Dendritic cell progenitor is transformed by a conditional v-Rel estrogen receptor fusion protein v-ReIER

Cell, Vol. 80, 341-352, January 27, 1995, Copyright © 1995 by Cell Press

Dendritic Cell Progenitor Is Transformed by a Conditional v-Rel Estrogen Receptor Fusion Protein v-RelER Guido Boehmelt,*t Jaime Madruga,* Petra D6rfler,* Karoline Briegel,* Heinz Schwarz,~ Paula J. Enrietto,§ and Martin Zenke* *Institute of Molecular Pathology Dr. Bohrgasse 7 A-1030 Vienna Austria ~:Max-Planck-lnstitut fLir Entwicklungsbiologie Spemannstrasse 35 D-72076 TObingen Federal Republic of Germany §Department of Microbiology State University of New York at Stony Brook Stony Brook, New York 11794

Summary A conditional v-Rel estrogen receptor fusion protein, v-RelER, causes estrogen-dependent but otherwise unaltered v.rel-specific transformation of chicken bone marrow cells. Here, we demonstrate that such v-relERtransformed cells exhibit B lymphoid determinants in line with earlier studies on v.rel.transformed cells. However, following inactivation of v-RelER oncoprotein activity by administration of an estrogen antagonist, cells differentiate into antigen-presenting dendritic cells as judged by several morphological and functional criteria. Additionally, under yet different culture conditions, v-relER cells differentiate into cells resembling polymorphonuclear neutrophils. Our studies therefore suggest that the conditional v-RelER, and probably also the authentic v-Rel, transform a common progenitor for neutrophils and dendritic cells. Introduction Differentiation of hematopoietic cells involves the highly ordered and controlled proliferation of immature progenitor cells and their commitment and differentiation into fully mature cells of various lineages. While a number of retroviral oncogenes efficiently bypass such normal control mechanisms and cause leukemia, they also provide invaluable tools to study mechanisms of normal hematopoietic cell differentiation on the molecular level. In the avian system, oncogene-transformed, nonestablished cell strains can be obtained in vitro under conditions in which they retain their capacity to undergo apparently normal terminal differentiation (reviewed by Beug and Graf, 1989). in this system, conditional oncogene versions have proven extremely useful, since cells transformed by such mutants can be grown when the respective oncoprotein is active, while "switching off" oncoprotein activity induces their terminal differentiation. In this paper, we describe the use iPresent address: OntarioCancer Institute,PrincessMargaretHospital, 500 SherbourneStreet, Toronto, Ontario M4X 1K9, Canada.

of this approach to assess the differentiation potential of chicken bone marrow cells transformed by a conditional v-Rel estrogen receptor fusion protein v-RelER. v-rel, the oncogenic version of c-rel transduced by the avian retrovirus complex REV-T/REV-A, belongs to the NF-KBIrelldorsal transcription factor family (reviewed by Gilmore, 1991 ; Bose, 1992). Members of this protein family are versatile regulators involved in growth control, differentiation, and pattern formation, v-rel encodes a 59 kDa protein that forms multiple complexes with several other cellular proteins and transforms avian hematopoietic cells both in vivo and in vitro. Several groups have demonstrated that v-Rel acts as a transcriptional repressor of Rel- and/or NF-KB-responsive genes in transient transfection assays (reviewed by Bose, 1992). Studies with a conditional hormone-inducible v-Rel estrogen receptor fusion protein (v-RelER), however, provided the first clues of v-Rel acting as a transcriptional activator of Rel- and/or NF-~:B-responsive genes (Boehmelt et al., 1992). Such a hormone-inducible v-RelER caused estrogen-dependent v-Rel-specific transformation of chicken bone marrow cells in vitro. Initial evidence suggested that v-rel contained within the REV-T/REV-A virus induced a disease of lymphomatous origin (Sevoian et al., 1964). This is in line with the observation that oncogenic activation of other members of the NF-~:Blrelldorsal family (e.g., Lyt-10/NFKB2) has been implicated in lymphoid tumor formation in humans (Fracchiolla et al., 1993, and references therein). The in vitro target cell for v-reltransformation was also classified as lymphoid (Beug et al., 1981; Lewis et al., 1981; Barth and Humphries, 1988; Benatar et al., 1991). Other studies, however, suggested that the v-rel REV-T/REV-A-induced disease was reticuloendotheliosis (Theilen et al., 1966; Olson, 1967), affecting cells associated with endothelia of blood vessels and with sinusoids of spleen, kidney, liver, and lymphoid organs. However, there are few studies in line with such a characterization. Cells derived from liver or spleen tumors of v-rel REV-T/REV-A-infected chickens exhibit either T lymphoid or myeloid determinants (Barth et al., 1990). Moreover, by using a replication-competent v-rel virus, Morrison et al. (1991) demonstrated that v-rel-transformed chicken bone marrow cells coexpress surface antigens specific to both lymphoid and myeloid cells. Thus, until now, the true target cell for v-rel transformation remained obscure. The availability of conditional v-Rel variants (e.g., v-RelER or a temperature-sensitive v-Rel; Boehmelt et al., 1992; Capobianco and Gilmore, 1993; White and Gilmore, 1993) now allows us to examine the differentiation potential of v-rel-transformed cells by releasing the v-rel-induced differentiation arrest. Identification of the differentiated cells obtained should permit us to determine the nature of the target cell for v-rel-specific transformation. In this paper, we demonstrate that v-relER-transformed chicken bone marrow cells express B lymphoid determi-

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Figure 1. Morphologyof Differentiatedv-relER Cells (A) v-relER and v-tel cells (clones 25 and C8, respectively) were cultured in EBM (a and b), in medium I and II (c-h) plus estrogen or ICI as indicated. Cells were cytocentrifugedonto slides and stained with May-Gn3nwald/Giemsa. In medium I, >85% of the cell population showed the elongated, bipolar morphology within 2-3 days. Differentiationin medium II for 4-5 days yielded about 75% of cells exhibiting the characteristicpolymorphonuclearphenotype, about 15% of elongated cells and a minor proportion of undifferentiatedcells. The characteristic morphological changes shown here for v-relER clone 25 were also observed for other v.relER cell clones. (B) Medium I- and II-differentiated v-relER cells at higher magnification.

