1 Department of Immunology, Erasmus MC, University Medical Center, PO Box 1738, 3000 DR Rotterdam, The Netherlands 2 Department of Dermatology, University of Erlangen, D-91052 Erlangen, Germany 3 Present address: Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DC, UK
The first two authors contributed equally to this work
Correspondence to: T. Nikolic; E-mail: t.nikolic{at}erasmusmc
Transmitting editor: P. W. Kincade
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Abstract |
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Keywords: bone marrow, dendritic cells, myeloid development
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Introduction |
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The developmental plasticity inherent to the DC system has been demonstrated in different studies, showing that both common lymphoid and common myeloid progenitors are able to differentiate into different types of DC (1416). The studies by Manz et al. and Mebius et al. indicated that both types of progenitors give rise to both CD8+ and CD8
DC in vitro and in vivo (14,15), while Izon et al. showed a previously unrecognized link between early B cell and DC ontogeny (16). Furthermore, recent experimental evidence suggests the existence of a common DC precursor in the BM (17) as well as in the peripheral blood (18) for CD8
+, CD8
and presumed PDC.
The studies cited above were mainly directed to define the origins of the different DC types and to identify their precursors in the BM. However, the identification of the different developmental stages of DC precursors in the BM still remains unclear. In this study, we have investigated in mouse BM the in vivo differentiation pathway of myeloid DC progenitors identified by differential ER-MP12/CD31 and ER-MP20/Ly-6C expression. In addition, we addressed the developmental relationship of myeloid DC precursors with macrophage precursors.
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Methods |
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mAb and conjugates
mAb used for cell sorting, flow cytometric analysis, immunocytochemistry and read out of T cell activation in the mixed leukocyte reaction (MLR) were either generated in our laboratory or obtained from BD PharMingen (Alphen aan de Rijn, The Netherlands). These were: biotinylated ER-MP12 (anti-CD31); FITCER-MP20 (anti-Ly6C); undiluted hybridoma culture supernatants ER-MP58 (19,20), ER-MP21 (anti-CD71/transferrin R) and M5/114 (anti-MHC class II), obtained in our laboratory; and 53-6.7FITC (anti-CD8), RM4-5PE (anti-CD4) and HL3phycoerythrin (PE) (anti-CD11c) purchased from BD PharMingen. Biotinylated ER-TR3 (anti-MHC class II) antibody was kindly provided by BMA Biomedicals (Augst, Switzerland). PEstreptavidin, TriColorstreptavidin and PEgoat anti-rat IgG (mouse absorbed) were used as conjugates (Caltag, San Francisco, CA).
Cell suspensions
Single-cell suspensions of BM, isolated from femora and tibiae, were prepared as described previously (20,21). Similarly, single-cell suspensions of spleen and lymph nodes were prepared and used for T cell isolation as described (22). Cultured BM-derived DC were isolated from Teflon culture bags or from culture plates. Cells were washed in PBS supplemented with 5% FCS (heat inactivated), 60 µg/ml penicillin and 100 µg/ml streptomycin when sterile suspensions were required. Prior to phenotypic analysis, cultured cells were washed with PBS supplemented with 0.5% (v/v) BSA (Organon Teknika, Boxtel, The Netherlands) and 20 mM sodium azide.
Cell sorting and flow cytometric analysis
For cell sorting, BM cells were labeled with two (ER-MP12 and ER-MP20) or three (ER-MP12, ER-MP20, and ER-MP58) mAb as described previously (20,21). Prior to sorting (FACS Vantage; Becton Dickinson, Amsterdam, The Netherlands), cell suspensions were filtered over a 30-µm sieve (Polymon PES; Kabel, Amsterdam, The Netherlands) to avoid clogging of the nozzle. After sorting, the purity of the cell suspensions was checked by re-running sorted samples. Purity was >95%, unless stated otherwise. Suspensions were kept at 4°C throughout the staining and sorting procedure. Sorted cells were counted in a Bürker hemocytometer.
