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Stem Cells, Vol. 15, No. 2, 144-153, March 1997
© 1997 AlphaMed Press

Contrasting Effects of TGF-ß1 and TNF-{alpha} on the Development of Dendritic Cells from Progenitors in Mouse Bone Marrow

Yasunori Yamaguchia,b, Haruhiko Tsumuraa, Mitsuru Miwab,c, Kayo Inabab

a Pharmaceutical Research Laboratory, Kirin Brewery Co. Ltd., Gunma, Japan;
b Department of Zoology, Graduate School of Science, Kyoto University, Sakyo, Kyoto, Japan;
c Department of Dermatology, Graduate School of Medicine, Kyoto University, Sakyo, Kyoto, Japan

Key Words. Maturation • Inhibition • B7-2 • MHC class II • ICAM-1 • Stimulatory activity

Dr. K. Inaba, Department of Zoology, Graduate School of Science, Kyoto University, Sakyo, Kyoto 606-01, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Dendritic cells (DC) are a distinct population of leukocytes and specialized antigen-presenting cells for T cell responses. Prior work has shown that GM-CSF can induce the development of large numbers of DC from proliferating progenitors in mouse bone marrow. We have monitored the effects of potentially enhancing and suppressive cytokines in these cultures. In this system, many immature DC develop from proliferating precursors during the first six days of culture, and between days 6-8 maturation of typical nonadherent and nonreplicating DC takes place. The maturation is accompanied by a large increase in the expression of major histocompatibilities complex class II (MHC II) and B7-2/CD86, and in mixed leukocyte reaction stimulating activity. Tumor necrosis factor-{alpha} (TNF-{alpha}), previously shown to be required for development of human DC, was found to enhance the maturation of mouse DC in the last two days of culture. Transforming growth factor-ß1(TGF-ß1), on the other hand, almost totally blocked DC maturation, but it had to be given in the first six days of culture when the DC were actively proliferating. TGF-ß1 did not block the production of immature, MHC II-positive but B7-2/CD86-negative DC. Maturation would take place between days 6-8 as long as the cultures were depleted of Fc-receptor-bearing cells, or if TNF-{alpha} were added. In both instances, maturation was not blocked even when TGF-ß1 remained in the culture. We conclude that the development of DC, in response to GM-CSF, can be modified by other cytokines. TGF-ß1 is suppressive but only indirectly via Fc-receptor-bearing suppressive cells, presumably suppressive macrophages, while TNF-{alpha} enhances the final maturation of DC.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Dendritic cells (DC) are distinctive leukocytes that are specialized antigen-presenting cells (APC) for T cell mediated immune responses [1]. It is well known that DC are derived from bone marrow myeloid progenitors. In mouse bone marrow suspensions, DC development can be induced over a seven- to eight-day culture period by applying GM-CSF [2]. Mixed colonies of DC, granulocytes and macrophages are evident in semisolid culture systems with GM-CSF [3]. A prior method that described the DC-stimulating activity of GM-CSF also described some approaches for separating the DC progeny from granulocytes and macrophages [4]. Although only a trace number of DC could be obtained from normal tissues with many processing steps, these methods enabled many people to generate highly purified and large numbers of DC for different immunological studies.

In the development of DC in mouse bone marrow, however, there are two important unknowns. First is the role of tumor necrosis factor-{alpha} (TNF-{alpha}) which was clearly shown to enhance the development of mature DC, together with GM-CSF, from human progenitors [5, 6]. In the murine system, the effect of TNF-{alpha} has mainly been shown for the maturation of Langerhans cells [7-9] and their emigration from the epidermis [10-12], but there is little information on DC from bone marrow progenitor cells. Second is the information of suppressive cytokines for the development of DC. This information is the clue to understanding the negative regulation of the development of DC from hematopoietic progenitors which has significant influence on the T cell-mediated immunity in the body.

As a suppressive cytokine, we chose transforming growth factor-ß1 (TGF-ß1) for several reasons: TGF-ß1 has been found in bone marrow and fetal liver where active hematopoiesis occurs [13] and it is known to affect differentiation and proliferation of hematopoietic progenitors, including the differentiation of granulocytes and macrophages that is induced by GM-CSF [14, 15]. This cytokine is also a potent immunosuppressive agent. Although the latter is mainly ascribed to T cells, a few studies have described the capacity of TGF-ß1 on the function of Langerhans cells [9, 16, 17]. However, there have been no studies of the effects of TGF-ß1 on DC development from less mature progenitors.

