In VitroCell. Dev.Biol.26:682-690,July 1990 9 1990TissueCultureAssociation 0883-8364/90 $01.50+0.00

T R A N S F O R M I N G G R O W T H FACTOR-/31 M O D U L A T E S T H E E F F E C T O F 1 a , 2 5 - D I H Y D R O X Y V I T A M I N D3 ON L E U K E M I C C E L L S MINORU MORIKAWA, NAOKI HARADA, GEN-ICHIRO SOMA, ANDTAKESHI YOSHIDA

Tokyo Institute for Immunopharmacology, Inc., 3-41-8Takada, Toshima-ku, Tokyo 171, Japan (M. M., N. H., T. Y.); and BiotechnologyResearch Center, Teikyo University, Sagamiko-cho, Kanagawa 199-01,Japan (G-L S.) (Received 27 December 1989; accepted 19 March 1990)

SUMMARY The human leukemic cells HL-60, U937, KG-1 and THP-1 incubated with transforming growth factor-{Jl (TGF-{JD were studied by examining cell surface antigens and macrophuge-specific activities. The addition of 0.5 ng/ml (20 pM) of TGF-/H with la,25-dihydroxyvitamin D3 [la,25(OHhD3] induced more Leu-M3 (CD14}-positive cells (approximately 80%~ than 5 X 10-s M la,25(OH}2D3 alone did (30 to 50%}, although original HL-60 cells did not express any Leu-M3 antigen at all. Tumor necrosis factor-a (TNF-a} !with TGF-/31 and 1a,25(OH~D3 was found to potentiate the expression of these surface antigens. i Furthermore, the phagocytic activity was also induced strongly. The expression of CR3 (CDllb~ antigen was also increased, and all Leu-M3-positive cells were found CR3-positive when HL-60, U937, and THP-1 cells were treated with these stimulants. In contrast, CR3 but not Leu-M3 was induced in KG-1 cells alter the same treatment. This may indicate that the responsiveness of leukemic cells to TGF-/3I and la,25(OH)2D3 might vary depending on a differentiation stage of the target cells. Furthermore, K562 cells originated from a more undifferentiated precursor, were not able to respond to these two inducers. These results suggested that some of TGF-/3 supedamily proteins might represent potent modulators in hematopoiesis, especially in the development of monocytes-macrophages or their precursors.

Key words: HL-60 cell differentiation to macrophage; TGF-f~I; la,25(OHhD3; TNF-a phagocytosis; chemiluminescence. (25,28}. These considerations have prompted us to perform a present series of experiments investigating a possible effect of TGF-/31 on the differentiation of macrophage lineage cells. In this paper, we will report results indicating that TGF-~I is a potent co-factor to differentiate HL-60 and other related leukemic cells to mature macrophagelike cells in the presence of la,25(OHhD3. The activity of TGF-/31 was synergistic with la,25(OHI2D3 and was further attgumented by the addition of TNF-a.

I NTRODUCTION Human leukemic cell lines have proved to be useful for studying factors and conditions associated with a differentiation pathway to macrophages from their precursors (31. For example, la,25-dibydroxyvitamin D,, [la,25(OHhD3] has been shown to differentiate HL-60 cells into macrophagelike cells (12 ~. Recently it has been reported that tumor necrosis factor-or (TNF-a~ in the presence of la,25(OHJ2D3 could regulate the differentiation of HL-60 cells into macrophagellkc cells (30). In vivo differentiation of bone marrow precursor cells to macrophages, however, may be regulated by additional unknown cytokines. In this context, transforming growth factor-fH (TGF-fJl) is a molecule of considerable biological interest, belonging to a large gene family, most members of which have regulatory actions on cell growth and differentiation (1,6,29). Biologically inactive precursor substances of these gene products are known to be produced and secreted by virtually all types of cells. Antiproliferative effects of TGF-/31 have been reported on many types of cells including those associated with the immune system. Thus, TGF-~I could suppress the activities of T cells, B cells, as well as N K cells (7,8,16,27L It has been shown too that monocyte functions could be modulated by TGF-/Jl