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nants consistent with initial studies on v-rel-transformed cells. However, following inactivation of v-RelER activity, cells differentiate into either antigen-presenting dendritic cells or cells reminiscent of polymorphonuclear neutrophils. These experiments support the notion that. v-RelER, and presumably also v-Rel, transform an at least bipotent hematopoietic precursor cell. Finally, we describe the development of a powerful in vitro model system for the analysis of dendritic cell function.

Results Differentiation Potential of v-relER Cells Previous work established that a conditional v-Rel estrogen receptor fusion protein (v-RelER) transforms chicken bone marrow cells in vitro in a strictly estrogen-dependent manner (Boehmelt et al., 1992). Several v-re/ER-transformed bone marrow cell clones were isolated and expanded in liquid culture in the presence of estrogen. On the basis of morphological criteria and expression of a series of cell type-specific surface markers, such cells appeared to be identical to cells transformed by wild-type v-re~ (Figure 1; Boehmelt et al., 1992, 1995). Additionally, all v-re/ER cell clones studied expressed surface immunoglobulin M (IgM) and high levels of major histocompatibility complex (MHC) class II antigen (see also below), as described before for v-re/-transformed cells (Barth et al., 1990; Benatar et al., 1991; and references therein). To determine the differentiation potential of such cells, v-RelER oncoprotein activity was experimentally "switched off" by addition of the estrogen antagonist ICI 164.384

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(ICI). After testing a variety of media and culture conditions that were expected to allow growth and differentiation of myeloid, erythroid, or lymphoid cells (data not shown), conditions were established that reproducibly supported differentiation of v-relER cells into two morphologically distinct phenotypes. First, incubation of v-relER cell clones in medium I (a modified standard growth medium supplemented with conalbumin and insulin) plus ICI resulted in the appearance of characteristic elongated cells (Figure 1). MayGr0nwald/Giemsa staining of these cells revealed a polarized morphology, characterized by vacuolized, reddish cytoplasm confined to one side of the cell body and smooth, bluish cytoplasm at the opposite side (Figure 1). Secondly, the same v-relER cell clones cultured with ICI under different media conditions (medium II, modified from Radke et al., 1982) yielded a second morphologically distinct cell type: these cells were spherical and unpolarized and contained segmented, polymorphic nuclei. The cytoplasm was without preference for basophilic or eosinophilic dyes (Figure 1). This differentiation potential was found for all v-relER clones tested (8/8). Additionally, the clonality of the starting cell population was demonstrated by subcloning experiments and analysis of the retroviral integration site (data not shown). As expected, cells transformed by wild-type v-rel did not respond to ICI treatment and remained virtually unchanged morphologically under all conditions tested (Figure 1A). We noted, however, that several v-rel and all v-relER clones (plus estrogen) contained about 0.5% of elongated, bipolar cells when grown in standard growth medium (data not shown). This indicates that the differentiation block

v-relER Transforms Progenitor of Dendritic Cells

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Figure 2. Ultrastructure of Differentiated v-relER Cells Electron microscopy of undifferentiated v-relER cells (A) and of v-relER cells differentiated in medium I (B, D-F) and II (C). (A)-(C) depict cells at identical magnification (3800 x ). Note the intracellular polarization and long cytoplasmic processes in longitudinal sections of differentiated dendritic v-relER cells (B and D). The basal side cf a dendritic v-relER cell (E and F) contains mitochondria, Golgi-apparatus, vesicles, microtubuli (black arrows in F), membranous vacuoles, ribosomes and bundles of intermediate filaments (open arrows in E and F). The cytoplasm on the opposite side is devoid of subcellular structures besides ribosomes (D). (D) and (E) show details of the cell depicted in (B) at higher magnification (8400 x ); the basal cytoplasm shown in panel F (11400x) refers to another dendritic v-relER cell. Black triangle indicates budding virus•

achieved by v-Rel or hormone-activated v-RelER is incomplete. Taken together, these experiments demonstrate that clonal populations of v-relER cells can be efficiently induced to differentiate into two different cell types depending on the culture conditions employed.

v.relER Cells Differentiate into Dendritic Cells To identify the nature of the differentiated v-relER cells obtained, several parameters were investigated• First, cellular ultrastructure was studied by electron microscopy. Second, the expression pattern of a panel of cell t y p e specific and lineage-specific markers was investigated. Third, the biological activity of the differentiated cells was assessed in functional assays. Electron microscopy of the elongated, bipolar v-relER cells obtained in medium I revealed that all organelles,

vacuoles, and other intracellular compartments localize to one side of the cell body (arbitrarily referred to as the basal side), while the opposite side contains a largely homogeneous cytoplasm with characteristic branching protrusions (Figure 2). Extended intermediate filament bundles were exclusively found on the basal side and might be important for the establishment and/or maintenance of the bipolar cell structure (see below). Some of these morphological features are characteristic for dendritic cells and thus provided the first hint that medium I-differentiated v-relER cells might represent avian dendritic cells. Dendritic cells represent professional antigen-presenting cells, characterized by their dendrite-like cytoplasmic protrusions, high expression of M HC class II antigens, moderate phagocytic activity, and low capacity to adhere to tissue culture plastic (reviewed by Steinman, 1991). Additionally, human skin dendritic (Langerhans) cells and

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Figure 3. Vimentin Expression in Differentiated v-relER Cells v-relER cells induced to differentiate in medium I and II (A-C and D-F, respectively) were subjected to immunofluorescence analysis employing a vimentin-specific antibody (see Experimental Procedures). Nuclei were stained with DAPI. Nuclear (A and D) and vimentin (B and E) staining is shown; in (C) and (F) individual records were superimposed. Please note that vimentin expression endows dendritic v-relER cells with a characteristic polarized appearance (C).

