donkey anti-rabbit IgG. The coverslips were

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Fig. 1. M-CSF up-regulated cell surface MHC-II but did not enhancethe allostimulatory activity of DC. (A) Monocytes were cultured for7 days with GM-CSF and IL-4 to obtain immature DC. These cellscould further maturate in the presence of TNF-α or LPS for 24 hoursand we compared the effects of M-CSF treatment to those of DCmaturation. (B) The expression of cell surface markers after 24 hoursM-CSF treatment was compared to the expression of immature and24 hours TNF-α-activated DC. Similar results were observed forLPS, although CD83 and CD86 were more strongly enhanced underLPS treatment (not shown). These data are representative from morethan 4 separate experiments using cells from distinct donors. (C) Theproliferation of peripheral blood lymphocytes cocultured for 5 dayswith immature DC (iDC) or M-CSF-, TNF-α- or LPS-treated DCwas quantified by measuring the [3H]thymidine incorporation asdescribed in Materials and Methods. Results are presented as thepercentage [3H]thymidine incorporation adjusted to 100% for T cellproliferation obtained with 10,000 immature DC of each donor andeach point is a triplicate mean. These T cell proliferations obtainedwith 10,000 iDC ranged from 62,987±25759 cpm of [3H]thymidineincorporation. DC preparations were obtained from 5 differentdonors.

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mounted and viewed under a confocal microscope (TCS4D, LeicaLasertechnic, Heidelberg, Germany).

Rate of MHC-II biosynthesisDC (106 /ml) were stimulated with M-CSF or LPS for differentperiods of time, washed twice in RPMI and transferred for 20 minutesat 37°C into RPMI without methionine or cysteine but containingGM-CSF, IL-4 and M-CSF or LPS. These cells were then labelledwith 100 µCi/ml [35S]methionine/cysteine (Promix, AmershamFrance, Les Ullis, France) for 20 minutes at 37°C in complete mediumwithout methionine or cysteine. The cells were washed twice in coldPBS and lysed in a solution containing1% Triton X-100, 150 mM NaCl, 20mM Tris-HCl, 5 mM EDTA, 0.2% BSA(Buffer I) and protease inhibitors, aspreviously used (Saudrais et al., 1998).Lysates were precleared twice for 2hours with Protein G-SepharoseTM

(Pharmacia, Uppsala, Sweden) andimmunoprecipitated overnight withDA6.147 or HB15A previously bound toProtein G-Sepharose. The samples wereboiled in Laemmli buffer, separated bySDS-PAGE under reducing conditions(100 mM DTT) on a 10-15% gradientgel and processed for fluorography andautoradiography.

Pulse-chase labellingDC (106 /ml) were washed once inRPMI, transferred into RPMI withoutmethionine or cysteine for 30 minutes at37°C and then incubated in the pulsemedium. After long term labelling (15hours) for analysis of the transport ofnewly synthesized MHC-II to the cellsurface, the cells were chased orstimulated for 5 hours with M-CSF,LPS or TNF-α in complete mediumcontaining GM-CSF and IL-4. After ashort term pulse (20 minutes) foranalysis of the MHC-II turn over, thecells were chased for various periods oftime in complete medium containingGM-CSF and IL-4, alone or in thepresence of M-CSF or LPS.

Cell surface biotinylation andimmunoprecipitationAfter pulse chase labelling, the cellswere washed once in cold PBS, surfacebiotinylated for 7 minutes on ice with25 mg/ml PBS/NHS-SS-biotin (PierceChemical Co., Rockford, IL), quenchedwith 50 mM glycine and lysed inBuffer I containing the usual proteaseinhibitors. Postnuclear lysates wereprecleared twice for 2 hours with ProteinG-Sepharose and immunoprecipitatedwith Protein G-Sepharose bound L243(detects αβ dimers, empty or associatedwith a peptide) and DA6.147 (detectsthe residual α chain, associated withIi and β). Immunoprecipitates werewashed as described elsewhere(Humbert et al., 1993) and eluted in 10%SDS, either at 95°C for 5 minutes or at

room temperarure for 30 minutes. SDS-resistant compact dimersmigrating at 60 kDa, corresponding to mature peptide-loaded MHC-II, were detected without previous boiling. After boiling, the SDS-resistant MHC-II dissociate into α and β chains. The eluted materialwas then resuspended in 100 µl of Buffer I without BSA. Biotinylatedproteins were recovered with streptavidin agarose from 90% of theeluted materials (Saudrais et al., 1998) and the remaining 10% wasleft untreated (total). Except in MHC-II transport analyses, thesamples were boiled in Laemmli’s buffer, separated by SDS-PAGEunder reducing conditions (100 mM DTT) on a 10-15% gradient geland processed for fluorography and autoradiography. Quantification

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1003M-CSF and MHC class II molecules in DC

was performed using a video camera (Bioprint System, Vuilbert-Lourmat, Marne la Vallée, France) and optical densitometry using theBio1D sofware (Vuilbert-Lourmat).