MATERIALS AND METHODS

Cell culture conditions. Human leukemic cell lines, HL-60, U937 and KG-1, were kindly provided by Dr. Akagawa (National Institute of Health, Tokyo, Japan1 and mycoplasma-|ree THP-1 (clone N) was obtained from Dr. H. Hemmi (Sagami Chemical Research Center, Kanagawa, JapanL K562 cells were supplied by Chugai Pharmaceutical Co., Ltd. (Tokyo, JapanL The HL-60 cells were used in most experiments and others were used in some experiments as specified in Results. The cells were maintained in R P M I 1640 supplemented with heatinactivated 10% fetal bovine serum (FBS}, penicillin (100 U/ml}, and streptomycin (100/~g/mll at 37~ C under 5% CO2 in a humidified incubator. The initial cell density 682

DIFFERENTIATION OF HL-60 CELLS BY TGF-f/1

was 1 X l0 s cells/ml in a 10-cm-diameter dish containing 10 ml of the culture medium and appropriate amounts of differentiation-inducers, as indicated in Results, were added at 0 time. After 3 d ineubation, the cells were usually harvested by using a rubber policeman, washed, and used for experiments. Biologicals and chemicals. Mouse monoclonal anti-trinitrophenol (TNP) antibodies (IgG2a and IgG2b) were kindly supplied by Drs. Majima and Ishida (Tohoku University, Sendal, Japan). Monoclonal antibodies, anti-Leu-M3 (fluorescein-conjugated and phyeoerythrin-conjugated) and anti-CR3 (phycoerythrin-conjugated), were purchased from Beeton Dickinson Co. (Mountain View, CA), intederon-y (IFN-y) from Genzyme Corp. (Boston, MA), porcine TGF-fJ1 and human TGF-f~I from R & D Systems, Inc. (Minneapolis, MN), and Polybead fluorescent microspheres (0.75-~ diameter) from Polysciences, Inc. (Warrington, PAL FBS was obtained from Bocknek Laboratories Inc. (Canada). 1a,25(OHhD3 was supplied by Chugai Pharmaceutical Co., Ltd. Retinoie acid (RA) was obtained from Sigma Chemical Co. (St. Louis, MO). The 1a,25(OHhD3 and RA were dissolved in ethyl alcohol. Porcine and human TGF-f31s were dissolved in 4 m M HCI containing 1 mg/ml crystalline bovine serum albumin (BSA). Recombinant human TNF-a was dissolved in RPM11640 containing 10% FBS. Detection of cell surface antigens. The cells were removed from dishes using a rubber policeman, washed twice with cold phosphate buffered saline (PBS) containing 0.3% BSA and 0.05% sodium azide (designated, BSA-PBS), and resuspended in 50 ~1 of BSA-PBS. The cells were treated with 5 /al of appropriate monoclonal antibodies. The cell suspension was incubated on ice for

683

30 min. After the incubation the cells were washed twice with BSA-PBS. Immediately after labeling the cells with fluorescent antibodies, they were fixed with 2.5% formaldehyde. If necessary the cells were treated with the second antibodies in the same manner. Among the monoclonal antibodies used in a series of experiments, Leu-M3 is a typical surface antigen of monocytemacrophage (24) and CR3 is linked with the macrophage functions {10). Fluorescence was determined with a flow cytometer, FACStar (Becton Dickinson Co.). Measurement of phagocytic activity. The cells treated with the indicated inducer(s) were suspended in R P M I 1640 containing ]0% FBS and 0.02% Polybead fluorescent microspheres, transferred to a culture tube, and incubated for several hours to equilibrate the medium with 5% CO2:95% air. Then the tubes were sealed and incubated at 37 ~ C overnight while they were gently shaken. After incubation, the cells were washed 3 times with cold BSA-PBS to remove the excess amounts of latex beads. The number of the cells incorporating the fluorescent beads was determined using a flow cytometer FACStar, and the percentage of cells ingesting the beads was obtained.