dendritic cells from chicken bursa contain bundles of vimentin-type intermediate filaments (Rappersberger et ai., 1990; Olah and Glick, 1992; Olah et al., 1992a, 1992b). Because the electron micrographs in Figures 2E and 2F clearly show bundles of intermediate filaments in longitudinal sections of dendritic v-relER cells, we analyzed these cells for vimentin expression using indirect immunofluorescence. Figure 3 shows that dendritic v-relER cells express vimentin in a characteristic polarized fashion, very similar to vimentin organization in dendritic cells of chicken bursa (Olah et al., 1992a, 1992b). Moreover, the polarized expression pattern appears to be characteristic for the dendritic cell type, since it was not detected for a number of other hematopoietic and nonhematopoietic cell types (data not shown). Rather, vimentin formed a "cage"-iike structure surrounding the nucleus, as also observed for v-rel-transformed cells or undifferentiated v-relER cells (grown in the presence of estrogen). This cage-like structure was reduced to a small vimentin aggregate in v-relER cells differentiated in medium II (Figures 3E and 3F). Since dendritic cells are involved in M HC class I I-dependent presentation of antigens to resting T helper cells (Steinman, 1991, and references therein), we next investigated MHC class II expression on differentiated dendritic v-relER cells. Cells were stained with a monoclonal antibody specific for the nonpolymorph region of the chicken B-L (MHC class II) J~chain and subjected to fluorescenceactivated cell sorting (FACS) analysis. All v-relER cell clones tested express high levels of MHC class II, which are however, independent of their differentiation state (see Figure 7A). Finally, a monoclonal antibody raised against dendritic cells of chicken spleen (CVI-ChNL-74.3; Jeurissen et al., 1992) revealed a spotted cytoplasmic staining pattern confined to the basal side of dendritic v-relER cells. A similar

2.5 x 105 primary spleen derived T lymphocytes (of Lohmann Brown chick) were incubated for 5 days with 103, 5 x 103, 104, 5 x 104, and 10~ mitomycin C-treated v-relER cells. T cell proliferation was determined by [3H]thymidine incorporation. Mitomycin C-treated autologous (Lohman n Brown) spleen cells induced no response, whereas spleen cells derived from the same chicken flock as v-relER cells (allogeneic, White Leghorn) induced a proliferative response only at high cell doses (5 x 105 cells). For v-relER cells, estrogen or ICI was added to the MLR at day 1 of the experiment to keep the v-RelER protein in the active or inactive state, respectively. Neither estrogen nor ICI had an effect on responder cells.

pattern was found in bona fide dendritic cells in chicken bursa. Staining of undifferentiated v-relER cells was perinuclear and weaker. The antibody also stained other hematopoietic cell types, albeit less efficiently (data not shown). In summary, these results support the notion that v-RelER transforms a hematopoietic progenitor for dendritic cells.

Dendritic v.relER Cells Are Functionally Active To assess functional properties of differentiated v-relER cells, three assays were used. First, the phagocytic activity of v-relER cells was measured in order to discriminate dendritic cells (low activity) from macrophages (high activity). Second, their ability to stimulate T cell proliferation in a primary mixed lymphocyte reaction (MLR) was assessed. Third, since dendritic cells are highly mobile, the behavior of individual v-relER-transformed cells in culture was followed by time-lapse cinemicroscopy. To measure phagocytic activity, both differentiated and undifferentiated v-relER cells were investigated for uptake of TRITC-labeled latex beads. Under the experimental conditions used, up to 20% of dendritic v-relER cells endocytosed 2-5 beads/cell within 1 hr. Similar results were obtained for undifferentiated v-relER cells and v-rel-transformed cells. These results are in accord with the early work of Lewis et al. (1981) in which the phagocytic activity of REV-T/REV-A-transformed v-rel cells was measured. Bone marrow macrophages, used as an experimental control, took up significantly more beads in the same period of time (85% of the cells contained 10-20 beads/cell), while T cells and erythroid cells were negative. Thus, dendritic v-relER cells exhibit a moderate phagocytic activity, as reported for normal dendritic cells (Steinman, 1991). As an additional property to distinguish dendritic cells from macrophages, acid phosphatase activity was determined. Dendritic v-relER cells were consistently found to be negative, while control macrophages were highly posi-

v-relER TransformsProgenitorof DendriticCells 345

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tive (data not shown), in line with studies on dendritic cells in mammals (Zucker-Franklin et aL, 1988). Among the antigen-presenting cells, dendritic cells are by far the most potent in inducing T cell responses (Steinman, 1991, and references therein). Therefore, undifferentiated and dendritic v-relER cells were investigated for their ability to stimulate proliferation of spleen T cells in primary dose-response MLR assays. Figure 4 shows that dendritic v-relER cells are potent inducers of allogeneic T cell proliferation. 10 x 103 mitomycin C-treated dendritic v-relER cells were as efficient as 500 x 103 spleen cells, derived from the same chicken flock used to generate the v-relER cells. Maximal stimulation was obtained using 50 x 103 dendritic v-relER cells. As expected, autologous spleen cells failed to stimulate [3H]thymidine incorporation of responder T cells. Surprisingly, undifferentiated v-relER cells (grown in the presence of estrogen) stimulated [3H]thymidine incorporation as efficiently as dendritic v-relER cells (Figure 4; see also Discussion). One of the most striking features of dendritic cells is that they constantly generate, bend, and retract cell processes that assume various shapes like spiny dendrites, bulbous pseudopods, and large thin cytoplasmic sheets or veils (reviewed by Zucker-Franklin et al., 1988; Steinman, 1991). To assess this property, we first established conditions in which dendritic v-relER cells efficiently adhered