RESULTS

M-CSF induced MHC-II up-regulation at the cellsurfaceWe studied the early effects of adding M-CSF to 7 days-

immature DC in a culture mediumcontaining GM-CSF and IL-4(Fig. 1A). Although a significantproportion of the cells becameadherent under these conditions,they could be recovered bypipetting or short term incubationin PBS/EDTA solution. Hence, itwas possible to perform acomparative analysis of immatureDC, LPS- or TNF-α-activatedDC and M-CSF-treated DC.Furthermore, we kept the cells in

the original medium before addition of M-CSF, TNF-α or LPSto make the parallel with a microenvironment of a DCprecursor in vivo which most probably contains a mixture ofcytokines rather than one cytokine. Treatment with M-CSF for24 hours induced a significant up-regulation of MHC-IIexpression at the cell surface (Fig. 1B), which was similar tothat obtained using TNF-α (Fig. 1B) or LPS (data not shown).This increase in MHC-II expression induced by M-CSF wasspecific to the combination of GM-CSF/IL-4/M-CSF since, as

Fig. 2. Accumulation of MHC-II atthe plasma membrane, in MIIC andin electron lucent compartments inM-CSF-stimulated DC. Ultrathincryosections of immature DC (A andD) and DC treated for 24 hours withLPS (B and E) or M-CSF(C and F) were double-immunogoldlabelled with anti-CD63/Lamp-3(PAG 15) and anti-MHC-II (PAG 10).Very little plasma membranelabelling of MHC-II was observed inimmature DC (A), whereas LPS(B) and M-CSF (C) stimulated cellsshowed intense labelling in newlyformed interdigitations andmembrane ruffles. In M-CSF-treatedcells, MHC-II (PAG 10) were oftenseen with CD63 (PAG 15) in electronlucent compartments having themorphology of early endosomes(C insert). In immature DC, mostMHC-II (PAG 10) colocalized withCD63 (PAG 15) in multilaminarMIIC (D). In cells treated for 24hours with LPS, the majority ofcytoplasmic compartments wereelectron dense with no apparentmembrane sheets and containedCD63 (PAG 15). MHC-II were onlyoccasionally detected intracellularlyin these cells (E). Conversely,multilaminar MIIC containing MHC-II and CD63 were still present in thecytoplasm of M-CSF treated cells.These compartments were oftenelongated and presented continuitieswith electron dense tubules (arrows)which frequently displayed apericentriolar distribution. Bars: 500nm (A,B,C; inset, 250 nm); 250 nm(D); 500 nm (E,F).

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previously reported (Hausser et al., 1997),MHC-II expression rapidly decreasedwhen GM-CSF and IL-4 were removedbefore addition of M-CSF (data notshown). We also noticed a small increaseof the CD1a expression with both M-CSFand TNF-α treatment (Fig. 1B).

M-CSF was not responsible for ageneral increase in cell surface moleculessince, as expected, c-fms expression wasdownregulated. Furthermore, expressionof the MΦ marker CD14 was slightlyupregulated by M-CSF. Thus, the effectsof M-CSF on MHC-II and c-fms were nota consequence of DC maturation due toendotoxin contamination, as endotoxinslike LPS, cannot increase CD14expression in DC. This was confirmed bythe observation that unlike TNF-α (Fig.1B) or LPS (data not shown), M-CSF didnot increase expression of the DCactivation marker CD83 or the expressionof the costimulatory molecule CD86. Theinfluence of M-CSF on the priming of Tlymphocytes by DC was then tested usingan MLR assay with allogeneic responder T lymphocytes.Whereas treatment with TNF-α or LPS for 24 hours increasedthe allostimulatory function of DC, M-CSF did not (Fig. 1C).This was quantified for 5 differents experiments and the resultswere normalized to 100% for the proliferation obtained for10000 immature DC (Fig. 1C, iDC) of each donor. Theseresults were consistent with the observation that M-CSF didnot up-regulate CD86 expression at the plasma membrane,which is essential for APC allostimulatory activity.