Here we report that TNF-{alpha} and TGF-ß1 can increase and decrease DC development, respectively, in mouse marrow cultures along with GM-CSF. In both cases, the cytokines can affect the final maturation stages of DC development rather than the initial proliferative steps. The inhibition by TGF-ß1 seems to be exerted indirectly via Fc-bearing cells, presumably suppressive macrophages. TNF-{alpha} can not only stimulate the maturation of DC, but can also overcome the suppressive effect of TGF-ß1.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice
Female BALB/C and C57BL/6 mice, seven- to eight-weeks-old, were purchased from Charles River Japan Inc. (Kanagawa, Japan), and BALB/C x DBA/2 F1 (CD2F1) from Japan SLC (Hamamatsu, Sizuoka, Japan).

Reagents
The culture medium was RPMI 1640 (GIBCO Laboratories; Grand Island, NY) supplemented with 10% fetal calf serum, 50 µM 2-mercaptoethanol and 20 µg/ml gentamicin. Purified recombinant murine GM-CSF and purified recombinant human TGF-ß1 were produced in a laboratory of Kirin Brewery (Gunma, Japan). Purified recombinant mouse TNF-{alpha} was purchased from Genzyme (Cambridge, MA) or was a generous gift from Dainippon Pharmaceutical Co., Ltd. (Osaka, Japan). Purified human {gamma} globulin (Organon Teknika Corp., Durham, NC) was used to pan Fc-receptor-positive (FcR+) cells.

Bone Marrow Cultures
A modification and combination of the methods of Inaba et al. [2] and Chen-Woan et al. [18] were used. In brief, marrow cells were flushed from the femurs of BALB/C or CD2F1 mice. Red cells were lysed with ammonium chloride and the cells washed twice with RPMI 1640. FcR+ cells were depleted by panning on dishes coated with human {gamma} globulin, or the cells were treated with a cocktail of monoclonal antibodies (mAbs) plus rabbit complement to remove CD4+, CD8+, MHC class II+ and B220+ cells; 0.5-1.0 x 106 cells were placed in 24-well plates (Nunc; Naperville, IL) in 1 ml medium supplemented with 10 ng/ml GM-CSF in the presence or absence of 100 ng/ml TGF-ß1. The culture medium was changed every two days by gently swirling the plates, aspirating 75% of the medium, and adding back fresh medium with cytokines. During these washes many of the growing granulocytes are removed. On day 6 the cells were dislodged by gently pipetting. The cells were then cultured in bulk, or following removal of FcR+ cells by panning. Only about 25% of the cells were recovered from the pans. The cells were then cultured an additional two days with 10 ng/ml GM-CSF, with or without 250 U/ml TNF-{alpha}, to allow further maturation of the DC. Most of the cells in the culture could then be dislodged by Pasteur pipetting and examined for cell surface markers and mixed leukocyte reaction (MLR) stimulating activity. Mature DC are cells with high levels of major histocompatibility (MHC) class II and B7-2/CD86, and strong MLR stimulating activity.

Phenotyping with mAbs
Two-color immunofluorescence was used to phenotype the cells at days 6 and 8 of marrow culture. Immature and mature DC stain moderately and intensely, respectively, with a biotin-modified mouse anti-I-Ad mAb (AMS-32.1, Pharmingen; San Diego, CA). The surface markers of these two groups of DC were determined with a panel of hybridoma culture supernatants (obtained from the American Type Culture Collection; ATCC; Rockville, MD). Control rat IgG2a was from Jackson ImmunoResearch Laboratories (West Grove, PA). The mAbs were applied at saturating levels for 30 min in phosphate-buffered solution-1%, fetal calf serum-0.02% azide on ice, washed, and then stained with fluorescein isothiocyanate (FITC)-mouse antirat IgG (Boehringer-Mannheim Biochemicals, Indianapolis, IN) for 30 min, washed and stained with the mouse anti-MHC II mAb followed by phycoerythrin (PE)-streptavidin (Tago Inc.; Burlingame, CA). Flow cytometry was carried out on a FACScan instrument (Becton Dickinson; Mountain View, CA), which had been calibrated with Becton Dickinson Calibrate Beads. Viable cells were selected using forward and side scatter analysis.