For flow cytometric analysis, samples of cultured cells (2 x 104 cells) were aliquoted into 96-microwell plates (round bottom; Nunc, Roskilde, Denmark) and labeled with antibodies as described before (21). Samples were analyzed on a FACSCalibur flow cytometer using CellQuest analysis software (Becton Dickinson).
Immunocytochemistry on poly-L-lysine-coated slides
For immunocytochemical analysis, aliquots of cultured cells were placed at 37°C, 7% CO2 in complete medium for 1 h to adhere to poly-L-lysine-coated microwell slides (Nutacon, Schiphol-Oost, The Netherlands). Next, cell preparations were gently washed once with PBS (37°C), fixed in 1% paraformaldehyde in PBS for 10 min at room temperature, and washed with PBS and with PBS supplemented with 0.5% BSA. Cells were incubated with primary mAb, followed by rabbit anti-rat IgG conjugated to horseradish peroxidase (HRP; Jackson ImmunoResearch, West Grove, PA) (both incubations 30 min at room temperature). Diaminobenzidine (DAB; Sigma, St Louis, MO) was used as substrate. Between incubations, cell preparations were washed in PBS supplemented with 0.5% (v/v) Tween 20. After the DAB reaction was completed (35 min at room temperature), the cell preparations were dehydrated and coverslipped using Entallan (Merck, Darmstadt, Germany). Antibody reactivity was determined under a light microscope. Culture supernatant of the Y3 myeloma followed by the second stage antibody was used as negative control.
Growth factors
Conditioned medium of LADMAC cells was used as a source of mouse macrophage colony stimulating factor (M-CSF) (23). Conditioned medium was prepared as described elsewhere (19). Recombinant mouse GM-CSF (rGM-CSF) was purchased from Biosource International (Camarillo, CA).
rGM-CSF- and M-CSF-stimulated BM cultures
DC were generated by culturing total BM or isolated subsets in Teflon culture bags (24) or 24 well plates (Nunc) in RPMI-1640 (no HEPES; Biowhittaker, Verviers, Belgium) supplemented with 10% FCS (heat inactivated; 0.2 µm filtered), 60 µg/ml penicillin, 100 µg/ml streptomycin and 20 ng/ml rGM-CSF. Cells were cultured at 37°C, 7% CO2 for various periods of time, as indicated, for up to 14 days. Differentiation of isolated BM subsets, to macrophages, was induced by culturing in Teflon culture bags (24) in IMDM (with glutamax I; Gibco, Invitrogen BV, Breda, The Netherlands) supplemented with 20% FCS (heat inactivated; 0.2 µm filtered), 15% LADMAC-conditioned medium as a source of M-CSF, 100 µM 2-mercaptoethanol, 60 µg/ml penicillin and 100 µg/ml streptomycin. Cells were cultured for 4 or 5 days at 37°C, 7% CO2 as indicated.
MLR
In vitro generated DC (stimulator cells) and T cells (responder cells) isolated from the spleen and lymph nodes were resuspended in RPMI 1640 supplemented with 25 mM HEPES, 10% FCS (heat inactivated; 0.2 µm filtered), 60 µg/ml penicillin, 100 µg/ml streptomycin, 20 mg/l sodium pyruvate and 50 µM 2-mercaptoethanol (further referred to as MLR medium). T cells were resuspended at a concentration of 2 x 106/ml and the concentration of DC varied depending on the desired ratio of DC:T cells. The cells (100 µl of each cell suspension) were incubated in round-bottom 96-well plates for 4 days at 37°C, 7% CO2. DC and T cells, incubated separately in MLR medium, were used as negative controls. Mitogenic stimulation of T cells by concanavalin A (final concentration 1.25 µg/ml) (Sigma) was used as a positive control.