Figure 5. Time-LapseCinemicroscopyof Adherent v-relER Cells (A)v-relERcellsadhereto tiesueculturedishes, if cultured in CEF-conditionedmedium (CCE medium), both in the presenceof estrogenor ICI, With estrogen, spindle-shapedcells with long dendriticprocessesare also observed.In the presenceof ICI,cellsacquirethe elongated morphologyobservedfor cells grown in suspension in medium I. Cells cultured for 18 hr in CCE mediumare shown. (B)v-relERcellsincubatedin CCEmediumplus ICI for 2 days are highly motileas revealedby time-lapsecinemicroscopy.Cellsmigrateabout 4-5 timestheirbodylengthwithin10 rain(arrow pointsto one representativecell).Cellsallowed to adherein the presenceof estrogendo not migrate (data not shown). (C) Time-lapse cinemicroscopy of spleenderived dendriticcells.

to the surface of the culture dish. Conditioned medium from chicken embryo fibroblasts (CCE medium) was found to be most potent. While medium I-differentiated v-relER cells are nonadherent, incubation in CCE medium plus ICI caused cells to adhere to the culture dish within hours (Figure 5A). Most importantly, adherent dendritic v-relER cells were found to be highly mobile, as revealed by time-lapse cine= microscopy (Figure 5B). Cells exhibiting the elongated morphology continually contracted to form rounded cells, which then showed sheet-like processes (veils) and acquired again the elongated phenotype. This process took about 5-10 min, while cells moved an average of 3-4 times their body length. The movement, however, was not directed (at least under the culture conditions employed so far). As expected, no cell divisions were observed during the experimental period (24-48 hr). Undifferentiated v-relER cells incubated in CCE medium (plus estrogen) also adhered to the culture dish (Figure 5A). However, cells were still actively dividing, retained their round morphology, and were virtually not motile. A minor cell population formed long spiny dendrite-like processes (Figure 5A), but was still fully competent in undergoing cell divisions, v-rel-transformed cells behaved identically. Whether this subpopulation of cells represents more resident dendritic cells, as opposed to the motile "veiled"

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Figure 6. DifferentiationProfileof v-relER Cells in Medium II (A) Maturationof v-relER cells into neutrophilsrevealedseveralintermediatestages.Shownareimmature cellswith a roundnucleus(stage 1), cellswith a notchedor horseshoe-shapednucleus(stage2), stage 3 cellswithan indentednucleusand cellswitha segmentedmultilobed nucleus (stage 4). (B) Chicken bone marrowcontainsneutrophils,which are phenotypically similarto stage 3 and 4 v-relER cells. (C) Time course of differentiation.Cytospin preparationsof v-relER cells inducedto differentiatein medium II were used to evaluatethe proportionof differentiationstages as depicted in (A). (D) Periodicacid Schiff (PAS) reagentrevealeda coarsestainingpattern in mediumII-differentiatedv-relER cells (ICI), whereasestrogentreated(estrogen)or dendriticv-relERcellswerenegative(notshown). Cells in (A) and (B) were photographedwith the same magnification, while cells in (D) are shown with a lower magnification.

dendritic cells, is not clear at present. Cells were, however, devoid of acid phosphatase activity, thereby excluding that they represent macrophages (data not shown). In conclusion, the high motility observed for veiled dendritic v-relER cells is very specific for this cell type and was not observed for other hematopoietic and nonhematopoietic cells, suggesting that it reflects normal behavior of functional dendritic cells. This idea is strengthened by our finding that chicken spleen cell preparations enriched for dendritic cells contained veiled cells that exhibited the same motility and characteristic way of moving as veiled dendritic v-relER cells (Figure 5C). v-relER Cells Differentiate into Cells

Resembling Neutrophils As shown in Figures 1-3, v-relER cells induced to differentiate in growth medium II exhibit a segmented polymorph nucleus. Such cells are also found in chicken bone marrow and peripheral blood enriched for leukocytes (Figure 6B; data not shown). They resemble a cell type that in birds

is referred to as heterophils (Lucas and Jamroz, 1961). Interestingly, during v-relER cell differentiation in medium II, several intermediate nuclear forms were observed (Figure 6A), reminiscent of a distinct pattern of nuclear maturation associated with neutrophil differentiation in humans (Zucker-Franklin et al., 1988). The round nucleus (stage 1) present in undifferentiated v-relER cells becomes "horseshoe-like," more and more indented, and finally multilobed (stages 2-4, respectively). This process takes about 5 days and finally converts up to 75% of the cell population into polymorphonuclear cells (stages 3 and 4, Figure 6C). As during differentiation of human neutrophils, a progressive loss of euchromatin and an increase of heterochromatin was observed (see Figure 2). While mammalian neutrophils can readily be identified by established histological staining techniques (e.g., periodic acid-Schiff [PAS] reagent, Sudan Black staining; Zucker-Franklin et al., 1988), employing such stains for characterization of chicken neutrophils was less revealing. Yet PAS reagent (which detects glycogen, a major energy source of neutrophiis) clearly stained medium II-differentiated v-relER cells. The staining pattern was, however, coarse (Figure 6D) and apparently atypical when compared with that observed for normal human neutrophils (Zucker-Franklin et al., 1988). Undifferentiated and dendritic v-relER cells were PAS negative (Figure 6D; data not shown). Surprisingly, granules were not observed in medium II-differentiated v-relERcells, neither by histological staining nor by electron microscopy (Figures 1 and 2). Since mim-1, a protein distantly related to mammalian defensins, is expressed in normal and transformed chicken promyelocytes and presumably also in normal neutrophils (Ness et al., 1989; Introna et al., 1990; Graf, 1992), medium II-differentiated v-relER cells were investigated for mim-1 expression. By Western blot analysis, we found that neither v-tel- or v-relER-transformed cells nor medium I- or II-differentiated v-relER cells express mim-1 (data not shown). In summary, several morphological criteria support the idea that medium II-differentiated v-relER cells represent neutrophils. However, further experiments are clearly required to provide a more thorough characterization of this cell type. In particular, the culture conditions employed so far might not be optimal to achieve terminal neutrophil differentiation. Thus, mature granules might not have formed or, alternatively, are already discharged (see Discussion).