MLR
Cells from the bone marrow cultures were treated with mitomycin C (Sigma; St. Louis, MO) at 50 µg/ml for 30 min at 37°C, washed twice with RPMI 1640, and applied in graded doses to 2 x 105 allogeneic T cells in 96-well flat-bottomed culture plates for three days. The T cells were prepared by using mouse T cell enrichment columns (R&D Systems; Minneapolis, MN). Eighty-five percent to ninety-five percent of these cells were CD3+. The MLR was pulsed with 2.5 µCi/ml final concentration of [3H]thymidine (0.74-1.1 Tbq/mmol, Amersham) for 8 h prior to harvesting. The DNA synthesis values are expressed as mean cpm with standard deviations of triplicate cultures.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Experimental Design
Prior work [2] has described the capacity of GM-CSF to generate large numbers of myeloid cells, including DC, from mouse marrow progenitors. Briefly, to avoid contaminating B cells and other mature leukocytes, cultures of marrow cells that have been treated with antibodies to MHC class II, B220, CD4, CD8 and rabbit complement must be set up. By the fourth day, granulocytes begin to form in large numbers, but the majority are nonadherent and can be rinsed away. This reveals, attached to the culture surface, clusters of growing DC as well as more dispersed colonies of growing macrophages. By day 6, the DC clusters are numerous and large, and contain many cells incorporating [3H]thymidine [2]. Some single mature DC have begun to float free from the clusters at day 6. At this time, the many large clusters of growing cells are dislodged by Pasteur pipetting and transferred to a large petri dish. The total yield of cells at day 6 is roughly 100%-110% of the starting number of marrow cells that were plated, but large numbers of granulocyte progeny have been washed away (see Materials and Methods). Over the next two days, many single nonproliferating, nonadherent mature DC are released.

We used fluorescence-activated cell sorter (FACS) analyses and MLR-stimulating activity to assess the role of cytokines on the production of mature DC. The cytokines were added either during the initial six days of the culture when large numbers of aggregates of growing DC appear, or in the final two days (days 6-8) when the aggregates mature into typical DC. The antibodies that produce the strongest staining of mature DC are mAbs to MHC class II, B7-2/CD86 and ICAM-1/CD54. Also, mature DC are potent stimulators of the primary allogeneic MLR. As discussed in the Introduction, TGF-ß1 and TNF-{alpha} were of interest to us.

FACS Analyses for ICAM-1 and B7-2 Expression by Marrow Progeny Cultured in the Presence of GM-CSF and either TGF-ß or TNF-{alpha}
In Figure 1Go, we show typical one dimensional histograms for B7-2 and ICAM-1 staining of the MHC class II+ cells. These costimulator molecules are expressed at very high levels on mature DC [1, 19-21]. At the top of the figure are the results at day 6, i.e., when most of the cells are in large balls of proliferating, immature DC. The level of ICAM-1 is high on all the cells in the bulk population, but only a small fraction of the cells are B7-2high (Fig. 1AGo). If TGF-ß1 was present during the first six days of the culture, the total yields of cells were slightly higher than that with GM-CSF alone, but the staining for ICAM-1 and B7-2 was uniformly low (Fig. 1GGo). The FACS profiles of Fc-receptor-negative (FcR–) cells, which had been panned on Ig-coated plates, are also shown in Figure 1Go. The FcR– GM-CSF-treated populations had an increased frequency of B7-2high cells because of the depletion of FcR+, B7-2low cells during panning, but the staining was otherwise similar to nonpanned cells (Fig. 1A versus 1DGo). When the FcR– population was prepared from cells generated in the presence of GM-CSF and TGF-ß1, no differences were observed in the staining patterns for B7-2 and ICAM-1, but the cells with high autofluorescence intensity were reduced (Fig. 1JGo)