Ultrastructural organization of MHC-II andCD63/Lamp-3 containing compartments in M-CSF-stimulated DCWe examined the effects of 24 hours M-CSF treatment onMHC-II distribution and MIIC organization usingimmunoelectron microscopy. As in LPS-activated DC (Fig.2B) and unlike in immature DC (Fig. 2A), MHC-II labelling

was highly enriched at the surface of M-CSF-treated DC (Fig.2C), consistent with the FACS analysis (Fig. 1B). In addition,both M-CSF- and LPS-treated DC displayed plasma membranefolds, reminiscent of ruffles for M-CSF and of dendrits forLPS. Interestingly, electron lucent compartments containingMHC-II were frequently seen beneath the plasma membraneof M-CSF treated DC (Fig. 2C insert). These compartmentswere reminiscent of early endosomal-like compartments sincethey also contained significant labelling of the early endosomalprotein EEA-1 (Stenmark et al., 1996) (data not shown). Suchearly endosomal-like compartments containing MHC-II werehardly observed in immature DC and never in mature DC (Fig.2A and B, respectively).

As in immature DC (Fig. 2D) but not mature DC (Fig. 2E),M-CSF-treated DC (Fig. 2F) often displayed colocalization ofMHC-II with CD63 in intracellular compartments whichpresented the multilaminar morphology of MIIC with

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Fig. 3. Time course of MHC-II redistributionand late endosomal reorganization in DCstimulated with M-CSF. Immature DC werestimulated for the indicated times with M-CSF, fixed in PFA, permeabilized withsaponin and immunofluorescence stained asdescribed in the Materials and Methods.Double labelling with FITC-coupled L243(anti-MHC-II, left panels) and Cyanin-3-coupled H5C6 (anti-CD63, middle panels)was followed by confocal microscopyanalysis. Images are representative of 3separate experiments. (Bars: 10 µm). Arrowsshow nascent tubular lysosomes in 4 hour-treated cells and well established tubularlysosomes in 24 hour-treated cells. Insertscorrespond to a blowup of an area in whichthe CD63 and L243 positive tubular networkis visible in 4 hour-treated cells.

1005M-CSF and MHC class II molecules in DC

characteristic concentrically arranged membrane sheets (Fig.2F). As in immature DC (Fig. 2D), M-CSF treated DC (Fig.2F) also contained MHC-II in electron dense compartmentsenriched in CD63 which were lysosomal compartments alsoenriched in Lamp-1 and Lamp-2 (data not shown). In contrastto the pattern observed in immature or mature DC, these MIICand lysosomal compartments were somehow smaller, oftenelongated or tubular and arranged concentrically around thecentrosome (Fig. 2F). The tubular structures were frequentlyin continuity with multilaminar MIIC showing a disorganizedshape as compared to iDC (Fig. 2F, arrows).

M-CSF induced rapid redistribution of MHC-II to thecell surface and reorganization of lysosomalcompartmentsConfocal microscopy was employed to analyse theredistribution of MHC-II during stimulation of immature DCwith M-CSF. In immature DC (t=0), the majority of MHC-IIwere located in internal MIIC, which also containedCD63/Lamp-3 (Fig. 3). After treatment for 1 to 2 hours withM-CSF, MHC-II and CD63 were concentrated in theperinuclear area of the cells (Fig. 3, 2 hours). Subsequently,protrusions like pseudopodia appeared at the surface of thecells, which still contained MHC-II and CD63 colocalized invesicles and also in nascent tubulovesicles (3 hours, notshown). After 4 hours, most of the cells had spread anddeveloped larger protrusions. At this stage, MHC-II stainingwas enhanced at the plasma membrane, while the perinuclearCD63 staining was stronger. A thin tubular network positivefor CD63 and MHCII was also seen in some cells (Fig. 3, 4hours, arrows and insert). After 24 hours M-CSF treatment,

most MHC-II were localized at the plasma membrane instructures resembling lamellae and ruffles (Fig. 3, 24 hours).Interestingly, the CD63-positive tubules observed in some cellsas soon as 4 hours after addition of M-CSF, expanded and werefound in approximately 50% of the cells after 24 hourstreatment.