Detection of release of reactive oxygen intermediates mediated by human receptor for Fc portion of IgG {FcyR). FcyRs-mediated reactive oxygen intermediate release was assayed with the method of Majima et al. (11). Briefly, mouse monoclonal anti-TNP-IgG2a and anti-TNP-lgG2b bind with human FeyRI and FcyRII, respectively. When the differentiated HL-60 cells were mixed with the correspondent monoclonal antibody and TNP-conjugated sheep red blood cell (SRBC), the antibody-SRBC complex was incorporated into the cells through FcyRl or FcyRH. The

FIG. 1. Photomicrograph of a phase contrast preparation of HL-60 cells treated with TGF-/31 or la,25(OHhD3 or both. Cells (1 Xl06 cells) were cultured in 10 ml of RPMI 1640 containing (a) 10% FBS alone; supplemented with (b) 5 )< 10-s M la,25tOHhD3; tc) 1.0 ng/ml TGF-/~I and 5 X 10-s M la,25(OHhD3 at 37~ C for 3 d under 5% CO2:95% air, respectively. Differentiated cells were stained with anti-Leu-M3 antibody; their positive populations were determined by flow cytome!ry as shown in Fig. 2.

684

MORIKAWAET AL.

550

as Leu-M3 (18) and CR3 (24) increased remarkably when the markers were examined by measuring fluorescent intensity of the respective antibody-labeled cells with a flow cytometer. Among them, Leu-M3 was found most specifically reflecting the differentiation of HL-60 cells. Thus, while original HL-60 cells did not show any Leu-M3 antigen, the appearance and increase in the number of the cells labeled with anti-Leu-M3 antibody was well correlated with the extent of the morphologic changes described above. Therefore, in t h e next series of experiments, the change of Leu-M3 was employed as a parameter of the HL-60 cell differentiation.

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Leu-M3 expression FIG. 2. Induction of Leu-M3 antigen in HL-60 cells by TGF-fH in synergy with Ia,25(OH)~D3. Cells (1 X 10~ cells) treated with and without the indicated inducer(sJ were stained with fluorescein-conjugated anti-Leu-M3 antibody as described in Materials and Methods. Cell proportion was determined, counting 104 cells by flow cytometry. Line I, no addition (--J; line 2, 1.0 ng/ml TGF-/~I (' - "); line 3, 5 X10-8 M la,25(OH)~D~ ( .... J; line 4, 1.0 ng/ml TGF-/~Iplus 5 X 10-~M la,25(OH)~D~ (--L

treated cells (1 X 106 cells) were suspended in 1 ml Hanks' balanced salt solution containing 0.1% gelatin, 5 mM HEPES and 1 X 10-5 M luminol. The reaction started by the addition of a complex of SRBC-TNP-IgG2a or SRBC-TNP-IgG2b to the cell suspension. Release of reactive oxygen intermediates during the process was detected by luminol activation in the reaction mixture. Chemiluminescence was recorded using Biolumat LB9505 (Berthord, West GermanyJ equipped with a computer program analysis. Cytochemical assays. Nonspecific esterase activity was determined cytochemically using a-naphthyl acetate as the substrate (32J. Cells spread on a microscope slide using Cytospin 2 (Shandon Southern Product, Ltd., England) were fixed with cold formalin-acetone and incubated for 30 min at room temperature in a staining solution(13). Hemoglobin determination was carried out using o-dianisidine (14J.

by 5 X 10-s M of la,25(OH)2D3 and 1.0 ng/ml of TGF-fH. Although la,25(OHJ~D3 itself could induce only 30 to 50% of Leu-M3-pesitive cells, both inducers added together could increase the positive population up to about 80%, whereas TGF-/31 alone had little effect on the expression of Leu-M3 antigen. As shown in Fig. 3, the number of Leu-M3-positive cells increased in the presence of various concentrations of la,25(OHJ2D3. The addition d TGF-pl augumented the expression d the antigen and the effect was induced at a concentration as low as 0.1 ng/ml and reached a plateau at 0.5 ng/ml. Inasmuch as TNF-a and IFN-y could induce the differentiation of HL-60 cells (20,24,30J and express CR3 antigen and some other macrophage functions (24}, we examined their effects on the expression of Leu-M3 antigen. As shown in Table 1, TNF-a could also stimulate the HL-60 cell differentiation in the presence d la,25(OH)2D3, although the effect was weaker than that of TGF-/~I. The highest expression of Leu-M3 antigen, however, was obtained when the three inducers were added together in the culture medium. Although RA is a known difierentiation-inducer d HL-60 cells into granulocytelike cells (2), it could not replace 100