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  		Figure 1. Expression of ICAM-1 and B7-2 in MHC II+ cells produced by adding GM-CSF to marrow suspensions. The cells were cultured six days in the absence (left, A-F) or presence (right, G-L) of TGF-ß1, and were examined either as bulk populations (A-C and G-I) or following depletion of FcR+ cells on Ig-coated plates (D-F and J-L). The cells were washed and cultured an additional two days with GM-CSF in the absence or presence of TNF-{alpha}. In each case, the cells were double-labeled for MHC class II (see Figures 2 and 3GoGo legends below) and for either ICAM-1 or B7-2. The histograms for the expression of these costimulators are shown for MHC class II+ cells.

 
The aggregates of proliferating DC were then dislodged and cultured two more days either in GM-CSF or GM-CSF + TNF-{alpha}. TNF-{alpha} by itself did not support DC development unless GM-CSF continued to be present (data not shown). TGF-ß1 was omitted from the day 6-8 cultures. It can be seen that only in GM-CSF there was a slight increase in ICAM-1 staining, and a slight increase in the frequency of B7-2high cells (Fig. 1A versus 1BGo). However, the addition of TNF-{alpha} substantially increased the frequency of cells with the mature DC phenotype, i.e., very high levels of ICAM-1 and B7-2. (Fig. 1CGo) If the marrow cells were cultured in TGF-ß1 initially, then there was a reduced yield of cells with high ICAM-1 or B7-2 (Fig. 1HGo). However, if FcR+ cells were removed at day 6, the remaining cells generated large numbers of presumptive mature DC even with GM-CSF alone (Fig. 1KGo), and a significant increase in the frequency of mature DC was noted upon the addition of TNF-{alpha} (Fig. 1LGo). Furthermore, the addition of TNF-{alpha} resulted in a significant increase in the frequency of ICAM-1high or B7-2high cells even in cultures without prior depletion of FcR+ cells (Fig. 1IGo). Therefore, the FACS studies suggested that TNF-{alpha} increased the yield of mature DC, and that TGF-ß1 induced a block in DC maturation that could be reversed, especially by removing FcR+ cells.

FACS Analysis for MHC Class II Expression by Marrow Progeny Cultured in the Presence of GM-CSF and either TGF-ß1 or TNF-{alpha}
Figure 2Go shows two color plots of MHC class II versus B7-2 staining of the different populations at day 6. Mature DC typically had mean fluorescence intensities of about 3,000 for MHC II and about 300 for B7-2. If the cells were cultured in GM-CSF only for six days, then there was only a small fraction (<15% of the cells) that had the typical mature DC phenotype (arrowheads in Fig. 2BGo). If the cells had been cultured in GM-CSF + TGF-ß1 for six days, then the levels of MHC II were lower, and high B7-2 expressing cells were rare (arrowheads in Fig. 2FGo). Therefore, consistent with the results in Figure 1Go, TGF-ß1 seemed to suppress the GM-CSF-induced maturation of DC. To show the evidence that TGF-ß1 works as a direct suppressor for GM-CSF-driven mature DC formation, the effect of anti-TGF-ß antibody was evaluated. The proportion of MHC-class IIhigh/B7-2high (mature DC) was 13.5% in GM-CSF (10 ng/ml) and 3.9% in GM-CSF (10 ng/ml) + TGF-ß1 (5 ng/ml), while it was 15.1% in GM-CSF + TGF-ß1 preincubated with pan-specific TGF-ß neutralizing antibody (62.5 µg/ml, R&D Systems). Thus, this antibody completely blocked the effect of TGF-ß1. According to the manufacture's instructions, the concentration of this antibody required to yield one-half maximal inhibition of porcine TGF-ß1 (0.25 ng/ml) in the assay using the TGF-ß-responsive cell line, HT-2, was 5 µg/ml. As the inhibitory effect of TGF-ß1 disappeared by the pretreatment with anti-TGF-ß neutralizing antibody, it was shown that the observed inhibition was caused by TGF-ß1 itself, but not by the potential contaminants in TGF-ß1 reagent.