After 4 days of co-culture, cells were harvested and analyzed by flow cytometry. Triple labeling of cells with anti-CD4, anti-CD8 and ER-MP21 antibodies was performed, and 15,000 events within the living gate was acquired. Analysis was performed using CellQuest analysis software. The percentage of ER-MP21 (CD71/transferrin R)+ cells, as a measure of proliferating cells, was determined within the CD4+ and CD8+ population. This method of analysis provides comparable results to those obtained with the [3H]thymidine incorporation method (unpublished results) and in addition allows us to analyze, in more detail, the characteristics of the responding T cell population
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Results |
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Three ER-MP12/20-defined subpopulations represent successive maturation stages of DC precursors in the BM
Morphological and functional analysis of DC cultures showed that different BM subpopulations develop with different kinetics into similar DC, which suggests that precursors in these populations might be in a different maturation stage at the time of isolation. To investigate this, we sorted the ER-MP12hi20, ER-MP12+20+ and ER-MP1220hi subpopulations from the BM, stimulated them with GM-CSF in vitro, and analyzed the expression of ER-MP12 and ER-MP20 by cultured cells at various time points (Fig. 4).
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In the same cultures we followed the development of DC by using the DC marker CD11c. A common pattern of in vitro DC development was observed in cultures from all three fractions: first CD11c expression was induced followed by a prompt down-regulation of ER-MP20 (Fig. 5). CD11c+ cells were present already at day 2 in the culture derived from the ER-MP1220hi subpopulation. CD11c+ cells were also present at day 2 in cultures derived from the ER-MP12+20+ subpopulation, but at much lower frequency. The percentage of CD11c+ cells was increased in both cultures at day 5 and continued to rise in cultures derived from the ER-MP12+20+ subpopulation to reach 91% at day 8 of culture. In contrast, in cultures derived from the ER-MP12hi20 precursors, CD11c+ cells were present only from day 5 (10%) and reached 45% at day 8 of culture, implying that these precursors are indeed the earliest precursors able to differentiate into DC when stimulated with GM-CSF.
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The earliest ER-MP12hi20 DC precursors have the phenotype of myeloid-committed cells
Previously, we showed that the same ER-MP12/20-defined subpopulations in the BM contain macrophage precursors as we found now for DC precursors (21). In addition, we also showed that within the ER-MP12hi20 subpopulation, early myeloid-committed cells could be purified from the other hematopoietic differentiation capacities on the basis of differential expression of a third marker, ER-MP58 (20). Since we found that DC could also be generated from the ER-MP12hi20 BM subset, we asked whether the earliest DC precursors also have a myeloid-committed phenotype by examining their ER-MP58 expression. For this purpose, ER-MP12hi20 BM cells were fractionated according to their level of ER-MP58 expression (Fig. 6) and cultured in the presence of GM-CSF. Cultures were examined microscopically for the presence of DC at different time points. Starting from day 7 (Table 2), morphologically mature DC could only be detected in cultures of the ER-MP58hi subset within the ER-MP12hi20 population. This onset of DC appearing in culture was similar to what was observed when the total ER-MP12hi20 subset was cultured (see above). No DC-like cells could be grown from the ER-MP58med and ER-MP58/lo subsets under these culture conditions. Together, these data show that, like macrophage precursors, GM-CSF-responsive DC precursors present in the ER-MP12hi20 BM subset are characterized by a high level of ER-MP58 expression and thus have a myeloid-committed phenotype.