Undifferentiated v-relER Cells Express B Lymphoid Determinants In early studies, v-rel-transformed spleen or bone marrow cells were classified as early pre-B or pre-B/pre-T lymphoid progenitors (Beug et al., 1981; Lewis et al., 1981), while subsequent experiments suggested that one of the v-tel target cells is an IgM-positive B cell (Barth and Humphries, 1988; Zhang et al., 1991; Benatar et al., 1991, and references therein). For these reasons, we investigated v-relER cells for B lymphoid determinants such as surface IgM expression, organization of the immunoglobulin genes,

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and expression of the B cell-specific activator protein BSAP. First, all v-relER clones analyzed and grown in the presence of estrogen express surface IgM, as shown by FACS analysis and live cell immunofluorescence with an IgMspecific monoclonal antibody (Chen et al., 1982; Figure 7A; data not shown). Treatment with the phorbol ester PMA further increased surface IgM expression to levels as high as that measured for the chicken B cell line RP9. However, IgM expression in such cells was still 5-fold lower than in cells from chicken bursa (data not shown). Most importantly, when v-relER cells were induced to differentiate into dendritic cells, expression of surface IgM was down-modulated. Surface IgM expression was also low in cells simultaneously treated with ICI and PMA (Figure 7A). Interestingly, such cells represented neither dendritic cells nor neutrophils, but exhibited a blast-like morphology and a round nucleus (data not shown). A more detailed analysis of this cell type is the subject of current investigation. Finally, MHC class II expression remained high and unaffected under all conditions tested, while erythroblast control cells were, as expected, negative for both IgM and MHC class II expression. Second, we investigated the status of the immunoglobulin genes in undifferentiated and differentiated v.relER cells by Southern blotting. The J-C-specific segment of the chicken ;L light chain gene (Figure 7B; Buerstedde and Takeda, 1991) was used as a probe. Figure 7B shows that undifferentiated v-relER cells have the ;L chain gene rearranged, since the probe detects, in addition to the 16 kb and 2.8 kb EcoRI fragments (specific for the unrearranged allele), a 14 kb EcoRI fragment that is specific for the rearranged allele. This 14 kb EcoRI fragment was also observed for cells of chicken bursa (Figure 7B), but was absent in chicken embryo fibroblasts (CEFs). As expected, immunoglobulin gene rearrangement was observed in both undifferentiated and differentiated v-relER cells, irrespective of whether differentiation was induced in medium I or II (Figure 7B). Finally, v-relER cells were investigated for the presence of the B cell-specific activator protein BSAP (Adams et al., 1992). We found that undifferentiated v-relER cells expressed chicken BSAP mRNA and protein, as demonstrated by RNase protection and electrophoretic mobility shift assay (data not shown). BSAP expression was, however, considerably lower (about 50-fold) than that detected

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Figure 7. B Lymphoid Determinants of Transformed v-relER Cells (A) v-relER cells express surface IgM. v-relER cells were cultured for three days in EBM plus estrogen (a), in medium I plus ICI (c) or in a medium containing phorbol 12-myristate 13=acetate (+PMA) supplemented with estrogen or ICI, respectively (b, d; see Experimental Procedure). Chicken cell lines RP9 (B cells; e, f) and HD3 erythroblasts (erbls; g, h) were grown in the absence (e, g) or presence (f, h) of PMA as indicated. Cells were stained with specific antibodies for the constant region of the chicken Cp_ heavy chain (grey) or the nonpolymorph region of chicken B-L (MHC class I]) ~ chain (hatched) and subjected to FACS analysis. Control cells incubated with FITC-labeled secondary antibody only (white).

(B) Rearrangement of the immunoglobulin light chain gene (Ig;L) in v-relER cells. Southern blot analysis of genomic DNA of transformed (TF), dendritic (DC) and neutrophil (NP) v.relER cells, of chicken bursa (BU), CEFs and the erythroblast cell line HD3 after digestion with EcoRl. Note that v.relER and v.rel cells, and cells of chicken bursa yield a doublet of specific DNA fragments derived from the rearranged (R) and unrearranged (UR) alleles, while CEF and HD3 control cells show a single band only, which corresponds to the unrearranged alleles. The probe used was a BamHI-Sall fragment spanning the JkC;L region (probe). Germline and rearranged configuration of the chicken Ig~, gene is depicted. '¥V, cluster of pseudogenes; L, leader sequence; V~,, variable region; J;% joining segment; C~, constant region; arrowheads indicate EcoRI restriction sites.

Cell 348

in bursa cells. Most importantly, no BSAP activity was found in dendritic v-relER cells. In summary, these experiments demonstrate that undifferentiated v-relER cells possess B lymphoid determinants, which, however, are down-modulated or lost when cells differentiate. As expected, the rearranged state of the immunoglobulin light chain gene is preserved in differentiated v-relER cells, indicating that immunoglobulin gene rearrangement does not interfere with their differentiation into neutrophils and dendritic cells.

Discussion The goal of the present study was to determine the differentiation potential of a hematopoietic target cell for v-relspecific transformation. This work led to the unexpected discovery that clonal cell populations transformed by the conditional v-RelER exhibit the potential to differentiate into fully competent dendritic cells and into cells that resemble neutrophils. Dendritic cells are found at various locations within the organism and represent a cell population whose function is to capture antigens, migrate to lymphoid organs, and present the processed antigens to lymphoid cells. Accordingly, several morphological and functional properties of differentiated v-relER cells support our conclusion. Dendritic v-relER cells exhibit an elongated, bipolar morphology, form lamellipodia or veils, and are highly motile in culture. They exhibit a low to moderate phagocytic activity, express high levels of MHC class II, and stimulate T cell proliferation in a primary mixed lymphocyte reaction. It was, however, surprising that also undifferentiated v-relER cells support T cell proliferation in this assay. Yet, our observation that these cells express high levels of MHC class II and exhibit properties of B-lymphoid cells (which are active in antigen presentation) might explain this finding. Alternatively, such cells might produce growth factors that promote T cell proliferation. Undifferentiated v-relER cells do indeed express an interleukin-8 (IL-8)-related mRNA (A. Petrenko and P. J. E., submitted), which could explain their stimulator activity, since IL-8 in mammals activates T cell proliferation. Differentiation of v-relER cells into cells resembling neutrophils is of particular interest, since neutrophil differentiation in vitro under well-defined culture conditions has not been achieved. In chicken, v.myb-ets-transformed bone marrow cells possess properties of neutrophil/macrophage progenitors, while cells transformed by specific v-myb variants resemble promyelocytes and are considered to represent precursors of chicken neutrophils (Golay et al., 1988; Introna et al., 1990; Graf, 1992). Both normal and transformed chicken promyelocytes as well as v-mybets myeloblasts express mim-1, a protein that is highly expressed in granules (Ness et al., 1989; Introna et al., 1990; Graf, 1992). In contrast, the medium II-differentiated v-relER cells described in this paper do not express mim-1 and lack granules. This might be because such cells were obtained in vitro and the appropriate culture conditions for granule formation have so far not been achieved. An alternative explanation provides our find-