Time course of MHC-II and CD63 cell surfaceexpression during M-CSF treatment FACScan was used to quantify MHC-II expression at the cellsurface during M-CSF treatment. As seen in Fig. 4A, MHC-IIexpression increased rapidly under M-CSF treatment (150%after 2 hours and 170% after 4 hours). However, unlike in TNF-α (not shown) or LPS-activated DC where surface MHC-II stillincreased after 4 hours, a plateau was attained at 4 hours. Incontrast to LPS, M-CSF further required a time lapse of about1 hour to induce an increase of MHC-II at the cell surface. Theexpression of CD83 was assessed in parallel to control DCmaturation, which was induced by TNF-α (data not shown) andLPS treatment (Fig. 4B) but not by M-CSF (Fig. 4B). As CD63mainly localizes with MHC-II in internal membranes of MIICin immature DC, we also looked for cell surface expression ofCD63 during M-CSF treatment. CD63 expression did notappear to be up-regulated by M-CSF, whereas LPS induced avery rapid increase in this molecule at the cell surface (Fig.4C).

This former result prompted us to determine whether CD63would be implicated in the early step of DC stimulation by LPSbut not by M-CSF. Therefore, in order to block CD63 functionby antibody cross-linking, immature DC were incubated for 4hours at 37°C with medium, anti-CD63 mAb or anti-c-myc

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Fig. 4. Quantification of specific up-regulation of cell surface MHC-II by M-CSF. (A-C) Time course of cell surfacemolecules. Immature DC were incubatedfor the indicated times with M-CSF (bolddiamond) or LPS (open square) inaddition to GM-CSF and IL-4. Cellsurface expression of MHC-II (A), CD83(B) or CD63 (C) was followed byimmunolabelling and FACScan. The datacorrected for isotype controls are themean fluorescence intensities in 3independent experiments using cells fromdifferent donors and are adjusted to 100%for the immature DC of each donor.(D) Blocking of MHC-II expression by ananti-CD63. Unstimulated DC and DCstimulated for 24 hours with M-CSF orLPS were incubated at 37°C for 4 hoursin the presence of medium, 50 µg/ml ofanti-c-myc mAb (as a negative control) or50 µg/ml anti-CD63 mAb. The cells werethen washed and the MHC-II plasmamembrane expression was labelled withL243 anti HLA-DR and a secondaryantibody coupled with FITC. Thefluorescence was analysed by FACS. Thedata corrected for isotype controls are themean of fluorescence intensities for 3independent experiments with distinctdonors and were adjusted at 100 forimmature DC (t=0) of each donor.

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mAb (as a negative control), in the presence of medium, M-CSF or LPS (Fig. 4D). In these conditions of fluid phaseendocytosis of H5C6, we first verified that the anti-CD63mAb accumulated in lysosomal compartments byimunofluorescence staining (data not shown). Second, thisblocking assay revealed that, in all conditions tested, the anti-CD63 mAb specifically inhibited more than 60% of theexpression of MHC-II at the plasma membrane of DC (Fig.4D). This result indicated that CD63 is required for MHC-IIexpression at the plasma membrane of DC under M-CSF orLPS treatment.

Early up-regulation of the transport of pre-existingMHC-II from internal compartments to the cellsurface and increased MHC-II biosynthesisThe rate of increase in MHC-II expression at the cell surfacesuggested a rapid redistribution of MHC-II from internalcompartments to the plasma membrane. Hence we determinedbiochemically whether the transport of pre-existing MHC-IIfrom internal compartments to the cell surface was enhancedunder M-CSF treatment (Fig. 5A and B). Immature DC were

metabolically labelled overnight and chased for 5 hours in thepresence of GM-CSF and IL-4 (0), alone or with M-CSF orLPS. Plasma membrane proteins were biotinylated and MHC-II were immunoprecipitated with L243 and analysed by SDS-PAGE and autoradiography. This procedure allowed us tofollow pre-existing MHC-II radiolabeled before the treatment.As expected from the half life of MHC-II in iDC (see also Fig.6), the total amount of preexisting MHCII αβ dimers did notvary significantly during the 5 hours chase, when M-CSF andLPS treated DC were compared to untreated iDC (Fig. 5A, lane0, 10% Total). However, as in the case of LPS, treatment withM-CSF for 5 hours led to an increase in pre-existing MHC-IIat the cell surface as compared to immature DC (Fig. 5A,biotinylated M-CSF and biotinylated 0). These data wereconfirmed by quantifying the ratio of MHC-II αβ dimersexpressed at the plasma membrane (Fig. 5A, biotinylated) tototal MHC-II αβ dimers (Fig. 5A, total). The ratio wascalculated for 4 separated experiments and normalized to 100%for immature DC (Fig. 5B).