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After 4 days in M-CSF-stimulated culture of the ER-MP12hi2058hi subset, 90% of the cultured cells expressed the ER-MP20 antigen at a high level (Fig. 7A), representing BM monocytes as previously shown (21). Changing the growth stimulus to GM-CSF at this point resulted in the appearance of DC only 1 day later as determined by morphological inspection. The more mature ER-MP12+20+ subset developed further along the macrophage pathway in vitro, since after 5 days of M-CSF-stimulated culture approximately half of the cells still expressed the ER-MP20 antigen, whereas the other cells in the culture had lost their ER-MP20 expression (Fig. 7B). When these cells were further stimulated by GM-CSF, similarly to ER-MP12hi2058hi-derived cultures, DC were visible after only 1 day of secondary culture and in increased numbers after 3 days of culture. Apart from DC, macrophages (ER-MP20) also developed in cultures from both precursor fractions as indicated by the presence of cells with (i) high forward and perpendicular light scatter (data not shown), indicative of a complex cell type like the mature macrophage with its numerous vacuoles, etc., and (ii) a high autofluorescence level, also characteristic of mature macrophages. After 9 days of GM-CSF-stimulated secondary culture, 28 and 71% DC developed from ER-MP12hi2058hi and ER-MP12+20+ derived cultures respectively. These cells expressed MHC class II molecules at a high level and had typical DC morphology, as determined in immunocytochemistry (Fig. 7C and D). Thus, myeloid precursors from the BM developing for 4 or 5 days along the macrophage lineage in M-CSF-stimulated culture still have the potential to generate DC.
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Discussion |
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In this study we have examined the different stages of BM DC precursor development in vivo, as defined by the expression of ER-MP12/CD31, ER-MP20/Ly-6C and ER-MP58 antigens. DC are derived in vitro with GM-CSF only from the ER-MP12hi20 (early blasts), ER-MP12+20+ (myeloid blasts) and ER-MP1220hi (BM monocytes) (21) subpopulations, while no cell growth could be detected from the other three sorted populations, ER-MP12med20, ER-MP1220 and ER-MP1220med. These populations represent subsets containing morphologically identifiable precursors and mature cells of the other hematopoietic lineages (lymphoid, erythroid and granulocytic respectively). Our results show that GM-CSF-responsive DC precursors reside in the same ER-MP12/20-defined BM subsets as M-CSF-responsive macrophage precursors (1921).
Earlier reports show that mature DC develop from the BM within 8 days in GM-CSF-stimulated culture (27). In agreement with this we observe that the majority of DC derived from any of the BM subpopulations express significant amounts of MHC class II molecules, which, together with their good potential to stimulate allogeneic naive T cells in vitro, indicates that mature DC develop in these cultures. However, in cultures derived from the ER-MP12hi20 cells, immature DC, expressing medium levels of MHC class II, were also present in significant numbers after 7 days of culture. These cells mature upon LPS stimulation, suggesting that they need an extended period of time to develop into mature DC since they develop from early precursors.
This delay in reaching a mature state is in agreement with the observation that during the in vitro development, a difference in time of appearance of CD11c+ cells exists in cultures grown from the ER-MP12hi20 versus the ER-MP12+20+ and ER-MP1220hi BM subsets (day 5 versus 2 respectively). This further implies that ER-MP12hi20 DC precursors are less mature than the other two subsets, and need more time to develop into DC than ER-MP12+20+ and ER-MP1220hi cells. In addition, the percentage of CD11c+ DC derived from the GM-CSF-stimulated ER-MP12+20+ cells is 2-fold higher than that found with the ER-MP12hi20 BM subset as a starting population (90 versus 45%). Probably the higher proliferative capacity of the ER-MP12hi20 population contributes to the observed difference in maturation kinetics. Both the size and extent of colony/cluster formation and culture recoveries indicate that ER-MP12hi20 cells have the highest potential to proliferate, which then decreases to be the lowest among ER-MP1220hi cells. The high recoveries obtained from the ER-MP12hi20 and ER-MP12+20+ fractions make it highly unlikely that contaminating populations (<5%) in the sorted fractions account for the observed results. Together, these findings reinforce the notion that ER-MP12hi20, ER-MP12+20+ and ER-MP1220hi populations represent different stages of DC development, since they show a reciprocal relationship between the ability to proliferate and the ability to develop into mature DC in a short period of time.
The successive expression of ER-MP12 and ER-MP20 antigens during the in vitro culture with GM-CSF strongly suggests that the three DC precursor populations, which we defined in the BM, differentiate along the pathway with the order ER-MP12hi20 ER-MP12+20+
ER-MP1220hi. Furthermore, this same sequence was previously shown for the macrophage precursors (21) suggesting that GM-CSF-responsive myeloid DC and macrophage precursors follow a similar ER-MP12/20-defined developmental pathway.