ing that v-relER cells express MIP-113 (A. Petrenko and P. J. E., submitted), which is known to cause neutrophil degranulation. Yet based on nuclear morphology, medium II-differentiated v-relER cells (stage 4) appear to be more advanced in neutrophil differentiation than any of the v-myb and v-myb-ets-transformed cell types described before. However, so far, it remains difficult to assess how these various cell types relate to each other, mainly due to the lack of functional neutrophil-specific markers. Our findings are consistent with the observation that, in mouse, dendritic cells and granulocytes arise from a common progenitor (Inaba et al., 1993). Furthermore, granulocyte/macrophage colony-stimulating factor (GMCSF), which supports growth of granulocytes and macrophages, leads to an outgrowth of dendritic cells from mouse peripheral blood and human CD34 ÷ progenitor cells (Inaba et al., 1992; Caux et al., 1992). However, the progenitor isolated by Inaba et al. (1993) is MHC class IInegative and acquires high MHC class II expression after differentiation into the dendritic phenotype. In contrast, the v-relER progenitor described in this paper expresses high levels of MHC class II both before and after differentiation induction. We envisage two possibilities to explain this finding. First, in undifferentiated cells, the active v-RelER protein might induce MHC class II transcription through multiple potential Rel/NF-KB-binding sites present in the chicken MHC class II promoter (Zoorob et al., 1990). Following v-RelER inactivation, MHC class II expression is then controlled by the dendritic cell differentiation program. Second, constitutive expression of MHC class II molecules on both transformed and differentiated v-relER cells could imply that MHC class II expression is independent of v-rel and v-relER expression. Thus, v-Rel proteins might transform a MHC class II-positive progenitor, which would be distinct from that described by Inaba et al. (1993). Finally, since the progenitor isolated by Inaba et al. (1993) also gives rise to macrophages, it will be interesting to determine whether culture conditions can be established that support differentiation of v-relER cells into macrophages. Such studies are presently being performed.

Does v-relER Transform a Pluripotent Hematopoietic Progenitor Cell? Several previous studies demonstrated that v-tel-transformed spleen or bone marrow cells express B or T cellspecific determinants as well as surface antigens found on myeloid cells (for references see Bose, 1992; Morrison et al,, 1992), Various v-tel-transformed B and T cell lines are currently being used to study lymphoid development in chicken (Schat et al., 1992; Benatar et al., 1991 ; Marmor et al., 1993). In accord with these reports, also the v-relER progenitor cells used in this study have their immunoglobulin genes rearranged and express surface IgM and the chicken homolog of the B cell-specific transcription factor BSAP. Interestingly, following induction of dendritic cell differentiation, surface IgM and BSAP are down-regulated. However, v-relER cells induced to differentiate in the presence of phorbol ester (PMA) also down-modulate surface IgM and BSAP expression, yet apparently do not exhibit prop-

v-relER Transforms Progenitor of Dendritic Cells

349

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Ig + Figure 8. Models of Differentiation Potential of v-relER Progenitors (A) v-relER transforms a common progenitor for dendritic cells, neutrophils and B cells. (B) v-relER transformation of a common progenitor for dendritic cells and neutrophils induces expression of B lymphoid determinants. (C) v-relER converts a B lymphoid progenitor into a progenitor for dendritic cells and neutrophils. Dark arrows indicate differentiation potential as described in this paper. Dashed arrows show that cells are expected to differentiate into mature B cells which, however, still remains to be shown. Open arrows, shift in differentiation program due to the transforming oncogene (v-relER). Normal differentiation capacity (line). Ig÷ indicates cells, where immunoglobulin genes are rearr&nged.

erties of dendritic cells or neut rophils. Since BSAP expression is undetectable in mature murine plasma cells, it seems possible that such v-relER cells undergo further lymphoid maturation. Currently, experiments aim at determining whether these cells perform immunoglobulin class switching. Thus, while transformed v-relER cells exhibit the potential to differentiate into dendritic cells and neutrophils, they simultaneously display determinants of B lymphoid cells. Several models can be envisaged to explain this finding. First, v-relER might transform a common progenitor for dendritic cells, neutrophils, and B cells containing already rearranged immunoglobulin loci (Figure 8A). This model would imply that at least a subset of normal dendritic cells has the immunoglobulin genes rearranged. Experiments to address this question are in progress. Second, v-relER might transform a common progenitor only for dendritic cells and neutrophils, yet v-relER oncogenic activity leads to induction of the lymphoid phenotype (Figure 8B). Whether such cells also acquire the capacity