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  		Figure 2. Expression of MHC II and B7-2 on bone marrow cells cultured for six days with GM-CSF in the absence (left, A-D) or presence (right, E-H) of TGF-ß1. Bulk cells, or populations depleted of FcR+ cells by panning on Ig-coated plates, were double labeled with anti-MHC II and a second antibody with FITC-anti-Ig, either a nonreactive isotype control or GL-1 mAb to B7-2/CD86. Mature DC (arrowheads) have very high levels of MHC class II and B7-2. Macrophages (low MHC II, high autofluorescence; arrows) are depleted by FcR panning.

 
If cells were cultured in GM-CSF with or without TGF-ß1 and then panned on Ig-coated plates, the disappearance of a small subset of cells (arrows in Figs. 2E and 2FGo) that had low MHC II and high autofluorescence was seen. We presume that these cells are macrophages.

Figure 3Go shows two color plots of MHC class II versus B7-2 staining of the different populations following culture with GM-CSF from days 6-8. If the cells had been cultured from days 0-6 in the absence of TGF-ß1 (left half of Fig. 3Go), then the yield of cells with the mature DC phenotype (high MHC II and B7-2) is about 40% (arrowheads, Fig. 3BGo). Most of the other cells have moderate MHC II and B7-2low, as is typical of immature DC [20]. When we prepare mature DC at day 8, one readheres the population to plastic and this depletes the immature DC [2]. If the cells were FcR panned, then the yield of mature relative to immature DC increased (Fig. 3D versus 3BGo).



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  		Figure 3. Expression of MHC II and B7-2 on bone marrow cells cultured in GM-CSF two additional days, days 6-8. Marrow cells were cultured in GM-CSF for six days in the absence (left, A-D) or presence (right, E-H) of TGF-ß1. The cells were then cultured from days 6-8 in the absence of TGF-ß1 but in the presence of GM-CSF. The cultures were of bulk cells or FcR+-depleted populations. The production of mature DC (arrowheads) was monitored by double labeling with PE-anti-MHC class II and FITC antibody to B7-2/CD86.

 
If the cells had been cultured from days 0-6 in the presence of TGF-ß1 (right half of Fig. 3Go), then the yield of cells with the mature DC phenotype (high MHC II and B7-2) was very small (arrowheads in Fig. 3FGo). Most of the cells had low MHC II and B7-2, as is typical of immature DC [20]. However, if the TGF-ß1-treated cells were FcR panned, then the yield of mature relative to immature DC increased markedly (Fig. 3H versus 3FGo).

Table 1Go summarizes the production of MHC class IIhigh cells (which are also B7-2 high, see Fig. 3Go) under all the different culture conditions. These cells are recognized as mature DC (1, 19-21). The yield of cells was not substantially different for all groups of panned (FcR–) and nonpanned (bulk) cells. It is clear that exposure to TGF-ß1 greatly reduced the yield of MHC II (or Ia)-positive and MHC IIhigh cells (compare the first and fourth groups of data). In contrast, exposure to TNF-{alpha} increased the yield of MHC IIhigh cells (Iahigh/total Ia). The enhancing effects of TNF-{alpha} were particularly striking in the cultures that had been treated with TGF-ß1 during the first six days.


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  		Table 1. Effects of TGF-ß1 and TNF-{alpha} on the frequency of MHChigh (IAhigh) DCs
 
In Figure 4Go we tested to see if the enhancing effect of TNF-{alpha}, or the suppressive effect of TGF-ß1, would predominate when the two cytokines were added together. To monitor DC maturation, we show staining for ICAM-1 and B7-2 for all the MHC class II cells in the culture. The day 6 cultures that had been in TGF-ß1 (top) had only trace numbers of mature DC. If these cells were then cultured two more days in GM-CSF with or without continued TGF-ß1, there was only a small incidence of ICAM-1high cells, and almost no increase in B7-2 expression (Fig. 4A versus 4B or 4CGo). The increased expression of not only ICAM-1 but also B7-2 was evident in the FcR panned population (Fig. 4B versus 4F and Fig. 4C versus 4GGo). It is suggested that TGF-ß1 had almost no effect on the maturation of DC committed precursors (immature DC). However, if TNF-{alpha} was added to the GM-CSF, again with or without continued TGF-ß1, the incidence of cells with high B7-2 and ICAM-1 increased markedly in the bulk population (Figs. 4D and 4EGo). Therefore, it appears that TNF-{alpha} is the dominant cytokine in mediating the development of a mature DC phenotype. These results indicate that TNF-{alpha} can overcome the suppressive effect of TGF-ß1 mediated by FcR+ cells.