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morphologic changes noted after the treatment either with 5 X 10-8 M of 1a,25(OH)~D~ alone or together with 1.0 ng/ml of TGFq31 for 3 d. Although 1a,25(OHJ2D3 alone could induce some morphologic changes (Fig. lb), the addition of TGF-pl had a remarkable synergistic effect inducing almost complete transformation of original HL-60 cells {Fig. laJ into a spreaded and spindle-shaped form as shown in Fig. lc. TGF-fH itself did not induce significant morphologic changes. In accord with the changes of cell morphology, the cells with surface marker antigens such

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FIG. 3. Induction of Leu-M3 antigen in HL-60 cells by various concentrations of TGF-fH and l~,25(OHJzD3. Cells were incubated at various concentrations of la,25(OH)2D3 (horizontal) in the presence of TGF-/31, and the proportion of the Leu-M3-positive cells was determined by flow cytometry. (Open circles), Ia,25(OH)~D3 only; Ia,25(OHJ2D3 with TGF-pl, 0.I ng/ml (open triangles); 0.25 ng/ml (solid circlesJ; 0.5 ng/ml (solid squares}; 1.0 ng/ml (open squares).

685

DIFFERENTIATION OF HL-60 CELLS BY TGF-/31

TABLE 1 INDUCTION OF LEU-M3 ANTIGEN IN HL-60 CELLS~ Additives Sample No.

la,25(OHb 2 D 3

1 2 3 4 5 6 7 8 9

TNF-a

TGF-f31

RA

Leu-M3-Positive Cells, %

+

43.5 0.1 0 0 62.9 71.5 1.2 94.5 0

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~ (1)< 106 cells) were cultured with the additive(s) indicated as (-t-) for 3 d. The concentrations of inducers were as follows: 5 X 10-s M Ia,25~OH)2D3, 12 U/ml TNF-a, 1.0 ng/ml TGF-f31;2 )< 10-6 M RA. Proportion of Leu-M3-positivecells was determined by flow cytometry. 1a,25(OH)~D3 for the induction of Leu-M3 antigen. Furthermore, the combination of RA and TGF-/31 did not show any effect on the HL-60 cells (data not shown). Next, we examined whether IFN-y could affect the induction of Leu-M3 antigen. On the other hand, the expression of the antigen was little influenced by the addition of IFN-y, as shown in Table 2. The combination of T N F - a and IFN-y was not able to induce Leu-M3 under the conditions used. To examine whether the synergistic effect of TGF-/3I with la,25(OH)2D3 require their co-existence in the cultures, the cells were incubated with one or two inducers for the first 2 d and for the subsequent 2 d they were incubated with other inducers as indicated in the Table 3. The extent of differentiation was determined by the expression of Leu-M3 antigen. Full differentiation was obtained only when the cells were incubated with TGF-/31 and la,25(OH)2D3 for 4 d, whereas other combinations, such as 2 d incubation with TGF-f~I followed by 2 d la,25(OH)~D~ incubation or 4 d with la,25(OH)~D3 alone, resulted in only partial induction of Leu-M3 antigen. These results indicated that both inducers were required at the same time for full differentiation of HL-60 cells.

The expression of HLA-DR was also examined by labeling HLo60 cells with anti-HLA-DR antibody after the treatments with these inducers (4~. It was found that HLA-DR was not expressed even after the treatments with any combination of TGF-/~I, la,25{OHhD3, and T N F - a . Functional properties of the differentiated cells. While nonspecific esterase is not detectable in the untreated HL-60 cells, it is known that la,25tOH)2D3 is able to induce the enzyme activity 122L We now found that the strong synergistic effect of TGF-/I1 and la,25(OH)~D3 was also observed in terms of this enzyme induction. After the incubation with 1.0 ug/ml TGF-f~I and 5 X 10-s M la,25(OH)~D3 for 3 d, 49.5% of the cells were nonspecific esterase-positive whereas only 26.5% of the cells were positive alter treatment with la,25(OHhD3 alone. TGF-/31 alone had no effect on the enzyme induction. The phagocytic activity of the treatment cells was determined by the use of fluorescent latex beads. The percentage of cells phagocytized beads was measured by flow cytometry. Both combination treatment with TGF-/31 plus la,25(OH)2D3 and that with T N F - a plus la,25(OH)2D3 could induce the phagocytic activities in a dose-dependent manner, although the effect of TGF-f31/ltt,25(OH}2D~ (Fig. 4 A) was significantly more prominent than that of

TABLE 2 LACK OF EFFECT OF IFN-y ON LEU-M3 ANTIGEN EXPRESSION IN HL-60 CELLS~ Additives Sample No.