The population that contains the earliest DC precursors in the BM, ER-MP12hi20, can be further divided into three subpopulations based on the expression of the early myeloid marker ER-MP58 (20). Of these three subsets, only ER-MP12hi2058hi BM cells are able to differentiate into DC in rGM-CSF-stimulated cultures. Since high level expression of ER-MP58 within the ER-MP12hi20 BM population has been shown to mark the earliest myeloid committed precursors, able to give rise to macrophages with M-CSF (20), we conclude that macrophages and myeloid DC most probably originate from the same precursors in the BM. Recently, Akashi et al. have described the existence of a common myeloid progenitor in mouse BM that gives rise to all myeloid lineages (10). The comparison of its colony forming ability and further characteristics with those of the ER-MP12hi2058hi population strongly suggests that the latter represents a similar, if not identical, multipotent myeloid precursor population.
In a recently published paper (7), Gilliet et al. show that the mouse PDC (CD11c+CD11bB220+) can be generated in vitro from the total BM, when Flt-3L is used as the growth factor. They also show that addition of GM-CSF to the culture medium completely blocks generation of PDC and increases myeloid DC development. Since we used GM-CSF alone, this is in agreement with the observation that in our cultures, no plasmacytoid, but only myeloid DC, were generated as all cells in the culture expressed high levels of CD11b (data not shown). An interesting question arises regarding the potential of our three populations to generate PDC with Flt-3L. For the BM monocyte population (ER-MP1220hi) we regard it unlikely that they may give rise to lymphoid-related PDC, as we have shown previously that BM monocytes are all CD11b+ and thus myeloid cells (21), Further, it has been reported that the CD31+/Ly-6C+ (i.e. our ER-MP12+20+) population contains precursor cells capable of differentiating into different cell types: CD8+ DC, CD8
DC, PDC and macrophages, depending on the experimental conditions (17). Our current results support the concept that developing myeloid DC, which might eventually express CD8
, and macrophages progress through the same precursor stages in the BM including the CD31+/Ly-6C+ stage. However, as we have shown earlier, the CD31+Ly-6C+ population in BM is phenotypically heterogeneous (21) and might contain cells with different developmental capabilities; clonal assays are essential to prove whether PDC and myeloid DC derive from single precursors. Finally, we define the DC precursor, at the stage prior to the one previously reported, which is capable of differentiating in both myeloid DC and macrophages (CD31hi/Ly-6C). As mentioned above, since GM-CSF directs cell development specifically to myeloid lineage, it is unclear from our data whether PDC might originate from the same population.
Taken together, in this report, we define different stages of GM-CSF-responsive DC precursors present in the BM and show their in vivo developmental pathway. Furthermore, we show that, throughout their BM development, myeloid DC precursors progress through the same stages as macrophages. Even when cells are induced in vitro with M-CSF to follow the macrophage developmental pathway, they can still be redirected to develop into DC when M-CSF is replaced with GM-CSF. This is in line with our hypothesis that over a prolonged stretch of development, progenitors of the mononuclear phagocyte system may either develop into macrophages or DC, depending on local conditions. However, additional clonal experiments are required to confirm this hypothesis. Finally, we feel that the ER-MP12/20/58 identification of distinct BM subsets provides a powerful means to study the developmental pathway of lymphoid-related PDC as opposed to the presently studied myeloid DC.
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Acknowledgements |
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Abbreviations |
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DCdendritic cell
Flt-3LFlt-3 ligand
GM-CSFgranulocyte macrophage colony stimulating factor
LPSlipopolysaccharide
M-CSFmacrophage colony stimulating factor
MLRmixed lymphocyte reaction
PDCplasmacytoid dendritic cell
PEphycoerythrin
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References |
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