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  		Figure 4. Effect of TNF-{alpha} on the expression of costimulatory molecules on bone marrow cells cultured in GM-CSF. Marrow cells were cultured in GM-CSF and TGF-ß1 for six days (A). At day 6, the cells were washed and recultured for two days in GM-CSF and the indicated cytokines. Both bulk cells (B-E), and FcR– cells (F-I), were studied. Shown are the fluorescence histograms for B7-2 and ICAM-1 staining of MHC class II+ cells.

 
MLR Stimulating Activity of Marrow Progeny that Have Been Exposed to both GM-CSF and other Cytokines, TGF-ß1 and TNF-{alpha}
We harvested the cells from marrow cultures that had been exposed to different combinations of cytokines, and tested their stimulating activity in the primary allogeneic MLR by applying them in graded doses to a constant number of 200,000 enriched allogeneic T cells. First we tested the effect of TGF-ß1 when added at varying times in the first six days of culture. GM-CSF was present throughout the six days. It is apparent that TGF-ß1 suppressed the development of MLR stimulating activity, but only if added during the first four days of the culture (Fig. 5AGo). When a dose response of the effect of TGF-ß1 was carried out using the presumptive DC in the six-day culture, significant blocking was seen with just 1 ng/ml TGF-ß1 (Fig. 5BGo). Consistent with the results on DC maturation using cell surface phenotype (above), the reduced MLR stimulating activity of TGF-ß1-treated cells was in large part reversed by the combination of FcR panning and TNF-{alpha} treatment between day 6 and day 8 (Fig. 5CGo).



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  		Figure 5. Primary MLR-stimulating activity of marrow cells following culture under different conditions. Marrow cells were cultured with GM-CSF in the absence or presence of 100 ng/ml TGF-ß1 for different times of the 6-day culture period (A) or varying doses (0.1-100 ng/ml) of TGF-ß1 for six days (B). At day 6, the cells were washed, panned and recultured for two days in GM-CSF with TNF-{alpha} (C). To remove suppressive macrophages and contaminating granulocytes, bulk cells were panned on Ig-coated plates before the onset of MLR culture. The graded dose of developing cells was added to 200,000 allogeneic purified T cells in 96-well flat-bottomed culture plates for three days. The MLR were pulsed with [3H]thymidine for 8 h prior to harvesting. DNA synthesis values are expressed as mean cpm with standard deviations for triplicates. A) The effect of adding TGF-ß1 at varying times in the first six days of culture. B) The effects of different doses of TGF-ß1 in the first six days of culture. C) The reversibility of the TGF-ß1 block in DC maturation.

 
Surface Phenotype of DC Generated with Different Cytokines between Days 6-8
As shown above, the yield of MHC II-high and B7-2-high DC was enhanced by TNF-{alpha}. Using a panel of mAbs, we tested if the surface markers of the DC were different depending on the cytokines that were applied. The phenotype of cells cultured with GM-CSF + TNF-{alpha} + TGF-ß1 is shown in Figure 6Go. The cell surface of the immature and mature DC was identical in bulk and FcR panned cells that had been cultured from days 6-8 in GM-CSF only, GM-CSF + TNF-{alpha}, GM-CSF + TNF-{alpha} + TGF-ß1, and GM-CSF + TGF-ß1 (not shown). Relative to the MHC II-moderate DC, the MHC II-high mature DC had higher levels of MHC I, B7-1, B7-2, ICAM-1, CD44, VLA-4/CD49d and DEC-205, and lower levels of 2.4G2 Fc receptor, Mac-1, and F4/80. Both types of DC had comparable levels of CD45 and heat stable antigen, with little or no CD4, CD8, B220 or Gr-1.