1 2 3 4 5 6 7 8

la,25(OH) 2 D 3

TGF-p I

-4-4-4+

-4+ + + + +

TNF-tr

RA

IFN-y

+ + +

+ + +

+ +

+ +

Leu-M3-P~sitive Cells, %

66.7 65.2 78.8 79.5 O.2 O.2 O.2 0.7

~ ~1 )< 10~ cells}were cultured with the additives indicated as IA-} for 3 d. Concentrations of inducers were as follows: 5 X 10-s M la,25(OH12D3, 1.0 ng/ml TGF-f31, 12 U/ml TNF-a; 2 )< 10-6 M RA, 200 U/ml IFN-y. Population of the Leu-M3positive cells was determined by flow cytometry.

686

MORIKAWA ET AL.

TABLE 3 REQUIREMENT OF CO-EXISTENCE OF TGF-/~I AND la,25(OH)~D~ FOR HL-60 CELL DIFFERENTIATION ~ First Culture With

Second Culture With

No additives TGF-/~1 Ia,251OH)~D~ TGF-/31 la,25(OH)~D~ TGF-/~I -4- la,25(OH)2D~

No additives TGF-~ 1 TGF-/~I la,25(OH)~D~ la,25~OH)~D~ TGF-/~I + Ia,25(OH)2D3

Leu-M3-Positive Cells, %

0 0.1 1.7 30.3 71.3 94.2

~ of inducers were as follows: 5 X 10-a M la,25(OH)2D~, 1.0 ng/ml TGF-f31; 1 X 1(r of HL-60 cells were incubated for 2 d with or without TGF-f~I and/or la,25~OH)2D~ and washed 3 times with RPMI 1640. Secondary incubation was carried out in the presence of the indicated indueer~s).

significant because it was reproducible in three independent experiments. When K562 cells were used as the target cells for the combination treatment with TGF-/31 and la,25(OH)2D3, they did not show any changes in morphology, nor in the cell surface antigens. D ISCUSSION TGF-/31, a multipotent growth modulator, was first reported by Sporn and his collaborators (15). They showed that TGF-/31 could stimulate reversibly the colony formation of N R K cells in agar in the presence of epidermal growth factor. Tucker et al. (26) showed that a polypeptide growth inhibitor from BSC-1 cells had high

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TNF-a/la,25(OH)2D3 (Fig. 4 B and Table 1). The combination of these three inducers together had no further additional effect on the pagocytic activity. As the phagocytic activity increased with cell differentiation, it is interesting to know the expression of CR3 and Fc?R on the cell surface. By two-color analysis with a flow cytometer, it was found that more than 90% of the cells expressed CR3 when they were treated with both TGF-f~I and la,25(OH)2D3. Interestingly, Leu-M3-positive cells were, without exception, CR3-positive as shown in Fig. 5. Next, to examine the Fo, R I on HL-60 cells, the release of the reactive oxygen intermediates was assayed after stimulating the cells with antibody (IgG2a)-sensitized SRBC (11). As shown in Table 4, the la,25(OH)~D3, or any combinations of 1a,25(OH)2D3, TGF-/~I and T N F - a , induced a significant amount of the reactive oxygen intermediate release, although the untreated cells could phagocytize little antibody (IgG2a)-SRBC complex, indicating the induction of reactive Fe),RI. As far as is concerned F c y R I I , no significant release of the reactive oxygen intermediates was observed when the cells were treated with antibody (IgG2b)-SRBC. Changes in other leukemic cells. It was next examined if TGF-f~I was able to affect the differentiation of not only HL-60 cells but other human leukemic cells such as U937, KG-1, T H P - I , and K562. Although U937 cells remained morphologically almost the same even after the incubation with la,25(OH)2D3, the cells showed a remarkable change when treated with both TGF-/31 and la,25(OH)2D3 together. Concomitantly with the morphologic change, Leu-M3 and CR3 cell surface antigens were induced by T G F - p l and 1a,25(OH)zD3 in a similar fashion as in HL-60. As shown in Table 5, a prominent effect on the phagocytic activity of U937 cells was obtained when they were treated with la,25(OH)2D3 and T G F - p l . More than 90% of the cells were able to incorporate the fluorescent latex beads. Furthermore, as shown in Fig. 6, KG-1 cells started to express a significant amount of CR3 after the incubation with la,25(OH)2D3 and T G F - p l , although LeuM3 was hardly induced. THP-1 cells, which have CR3 antigen but not Leu-M3 antigen before treatment, could induce the latter antigen by the addition of TGF-/31 alone to the culture medium (Fig. 7). This effect was considered