The effects of M-CSF and LPS on MHC-II synthesis werealso compared. DC stimulated with M-CSF or LPS for

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Fig. 5. M-CSF increased export of pre-existingMHC-II from intracellular compartments andneosynthesis of MHC-II/Ii complexes in DC.(A) Immature DC were radiolabelled for 15 hoursand chased for 5 hours in medium (0) or mediumcontaining GM-CSF, IL-4 and either M-CSF orLPS. Plasma membrane proteins werebiotinylated, the cells were lysed, and MHC-II αβdimers were immunoprecipitated with L243. Thebiotinylated fractions were separated from 90% ofthe total lysates using streptavidine agarose beads.Separation and electrophoresis were performedwithout boiling the samples to allow detection ofthe SDS-stable dimers of αβ chains. Theautoradiograph shown is representative of 4independent experiments using cells from differentdonors. (B) The ratio of biotinylated to 10%remaining total αβ dimers was calculated in 4independent experiments of MHC-II transportassay for immature DC (0) and DC stimulated byM-CSF or LPS. (C) To investigate MHC-IIbiosynthesis, DC were metabolically labelled for20 minutes before (t=0) and after M-CSF or LPStreatment for different times as indicated. Thecells were lysed, the MHC-II/Ii complexes wereimmunoprecipitated with DA6.147 (specific of theα chain of MHC-II) and total αβIi complexeswere separated by SDS-PAGE under reducingconditions and processed for autoradiography.P41, mp33 (mature p33), p35 and p33 are differentforms of Ii. (D) The synthesis of the β chain wasquantified and adjusted to 100% at t=0. The sameresults were obtained after quantification of Ii p33.(E) Lysates from DC metabolically labelled andlysed immediately (0), or after incubation with M-CSF or LPS for 48 hours, wereimmunoprecipitated with DA6.147 (anti HLA-DRα) and HB15A (anti-CD83). Results weresimilar for 3 DC preparations from differentdonors.

1007M-CSF and MHC class II molecules in DC

different times were short term metabolically labelled andMHC-II/Ii biosynthesis was analysed by immunoprecipitationwith DA6.147 (anti-HLA-DR α mAb). Unlike LPS, M-CSFdid not inhibit the biosynthesis and oligomerization of MHC-II/Ii complexes (Fig. 5C) but rather significantly increased theirsynthesis. Quantification of the β chain expression in a separateexperiment indicated that M-CSF induced up to 2-fold increasein MHC-II biosynthesis after 24 hours of treatment (Fig. 5D).As a control of the specificity of the effect on MHC-IIsynthesis, CD83 was immunoprecipitated in parallel fromlysates of DC unstimulated or stimulated for 48 hours with M-CSF or LPS. In contrast to LPS, M-CSF did not enhance CD83synthesis even after 48 hours (Fig. 5E).

M-CSF stimulation of immature DC increased MHC-IIturn overTNF-α and LPS are known to enhance the Ag presentingfunction of DC by stabilizing MHC-II/peptide complexes atthe cell surface (Cella et al., 1997). This stabilization impliesa decrease in the internalization of MHC-II and a resultantdecrease of their degradation in lysosomes. Therefore, wecompared the effects of M-CSF and LPS on MHC-II turnover.Immature DC were metabolically labelled for 20 minutes andchased for different periods of time in the absence (iDC) orpresence of M-CSF or LPS. Plasma membrane proteins werebiotinylated and MHC-II expressed at the plasma membrane(biotinylated) and total cellular MHC-II were quantified byimmunoprecipitation with L243. In contrast to LPS, M-CSFdid not stabilize MHC-II at the plasma membrane but ratherincreased their degradation (Fig. 6A). When the degradationof α chain was quantified (Fig. 6B), αβ dimers were found tobe twofold more rapidly degraded than in immature DC. Itshould be noted that the data presented here (Fig. 6B)correspond to experiments performed on cells provided by thesame blood donor. Taken into account that αβ dimergeneration is complete after only 4 hours of chase in iDC andthat minor differences may occur between different donors,

we calculated from the presented experiments and from otherexperiments (not shown) that the half lives of MHC II αβdimers were between 8 and 10 hours in immature DC, andgreater than 40 hours in mature DC. This would imply a veryrapid turnover of MHC II αβ dimers in M-CSF treated cellswhich we approximate around 4 hours. The same lysates werethen used for immunoprecipitation with DA6.147 (anti HLA-DR α-chain). Since all MHC-II not removed by L243 wereassociated with Ii and were immunoprecipitated by DA6.147,this procedure allowed us to evaluate the Ii degradationpreceding the peptide loading of MHC-II. Results indicatedthat M-CSF did not in fact modify the degradation of Ii andsubsequent formation of MHC-II αβ/peptide complexes (datanot shown).