to terminally differentiate into fully mature B cells still remains to be shown. Third, v-relER might transform a cell of the B lymphoid lineage and, due to its transforming capacity, cause a "shift" toward the differentiation program of a dendritic cell/ neutrophil progenitor (Figure 8C). This progenitor cell would still retain B lymphoid determinants. A change in the differentiation program of B lymphoid cells into another lineage due to ectopic oncogene expression is not without precedent. Klinken et al. (1988) reported that ectopic v-raf expression causes a switch of B cell lines into cells of the myeloid lineage that contain rearranged immunoglobulin genes. How Do v-Rel Proteins Bring about Transformation? While all our observations suggest that a hormoneactivated v-RelER (and most probably also the authentic v-Rel) transforms a dendritic celllneutrophil progenitor, the question emerges whether such an idea would be compatible with the phenotype of the disease caused by the v-re~ REV-T/REV-A virus. Both histological and cytological studies identified the v-re~ REV-T/REV-A-induced disease as reticuloendotheliosis. Transformed cells exhibited a histiocytoid, mesenchymal morphology with spiny processes and were found associated with blood vessels in liver, spleen, kidney, and the scaffold of lymphoid organs. Both thymus and bursa had undergone significant atrophy, due to massive tumor cell growth at the expense of the lymphoid tissue (Theilen et al., 1966; Mussman and Twiehaus, 1971). These findings are reminiscent of several properties of the v.re/ER-transformed bone marrow cells described above. First, undifferentiated v-re/ER cells and v-re~ cells grow in large aggregates, adhere to other cell types and to extracellular matrix proteins (G. B. and J. M., unpublished data). Second, cells with long dendrite-like protrusions were found in v-re~ and v-relER cultures grown in CCE medium (plus estrogen for v-relER cells). Such cells were stationary, but retained their capacity to proliferate. Thus, during development of reticuloendotheliosis, v-rel-transformed cells might get immobilized on locally restricted areas of blood vessels and/or the scaffold of lymphoid organs in which they proliferate to form large aggregates. Transformed cells might also pass the endothelium and proliferate perivascularly. Finally, a decrease in the number of neutrophils (heterophils) in v-rel REV-T/REV-A virus-infected animals was observed (Olson, 1967), indicating that v-rel perturbs normal neutrophit differentiation in vivo. However, it remains enigmatic how the v-Rel proteins transform dendritic cell/neutrophil progenitors, which also express B cell determinants. Surprisingly, dendritic cells present in lymphoid organs of mice express high levels of RelB, another member of the NF-KB/Rel transcription factor family (Carrasco et al., 1993). Thus, it is tempting to speculate that v-RelER (and v-Rel) mimic or interfere with normal RelB function in such cells or their respective progenitors. Moreover, expression of the v-Rel oncoproteins might simultaneously influence the activity of other members of the NF-~B/Rel transcription factor family that

Cell 350

are important for proper B lymphoid development. Such an idea would be in line with the finding that v-Rel proteins c o m p l e x to c-Rel and the chicken h o m o l o g of p105 (NFKB1; Morrison et al., 1989; Boehmelt et al., 1992). Additionally, alterations in the c-re~ locus or in the rel-related lyt-lO gene (NFKB2) were observed in B lymphoid tumors in humans (Fracchiolla et al., 1993, and references therein). Whether m e m b e r s of the NF-KB/Rel family of proteins are also important for normal neutrophil d e v e l o p m e n t remains to be shown. v . r e l E R Cells as a Model System for Studying

Dendritic Cell Differentiation While it is well established that dendritic cells capture, process, and present antigens, it is still a matter of debate, how the different types of dendritic cells (Langerhans cells in skin, veiled cells, interdigitating cells, or follicular dendritic cells) relate to each other. So far dendritic cells were obtained from peripheral blood, bone marrow, spleen (Inaba et al., 1992, 1993), and by in vitro differentiation from CD34 + h u m a n peripheral blood stern cells (Caux et al., 1992). A detailed analysis of their functional and biochemical properties has, however, remained difficult, mainly because pure and h o m o g e n o u s cell populations are not yet available and because of the limited cell numbers obtained. Very recently, a v-myctransformed immortalized m o u s e cell line with features of dendritic cells was described (Paglia et al., 1993), although it remains unclear whether or not the active v-Myc oncoprotein affects the dendritic p h e n o t y p e of these cells. Additionally, specific growth conditions were developed for h u m a n dendritic cells, which, however, rapidly ceased proliferation after 3 weeks in culture (Romani et al., 1994; Sallusto and Lanzavecchia, 1994). The v-relER cell differentiation system for dendritic cells described in this paper should help to o v e r c o m e some of the limitations. For example, clonal and h o m o g e n o u s cell populations of v-relER progenitors are readily available in large cell numbers. Such cells can be propagated in culture for several months and will give rise to fully functional dendritic cells. This now enables an analysis, by differential cDNA cloning strategies, of the gene expressional repertoire responsible for the dendritic differentiation program. Our work provides, therefore, the foundation for a thorough molecular analysis of a cell type that is considered to be critical for future i m m u n o t h e r a p y studies.

Experimental Procedures Cell Culture v-re~- and v-relER-transformed cells (Morrison et al., 1991; Boehmelt et al., 1992), bone marrow macrophages, chicken embryo fibroblasts (CEFs), and chicken cell lines (HD3, RP9, Beug et al., 1981; 1982) were grown in standard growth medium (EBM) containing Dulbecco's modified Eagle's medium (DMEM) supplemented with 8% fetal calf serum (FCS), 2O/ochicken serum (ChS), and 20 mM HEPES (pH 7.3). Medium I consisted of EBM supplemented with conalbumin (130 p~g/ml) and human recombinant insulin (0.1 p.g/ml). Medium II was DMEM supplemented with 8% FCS, 5% ChS, 8 mg/ml detoxified BSA, 1.9 mg/ml sodium bicarbonate, 0.12 mM l~-mercaptoethanol, 130 p.g/ ml conalbumin, and 0.1 I~g/ml human recombinant insulin. MLR medium contained DMEM supplemented with 1% heat-inactivated FCS, 2.5% heat-inactivated ChS, 2 mM glutamine, 0.05 mM

~-mercaptoethanol, and 10 mM HEPES (pH 7.3). The medium used to achieve high surface IgM expression in v-re/ER cells was DMEM supplemented with 8% FCS, 5% ChS, 130 i~g/ml conalbumin, 20 mM HEPES (pH 7.3) and human recombinant insulin (0.1 p.g/ml)containing 10-6 M 17-~ estradiol (Sigma) and 20 ng/ml phorbol 12-myristate 13acetate (PMA, Sigma). To obtain CCE medium (CEF-conditioned EBM), primary CEFs were grown for 2-3 days in EBM, the culture supernatant was recovered and sterile filtered (0.45 p.m). 17-13estradiol and IC1164.384 (ICI) were administered daily (Boehmelt et al., 1992).