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TNF-(x (U/ml) FIG. 4. Phagocytic activity of HL-60 cells induced by TGFd31,1a,25(OH)2D3, and TNF-~..4, cells were incubated at various concentrations of TGF-P] with la,25(OH)2D3, 1 X 10-8 M (open circles), 5 X 10-s M (solid circles) and 8 X 10-8 M" (solid squares). B, cells were incubated at various concentrations of TNF-a in the presence of 2.5 X 10-8 M lo,25(OHJ2D3 (open circles) or 2.5 X 10-8 M la,25(OH)2D3 plus 0.5 ng/ml TGF-/~I (solid circles). Cells treated with the indicated inducers for 3 d were incubated with fluorescent latex beads overnight. Bead-incorporated cells were counted by a flow cytometer as described in Materials and Methods and the percentage of cells ingesting the beads was shown on the vertical axis.

687

DIFFERENTIATION OF HL-60 CELLS BY TGF-~I

I!

TABLE 4

FcyRI-MEDIATED RELEASE OF REACTIVE OXYGEN INTERMEDIATES BY THE DIFFERENTIATED HL-60 CELLS

~q m

Additives"

None 0.03 • 0.014 la,25(OH hD~ 1.00 la,25(OHhD3 + TGF-fil 0.93 • 0.057 la,25(OHhD3 + TNF-a 1.76 • 0.028 la,25(OHhD~ -k TGF-f31 + TNF-a 0.95 • O.014 ~ were treated with the indicated inducer(s) under the same conditions described in the legend of Fig. 1. Concentration of TNF-a was 12 U/ml. bCells treated with la,25(OH)2D3 emitted 560 epm which was defined as 1 unit.

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homology with TGF-f~ which is now called TGF-fJ2. Since then various proteins were found to belong to TGF-f3 gene superfamily. They are known to have a bifunctional effect, either growth inhibitory or growth promoting, depending on target cell types and culture conditions {21i. Recently, TGF-fJI has been shown to be an extremely active immunosuppresslve agent (7,8,25,27) as well as a potent chemoattractant for human peripheral blood monocytes (28). Furthermore, various cells and tissues including macrophages (17}, T cells (27}, bone (19,31}, gonads O), and bone marrow (33) synthesize many proteins that belong to TGF-f3 gene superfamily. Mason et al. ~9} showed that /~ chain of inhibin, a specific and potent polypeptide inhibitor of the secretion of follicle-stimulating hormone of gonadal origin, is the product of TGF-/3 gene superfamily. They also clarified that activin, a modulator of inhibin, is also one of TGF-~ supedamily proteins because it is composed of two types of/~ chains of inhibin, f~A and fJB. Furthermore, two groups (5,33) demonstrated that activin was able to differentiate K562 cells into hemoglobin-producing cells. These facts suggested that TGF-/~ might be an important regulator of erythropoiesis. In the present series of experiments, we investigated the effect of TGF-fJ1 on the leukemic cells which are committed toward monocytes-macrophages. TGF-{~I alone had apparently some biochemical effects; for example, the weak suppression of Leu-M1 (CD15) antigen but virtually no growth inhibition on the HL-60 cells at 1.0 ng/ml under the conditions used. However, the remarkable morphologic changes were induced when both TGF-~l and la,25(OHhD3 were added to the culture medium (Fig. 1). These cells acquired the expression of monocyte-macrophage-specific antigen Leu-M3 on their