M-CSF increased internalization of MHC-IIThe fate of cell surface MHC-II was investigated by followingthe internalization of L243 (anti-MHC-II) prebound to theplasma membrane of immature, mature and M-CSF-treated DCfor 24 hours. The cells were incubated for 1 hour at 4°C in thepresence of FITC-coupled L243 and then either left on ice orfurther incubated for 20 minutes at 37°C. At 4°C, all the cellsbound L243 (Fig. 7) and specific binding was controled byincubating the cells with a FITC directly coupled mouse anti-CD1e mAb (not shown), a glycoprotein express exclusively inintracellular compartments in iDC and mDC (Angénieux et al.,2000). After 20 minutes at 37°C, most of the prebound L243remained at the plasma membrane in mature DC, consistentwith a stabilization of MHC-II αβ dimers at their plasmamembrane. In contrast, a small part of L243-FITC had beeninternalized in immature DC and partially colocalized with theearly endosome marker EEA1. In M-CSF stimulated DC,the vast majority of FITC-labelled complexes had beeninternalized into compartments which were also labelled byanti-EEA1. These results strongly suggest that M-CSFenhances the turn over of MHC-II at the cell surface byincreasing their internalization.

Fig. 6. M-CSF enhanced MHC-II turnover in DC. (A) DC were shortlymetabolically labelled and incubated inmedium containing or not LPS or M-CSF for different times. Plasmamembrane proteins were biotinylatedand MHC-II αβ dimers wereimmunoprecipitated with L243 andanalysed directly (10% total) or afterprecipitation of the biotinylatedmaterials with streptavidine agarose(biotinylated) from 90% of the totallysates. The band revealed in theabsence of chase (0) corresponds to Iithat is unspecifically detected by L243possibly due to its abundance. (B) Theexpression of the α chain (total) wasquantified for each point in 2 separateexperiments and adjusted to 100% at t=2hours since before (t=0), the α chainsare not detectable in any trated cells. Thelysates immunoprecipitated with L243were further immunoprecipitated with DA6.147 (anti-HLA DRα). This procedure allowed us to follow the disappearance of αβIi precursorforms of MHC II and hence the degradation of associated Ii that precedes appearance of αβ dimers and the peptide loading of MHC-II (notshown).

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DISCUSSION

M-CSF has been shown to affect the function of DC byperturbating their differentiation pathway (Akagawa etal., 1996; Caux et al., 1996; Gabrilovich et al., 1996;Hausser et al., 1997; Menetrier-Caux et al., 1998;Szabolcs et al., 1996). Here, we investigated the earlyevents occurring during incubation of immature DCwith M-CSF, in the continuing presence of GM-CSFand IL-4. We focused on the modifications of MHC-IItransport and distribution induced in these cultureconditions. We found that M-CSF rapidly up-regulatedMHC-II at the cell surface and induced theredistribution of another MIIC marker, CD63, intotubular compartments. The up-regulation of MHC-IIresulted first from increased export of pre-existingMHC-II from internal compartments and second fromincreased MHC-II biosynthesis. The upregulation ofMHC-II did not involved MHC-II stabilization at theplasma membrane. In contrast to DC exposed to LPS orTNF-α, these events were not associated with anincrease in the allostimulatory function of DC, probablydue to a faster internalization and degradation of MHC-II and the absence of CD86 up-regulation. Thus, ourresults suggest that short time treatment of DC with M-CSF triggers molecular mechanisms that are distinctfrom those involved in the maturation of DC and whichparticipate in the inhibitory effects of M-CSF on the DCfunction.

It is now established that during activation of DC byinflammatory molecules or bacterial products, the MHC-IIwhich are initially present in late endosomal/lysosomalcompartments are redistributed to the plasma membrane,while other lysosomal membrane proteins remainintracellular. This would suggest a regulated sorting oflysosomal proteins. During murine DC maturation, mostMHC-II are first detected in Lamp positive compartments andlater in peripheral non lysosomal vesicles which have beenproposed to be CIIV (Pierre et al., 1997). These vesiclesdisappear with complete maturation and MHC-II/peptidecomplexes are then found at the plasma membrane where theycluster with other molecules of importance for T cellstimulation, such as CD86 (Pierre et al., 1997; Turley et al.,2000). Our present study reveals a similar sorting of MHC-IIfrom CD63/Lamp-3 positive compartments. However, themorphological modifications, and delayed induction ofMHC-II cell surface expression under M-CSF treatment are

consistent with a new mechanism of regulation of the MHC-II transport.