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  		Figure 6. Cell surface phenotype of marrow cells (mainly DC) following culture between days 6-8 in a combination of GM-CSF, TNF-{alpha} and TGF-ß1.The marrow cells were harvested and stained for MHC class II (vertical axis, phycoerythrin-label) and for the different surface antigens indicated in each panel (horizontal axis, FITC-label). There are four sets of cells that can be identified on the basis of the level of MHC II staining. MHC II– cells are primarily granulocytes (see Gr-1 panel). MHC IIlow cells are likely to be macrophages (see Mac-1 panel). MHC IImoderate and MHC IIhigh cells are the immature and mature DC, respectively.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The production of mature DC from marrow progenitors provides an important means for increasing an individual's APC function for T cell-mediated immunity. Here we have considered cytokines that can increase or decrease the output of mature DC in a previously described mouse marrow suspension culture system. In this system, GM-CSF is the only exogenous cytokine that is added to observe the development of mature DC. The most critical feature of the system is to remove contaminating cells. Most granulocytes that are produced in the culture can be rinsed away because they are nonadherent. Most macrophages remain firmly affixed to the culture surface. Most B cells, which represent a major component of the starting marrow suspension, are best removed initially by depletion with antibody and complement. If not, a significant number of B cells persists in the final preparation (10%-20% of the cells) in spite of numerous washes, presumably because B cells are able to stick significantly to plastic or to adherent macrophages and stromal cells.

In the marrow culture system, the mature progeny DC are large, nonreplicating, nonadherent, motile cells that extend large lamellipodia or veils. The surface is rich in the molecules required for effective presentation to T cells, especially very high levels of MHC II, ICAM-1 or CD54, and B7-2 or CD86. Here we observed the development of mature DC simply by looking for the appropriate morphology by inverted phase contrast microscopy. However, better quantitative data were obtained using the FACS and MLR stimulating activity. Using these assays, we were able to show a significant enhancing effect of TNF-{alpha} on DC maturation, and a significant inhibitory effect of TGF-ß1. It is to be stressed that these cytokines did not alter the total yield of mononuclear cells in the marrow cultures. However, the state of maturation of the DC could be altered markedly. TNF-{alpha} from days 6-8 increased the yield of cells with very high MHC II and B7-2, i.e., typical mature DC, and TGF-ß1 greatly reduced the yield of mature DC when applied early, days 0-6.

The enhancing effect of TNF-{alpha} on DC maturation was predicted from prior studies. Caux and Banchereau reported that both GM-CSF and TNF-{alpha} were required to generate mature DC from CD34+ progenitors in human cord blood [5]. Young et al. made similar observations with CD34+ progenitors in human marrow [3, 6]. Sallusto and Lanzavecchia induced DC development from progenitors in adult human blood with GM-CSF and interleukin 4. They found that the cells with some properties of immature DC could be induced to mature with TNF-{alpha} to gain potent stimulatory activity in allogeneic (allo)-MLR [22]. However, all of these studies have been done in human cells. The epidermal Langerhans cells are mainly used to examine the effect of TNF-{alpha} in murine system, and little information is available so far in mouse DC progenitors.

We find that TNF-{alpha} likewise increases the maturation of DC in mouse marrow especially between days 6-8, when most of the maturation of DC takes place. It remains to be seen if endogenous TNF-{alpha} is contributing to the development of DC when exogenous GM-CSF only, but not TNF-{alpha}, was used in these cultures, since GM-CSF has been reported to induce TNF-{alpha} production in a murine macrophage cell line [23]. Studies with neutralizing anti-TNF- antibodies, or with TNF- receptor knockout animals, should resolve this issue. In all cases, however, it is essential that GM-CSF be added, not just TNF-{alpha}, to observe the development of mature DC. This combination, of course, only represents one known combination for DC development; others are under study.