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688

MORIKAWA ET AL.

as U937, KG-1, and THP-1. Among them KG-1 cells are known as less mature cells and THP-1 cells are more mature {personal communication, Dr. J. Minowada). Based on such differentiation stages, they could more or less respond to TGF-/~l/la,25(OH)2D~. Especially it is interesting that THP-1 cells were able to partially induce Leu-M3 antigen with the treatment of TGF-/~I alone {Fig. 7). These results certainly suggested a possible physiologic role of TGF-/~I in vivo for further differentiation of macrophage precursor cells at a certain maturation stage. Tessier and Hoang (23~ reported that the growth of acute myeloblastie leukemia was suppressed by TGF-/~ in the presence of g r a n u l o c y t e - m a c r o p h a g e colonystimulating factor. They suggested that TOF-/~ seemed to have an antiproliferative effect rather than the growthstiniulatory properties to acute myeloblastic leukemia. This taken together with our present results indicates that TGF-/~I may work as a "differentiation

TABLE 5 PHAGOCYTIC ACTIVITY OF U937 CELLS TREATED BY TGF-fll AND la,25{OH}~D~o Cells Incorporated Beads, %

Additives

None TGF-f~I l~,25(OH)~D~ TGF-/~I + 1tr,25(OH)~D3

46.0 50.9 77.4 92.5

~ cells were cultured under the same conditions as described in the legend of Fig. 1 for HL-60 cells, and were incubated with the fluorescent latex beads. The population of cells that incorporated the beads was determined by flow cytometry as described in Materials and Methods. cell surface, and gained stronger functional activities than the untreated cells. In addition to HL-60 ceils, we examined other monocyte-macrophage lineage cells such q. m ,,,..=

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FIG. 6. Expression of CR3 antigen on KG-1 cells treated by TGF-/~I and la,25(OH)~D3. Respective concentrations of inducers were 5 X 10-a M for la,25~OH)zD3 and 1.0 ng/ml for TGF-/31.

DIFFERENTIATION OF HL-60 CELLS BY TGF-f~I

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Leu-M3 expression FIG. 7. Expression of Leu-M3 antigen on THP-1 cells treated by TGF-{31 alone. Cells were incubated with t.0 ng/ml TGF-f~I for 3 d and the Leu-M3-positive cells were determined by flow cytometry as described in Materials and Methods. Dotted line, untreated cells; solid line, ceils treated with TGF-/31. modulator" of various differentiation or growth factors of hematopoietic cells, or both. The present result showed that the co-existence of TGF-/31 and 1a,25(OHhD3 in culture medium was required to induce strong differentiation of HL-60 cells as well as other human leukemic cells in macrophage lineage. Thus, sequential treatments with both stimulants, either la,25iOHhD3 pretreatment followed by TGF-fH or TGF-/31 followed by la,25(OHhD~, could not achieve the similar differentiation effects induced by the simultaneous treatment with both agents. The exact reason for this requirement is unknown at the present time, although there is a possibility that macrophage-differentiation activity of la,25(OHhD3 m a y be, at least partly, mediated by TGF-/31. Possible production of TGF-f31 by the cells treated with la,25~OH)~D3 is now under investigation. As mentioned above, it has been reported that activin is able to differentiate K562 cells {5,33). However, TGF-f~I, regardless of the presence or absence of la,25(OHhD~, had no effect on K562 cells in the present study. This indicates that TGF-f~I may have the activity with a different spectrum from that of activin on the bone marrow precursor cells. These results suggested that TGF-f~I in synergy with la,25(OHhD3 might facilitate the maturation of precursor cells in bone marrow, which were committed to a macrophage lineage. In conclusion, this is the first report to our knowledge showing a potentially important role of TGF-]31 played physiologically on the differentiation of macrophage lineage ceils. Further studies are being conducted to reveal the molecular mechanism of TGF-fH actions, the activation of Fc)' receptors, and a scope of its action in vivo. REFERENCES 1. Assoian, R. K.; Frolik, C. A.; Roberts, D., et al. Transforming growth factor/3 controls receptor levels for epidermal growth factor in NRK fibrohlasts. Ceil 36:35-41; 1984.

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