In addition to MHC-II redistribution, M-CSF treatmentinduced a major membrane reorganization of the CD63-positive compartments, as demonstrated by theirtubularization. Although the molecular mechanisms leading tothis reorganization are not known, it is attractive to speculatethat both events take place in a coordinated fashion. Our resultsfor M-CSF treatment of DC show that an initial cell retractionis accompanied by concentration of MIIC in the pericentriolararea. MHC-II then relocate to the cell surface simultaneouslywith the appearance of tubular lysosomes. One hypothesis isthat these tubular lysosomes could be directly involved in thetransport of MHC-II to the plasma membrane, possibly byfacilitating their sorting from other lysosomal molecules.Consistent with this hypothesis, small vesicles located in thevicinity of such tubular lysosomes have been proposed to actas intermediates in the transport of MHC-II to the plasmamembrane (Harding and Geuze, 1993). Moreover, we coulddetect multilaminar MIIC and electron dense structures incontinuity only in M-CSF-stimulated DC. Such close aposition

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Fig. 7. M-CSF increased anti-MHC-II internalisation at thesurface of DC. Immature DC were incubated for 24 hours inmedium containing GM-CSF, IL-4 and M-CSF or LPS, andthen for 1 hour at 4°C in the presence of 5µg/ml FITC-L243(anti-HLA-DR αβ dimers). The cells were washed and eitherfixed in 3% PFA on poly-L-lysine-coated coverslips (binding4°C) or further incubated for 20 minutes at 37°C beforefixation (Internalization 20 minutes, 37°C). After quenchingof PFA, the cells were permeabilized with saponin andincubated with anti-EEA1 rabbit antiserum (1/2000 dilution)to label early endosomes, followed by a Texas Red coupledsecondary antibody. Fluorescence was analysed by confocalmicroscopy and results were similar for DC preparationsfrom 2 different donors. Bars, 10 µm.

1009M-CSF and MHC class II molecules in DC

suggests that the tubular structures might derive frommultilaminar MIIC. Nevertheless, it cannot be excluded thatnewly formed tubular lysosomes associate with pre-existingMIIC and, like phagolysosomes or autophagosomes, induceMIIC acidification and increased degradation of MHC-II. Thiswould be consistent with our observation that M-CSF enhancesMHC-II degradation in DC and with the report of Harding etal that in IFN-γ-activated MΦ, early tubulovesicular lysosomes(Lamp-1 and Cathepsin D positive) contain high levels ofMHC-II, while late mature lysosomes contain less MHC-II(Harding and Geuze, 1993).

As in the case of LPS activation, the molecular effectors thatphysically control MHC-II transport to the plasma membraneduring M-CSF treatment are still unknown. One potentialcandidate is a multimolecular complexe containingtetraspanins (CD9, CD63, CD81 and CD82) which has beenshown to associated with MHC-II in MIIC (Hammond et al.,1998; Rubinstein et al., 1996). Although the precise functionof this complex is not known, it has been suggested that it couldbe involved in intracellular signal transduction or cellactivation (Wright and Tomlinson, 1994). Furthermore, CD81is thought to play a costimulatory role in B cell activation(Tedder et al., 1994). In this context, our data indicate that thecell surface expression of CD63 is up-regulated by LPS but notby M-CSF. This supports the view that the mechanisms ofredistribution of MHC-II from MIIC to the plasma membrane,induced by M-CSF and LPS, are distinct. Nevertheless, theinternalization for 4 hours of an anti-CD63 by fluid phaseuptake inhibited the transport of MHC-II in immature ormature DC, as well as in M-CSF-treated DC. We couldhypothesize that CD63 or the multimolecular complex oftetraspanins might be also required for the transport of MHC-II under M-CSF treatment. However, we cannot exclude thepossibility that a similar effect could be obtained by targetingother membrane components of late endosomal/lysosomalcompartments. Furthermore, since strong inhibition of MHC IIexpression was also seen in iDC treated with anti-CD63 mAb,this blocking effect obtained with antibodies against aconstituent of internal endosomal membranes may reflect amore general feature of an exocytic process from lateendosomes/lysosomes. Further investigations aimed atresolving these issues as well as to explain why plasmamembrane expression of CD63 is preferentially increasedduring LPS activation of DC are now in progress.