The suppressive effect of TGF-ß1 on the development of DC in response to GM-CSF was profound, with an almost complete block of mature DC development. Keller et al. [14] reported that TGF-ß1 enhances GM-CSF-induced colony formation and shifts production of progenitors to granulocytes. A dual activity of TGF-ß1 on colony-forming units-granulocyte/macrophage (CFU-GM) has also been described, i.e., an inhibition of early CFU-GM and a stimulation of late CFU-GM [24]. Fan et al. documented a stimulation by TGF-ß1 on the GM-CSF, but not M-CSF, that induced proliferation of bone marrow-derived macrophages by enhancing the expression of GM-CSF receptors [25]. We observed an increase in granulocytes in our cultures with TGF-ß1, and that TGF-ß1 also induced a substantial development of weak MHC II+ cells. The latter observation suggests that TGF-ß1 does not block the differentiation of bone marrow progenitor cells to DC. The critical effect of TGF-ß1 was on DC maturation. Recently, after the submission of this paper, Strobl et al. [26] reported that TGF-ß1 promoted GM-CSF, TNF-{alpha} and stem cell factor-induced DC development in serum-free culture condition from CD34+ hematopoietic progenitors in human cord blood. Although the system is totally different from ours, their observation of TGF-ß1 "promotion" of DC development seems to be opposite from our findings. Their DC developed by the addition of TGF-ß1, however, are CD1a+ and have significant but not strong alloMLR stimulatory activity. These characters coincide with typical fresh Langerhans cells (immature DC). As we discussed above, our findings are that TGF-ß1 does not suppress the differentiation of immature DC but does the maturation of DC. In addition, the block could be also reversed either by removing a suppressive cell type with FcR panning, or by adding TNF-{alpha} (Figs. 1 and 3GoGo). Both reversals could take place in the continued presence of TGF-ß1 (Fig. 4Go). As Strobl et al. added TNF-{alpha} in culture, it is difficult to conclude the effect of TGF-ß1 on DC development as suppressive or promotive. It will be necessary to elucidate the effect of TGF-ß1 from the viewpoint of mature versus immature DC.

Grusschwitz and Hornstein suggested that TGF-ß1, which is produced by Langerhans cells, might keep the cell in an immature stage [17]. However, TGF-ß1 did not act directly on the DC as discussed above, but instead seemed to activate FcR+ cells to inhibit DC development in our experimental system. This discrepancy may be due to the culture condition with excess presence of GM-CSF for the DC development. The cells removed by FcR panning expressed low MHC class II and high autofluorescence on FACS analyses (Fig. 2Go). A major population remaining on the Ig-coated plate was firmly adherent to the plastic surface. They were highly phagocytic to Latex beads and carbon particles, and were strongly positive for nonspecific esterase activity (data not shown). Therefore, we suggest that suppressor cell mediating inhibitory effect of TGF-ß1 is most probably a macrophage. It is interesting to note that TGF-ß1 can induce a macrophage suppressor activity [27, 28] much as we postulate it may occur in our cultures. TGF-ß1 gene knockout mice have multifocal inflammatory disease [29, 30], which suggests a role for TGF-ß1 in suppressing the immune response. Our observation of reduced DC maturation as a result of the effects of TGF-ß1 suggests another component to the well-known immunosuppressive effects of this cytokine.

Depletion of FcR+ cells resulted in the spontaneous induction of DC maturation with GM-CSF irrespective of the presence or absence of TGF-ß1 (Fig. 4Go). Allogeneic T cell proliferations by DC that develop in the continued presence and absence of TGF-ß1 from FcR– cells from days 6-8, were 191,500 ± 13,200 cpm and 223,100 ± 21,400 cpm at the ratio of 40:1, respectively. Thus DC in both these cultures are comparably potent in alloantigen-presenting capacity. This result supports the speculation that TGF-ß1 does not act directly on immature DC and is compatible with the results by Demidem et al. and Rabbe et al. showing that TGF-ß1 did not suppress the MLR-stimulating or antigen-presenting activity of fresh Langerhans cells [9, 16].

In conclusion, our studies indicate that GM-CSF is a major cytokine for inducing the growth of immature DC from proliferating progenitors in mouse marrow. However, the further maturation of these cells is enhanced by the major inflammatory cytokine TNF-{alpha}, which in turn supersedes potentially suppressive effects that are mediated by macrophages and TGF-ß1.


   Acknowledgments
 
The authors thank Dr. R.M. Steinman and Dr. A.S. Shimosaka for reviewing the manuscript prior to submission. This work was partly supported by Grants-in Aid for Science Research on Priority Areas and for General Scientific Research from the Ministry of Education, Science and Culture of Japan.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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accepted for publication October 15, 1996.




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