Histochemical Staining May-GrSnwald/Giemsa staining (Merck) was done on air-dried, methanol-fixed cytospin preparations. Sudan Black B and Periodic acidSchiff (PAS) staining (Sigma) were performed as described by the manufacturer. For acid phosphatase staining, see Barka and Anderson (1962).

FACS Analysis 106 cells were incubated with monoclonal antibodies M-1 (specific to the chicken ct~ heavy chain; Chen et al., 1982) or 2Gll (specific for the nonpolymorph region of the chicken B-L [MHC class II] I~ chain; Kaufman et al., 1990). Cells were then stained with FITC-conjugated goat anti-rabbit IgG and analyzed (FACScan, Becton Dickinson).

Immunofluorescence Analysis Nonadherent cells (7 x 104) were washed in PBS and allowed to adhere to adhesion slides (Bio-Rad, 5 min). Cells were then rinsed with PBS, fixed (3% paraformaldehydein PBS, 15 min), permeabilized with 0.5% NP-40 in PBS (15 rain), and washed with PBS. To detect vimentin, the monoclonal vim3B4 antibody (Boehringer Mannheim) was used, followed by incubation with FITC-conjugated goat-antimouse IgG and DAPI (0.5 pg/ml) to stain nuclei. Fluorescence was visualized by using a Zeiss Axiophot microscope equipped with a CCD camera. Images were processed with Gene Join, Enhance, and McRasQ programs.

Electron Microscopy Cells were washed in PBS, fixed in 2.5% glutaraldehyde in PBS, and subjected to electron microscopy (Reichmann et al., 1993).

Time-Lapse Cinemicroscopy Cells were recorded automaticallywith a Zeiss Axiovert35 microscope equipped with a small incubator (37°C, 50/o CO2) and a CCD video camera (Sony).

Phagocytosis Assay v-relER and v-rel cells, bone marrow macrophages, and the chicken T cell line MSB1 were grown in EBM. Differentiation of v-relER cells was induced as above. TGFct-dependent erythroid progenitor cells were obtained from bone marrow as described (Schroeder et al., 1993). Cells (2 x 106) were washed in EBM and incubated with TRITClabeled latex beads (diameter: 0.48 rim; Polysciences Inc.) in EBM (1 hr, 37°C). Cells were then washed twice with 10 ml of PBS, resuspended in 200 id of PBS, and fixed in I ml of methanol (5 rain). Following a wash with PBS, cells were centrifuged (1500 rpm), resuspended in 20 pl of mounting solution containing DAPI (0.5 p.g/ml),and mounted onto slides. Adherent cells were detached from the tissue culture dish by EDTA treatment prior to analysis of phagocytic activity. Finally, cells were analyzed by fluorescence microscopy by counting fields of 200-700 cells. The average percentageof positive cells was calculated from three independent experiments.

Mix Lymphocyte Reaction (MLR) Since the flock of White Leghorn SPAFAS chickens (maintained at the Institute of Molecular Pathology, Vienna) is now inbred for more than 15 years, it showed only a limited potential of alloreactivity. Therefore, spleen cells were prepared from Lohmann Brown chickens (obtained from a commercial breeder) for MLR. Low speed centrifugation followed by Ficoll Hypaque purification (Eurobio, density 1.077 g/ml) was used to remove erythrocytes. All washing steps were performed in EBM containing heat inactivated sara. Viability of cells was evaluated by trypan blue (Sigma) exclusion.

v-relER Transforms Progenitor of Dendritic Cells 351

For the MLR assay, 2.5 x 10s responder cells were incubated with an increasing number of mitomycin C-treated (25 p.g/ml; 45 min; 39°C) stimulator cells in 100 p.I of MLR medium in 96-well plates. Following incubation for 88 hr at 39°C, cells were labeled with 0.8 iiCi [3H]thymidine (29 Ci/mmol; Amersham) for an additional 18 hr and analyzed. To obtain normal dendritic cells, chicken spleen cell suspensions were prepared as described above. Cell suspensions were washed in EBM, followed by high speed centrifugation (20 rain, 4500 rpm) through a Percoll cushion (density 1.085 g/ml). The interphase fraction was washed in EBM, and cells were incubated at high density (3-5 x 106 cells/ml) for 8-18 hr. Subsequently, nonadherent cells were carefully removed. Adherent cells represented a mixture of strongly adherent macrophages, loosely adherent cells with large dendrite-like protrusions and highly mobile veiled dendritic cells.

Southern Blot Analysis Southern blot analysis was done according to standard procedures. Genomic DNA was prepared and digested with EcoRI. A 2.3 kb BamHI-Sall DNA fragment of the unrearranged chicken light chain gene spanning the JX-CZ region (Buerstedde and Takeda, 1991) was used as a probe. Acknowledgments Correspondence should be addressed to M. Z. We are very grateful to Drs. O. Vainio and J. Kaufman for their advice in chicken mixed lymphocyte reactions. Furthermore, we thank Drs. S. Jeurissen, C.-L. Chen, and M. D. Cooper as well as J. Kaufman, S. Ness, and T. Graf for providing monoclonal antibodies, J.-M. Buerstedde for piasmid DNA, and Dr. A. Wakeling for ICI 164.384. We thank Drs. H. Beug, M. Busslinger, and A. Peterenko for their support and Dr. M. Cotten for critical reading of the manuscript. For excellent technical assistance and photography, we wish to thank G. Stengl and H. Tkadletz, respectively. This work was supported in part by grants from the National Institutes of Health (CA 51792) and the Council for Tobacco Research (3196) to P. J. E. Received June 1, 1994; revised November 17, 1994.

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