In our study, a combination of M-CSF, GM-CSF and IL-4,upregulated the biosynthesis of MHC-II in DC. This is incontrast to a previous report (Willman et al., 1989) that in bonemarrow MΦ, M-CSF not only decreased the basal levels ofMHC-II gene and protein expression but also inhibited theinduction of MHC-II by IFN-γ and GM-CSF. Hence, the effectsof M-CSF on MHC-II expression would seem to be highlydependent on the cell type, the stage of differentiation of the cellsand the associated cytokines. It has also been reported that LPSfirst increases and then reduces MHC-II biosynthesis in DC(Cella et al., 1997). Although we were not able to detect anyearly up-regulation during LPS treatment, possibly due todifferences in experimental procedures, we confirmed a strongdecrease in MHC-II biosynthesis at later stage of LPS activationand conversely a long term increase under M-CSF treatment.This points to the existence of two distinct pathways regulatingMHC-II biosynthesis in DC. One leads to downregulation of

MHC-II synthesis and to synthesis of accessory molecules likeCD86 involved in the Ag presenting function of DC, while theother leads to MHC-II synthesis alone without synthesis ofaccessory molecules and without affecting the allostimulatoryactivity of DC. In support of this hypothesis, we found evidencethat the turnover of MHC-II was increased in M-CSF stimulatedDC. In our hands, LPS or TNF-α (not shown) treatment did notpromote as strong an increase of the half lives of MHC II αβdimers in monocyte derived DC as found by others (Cella et al.,1997). This was probably due to a rather inefficient activation ofDC in our assays. Indeed, in one isolated experiment for whichLPS was replaced by an activation system using CD40/CD40Lactivation, αβ dimers half life seemed to be further increased(not shown). Moreover, different LPS serotypes may have beenused in the different studies and to our knowledge, a comparativestudy of the effect of these different serotypes (E. coli serotype0128:B12 in the present report) on MHC II turnover remains tobe performed. Nevertheless, the dramatic differences in MHC IIhalf lives observed (T1/2= 4 hours and T1/2= 40 hours in M-CSF and LPS treated cells, respectively) are counterbalanced bythe increase of biosynthesis in M-CSF treated DC (10-fold whencompared to LPS treated DC at 24 hours). The lack of αβstability at the cell surface and the electron microscopy studiesrevealing the frequent occurrence of electron lucent andheterogeneous vesicles resembling early endosomes andcontaining both MHC-II and CD63 in M-CSF treated cellswould be consistent with internalized MHC-II passing throughearly endosomal compartments before their rapid degradation intubular lysosomes.

In contrast, within the first 2 to 4 hours of DC treatment, theincrease of cell surface MHC II induced by M-CSF or LPSwould appear as a more similar process. Indeed, within 6 hoursof treatment, M-CSF does not promote more than a 1.5-foldincrease of MHC II biosynthesis while no significant decreaseis observed in LPS treated cells. Since it takes about 4 hoursfor the maturation of αβ dimer to occur and for these dimersto reach the cell surface this cannot account for the rapid upregulation of plasma membrane expression observed witheither treatment. Instead, our biochemical analysis indicatesthat this is due to the rapid exit of preexisiting αβ dimerspresent in the MIIC of iDC before LPS or M-CSF treatment.

Although it remains to ascertain whether M-CSF displayssimilar effect in vivo, the present study indicates how locallyproduced M-CSF could affect the Ag presenting function ofDC by firstly emptying the internal pool of preexisiting MHCII αβ dimers and secondly by reducing the life time ofpresentation at the cell surface of a particular Ag/MHC IIcomplex. This would be relevant to pathological situationswhere high level of M-CSF are believed to inhibit this functionby deviating DC toward the MΦ differentiation pathway. Thisstudy also reinforces the notion that DC possess a high capacityto shape their internal membranes on demand since two typesof modifications, maturation or M-CSF stimulation, define twodifferent mechanisms of sorting and transport of MHC-IImolecules associated with different reorganizations oflysosomal membranes.

The authors are especially grateful to J. Mulvihill for excellenteditorial assistance and to Drs P. H. Gaillard and L. Johannes for theircritical reading of the manuscript. We also thank D. Morineau and D.Meur for photographic work, Schering-Plough for kindly providinghrIL-4, Novartis for hrGM-CSF and the Genetic Institute for hrM-

1010

CSF. This work was supported by grants from the EuropeanCommunity (ERB CHR XCT 940592), the ‘Association pour laRecherche contre le Cancer’, the Human Frontier Science Program,AMGEN SA (Paris, France), INSERM (EPI 99-08), the Etablissementde Transfusion Sanguine de Strasbourg and Institut Curie.

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