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Mouse parenchymal liver cells in culture secrete a growth inhibitor for myeloma cells
Journal of Hepatology 1996; 24: 225-229 Printed in Denmark . AN rights reserved
Copyright 0 European Association for the Sludy of the Liver 1996 Journal of Hepatology ISSN 0168-8278
Mouse parenchymal liver cells in culture secrete a growth inhibitor for myeloma cells Megumi Takahashi
and Kozo Yokomuro
Nippon Medical School, Department of Microbiology and Immunology, Tokyo, Japan
Methods: Growth inhibitory activity in the conditioned medium of mouse parenchymal liver cells was examined in three strains of myeloma cells. Results: ‘Ikvostrains of myeloma cells were highly sensitive to a low concentration of mouse parenchymal liver cell derived growth inhibitor, whereas one strain was resistant to the same concentration. Interferon-@ and transforming growth factor-j? activity were detected in mouse parenchymal liver cells, while interferon-y and tumor necrosis factor-a were not. The growth suppression exerted by mouse parenchymal liver cell derived growth inhibitor in the three myeloma strains was distinct from that exerted by transforming growth factor-j?, tumor necrosis
factor-a, interferon-up and interferon-y. The mouse parenchymal liver cell derived growth inhibitor was eluted with a peak activity in the 18 kDa range and focused into p1 values of 3.840, and it was lost when mouse parenchymal liver cells were treated with heat or trypsin. Conclusion: These results indicate that mouse parenchymal liver cell derived growth inhibitor differs from the well-characterized growth inhibitors, transforming growth factor-/?, tumor necrosis factor-a, interferon-a/? and interferon-y.
I
period of embryonic life in mammals, the liver plays a major role in the development of the hematopoietic system. In adults, the liver regulates not only the intermediary metabolism but also the participation of both the immune and hematopoietic systems in the host defense reaction. In recent years, there has been increased direct and indirect evidence of the immunological function of the liver; this includes phagocytosis and the processing of antigens passing through the intestine (l), the inducement of oral tolerance (2), the transport of polymeric immunoglobulin A into bile (3), the presence of antibody-forming cells (4,5), and cytokine production (6-8). It is well known that Kupffer cells are potent producers of inflammatory cytokines such as TNF-cc, IL- 1, and IL-6 (9). Hepatic sinusoidal endothelial cells and Ito cells are also able to secrete certain cytokines (10). Recently, it has been revealed that not only hepatic sinusoidal cells but also hepatocytes produce various cytokines. Sakamoto
et al. and Tsukui et al. have reported that the conditioned medium of mouse parenchymal liver cells (PLM) contains IL-l, IL-6, GM-CSF, and M-CSF (11-13). These cytokines found in PLM act as positive growth regulators, predominantly promoting cell proliferation and differentiation. In this study, we have examined the inhibitory effect of PLM on the proliferation of various established cell clones. PLM had no inhibitory effect on the proliferation of mouse lymphoma and L cell clones at a high concentration. However, we found that, among mouse myeloma cells, two strains were highly susceptible but one was resistant at a low concentration of PLM. To elucidate the characteristics of the growth inhibitor in PLM, we compared its active factor with well-characterized regulators of cell growth in the manner of suppression of myeloma cell growth. The factor was partially purified gel filtration and isoelectronic focusing.
Received 19 October 1994; revised 30 January; accepted 23 February
Materials and Methods Cell lines Non-secreting mouse myeloma P3/NS l/ 1-Ag4- 1, P3X63-Ag8.6563, and Sp2/0-Ag14 were routinely pass-
N THE EARLY
1995
Correspondence: M. Takahashi, Nippon Medical School, Department of Microbiology and Immunology, l-l-5, Sedagi, Bunkyo-ku, Tokyo 113, Japan.
Key words: Cytokines; Growth inhibitor; Myeloma cells; Parenchymal liver cells.
225
M. Takahashi and K. Yokomuro
aged in RPM1 1640 medium supplement with 10% fetal calf serum (FCS) and 50 PM 2-ME in our laboratory. Preparation of parenchymal liver cell conditioned medium (PLM) The liver cells were isolated from normal BALB/C mice according to the collagenase digestion method described by Sakamoto et al. (11). Briefly, the livers were perfused in situ with 0.05% collagenase (150-300 units/mg Wako, Tokyo, Japan). Parenchymal liver cells were separated from whole liver cells suspension by low-speed centrifugation and 5 ml of the cell suspension was seeded into lo-cm diameter culture dishes, at a concentration of 1X lo6 cells/ml, in RPM1 1640 medium containing 10% FCS. After 3 h, nonadherent cells were removed by washing, and only the adherent cells were cultured. Twenty-four hours later, PLM was centrifuged at 500 g for 10 min to remove dead cells and cell debris. According to morphological observation, cultured parenchymal liver cells consisted of 99% typical parenchymal liver cells. To investigate the characteristics of the active factors, we heated PLM to 55,65,75, and 85°C for 30 min and subjected the preparation to trypsin treatment; PLM was incubated with 1650 units/ml of trypsin (Mochida, Japan) for 3 h at 37°C. Trypsin digestion was stopped by adding 1 mg/ml of soybean trypsin inhibitor (Sigma, St. Louis, MO, USA).
ture plates. After 18-24 h of incubation, L929 cultures were replaced with serial dilutions of the test samples and medium containing Actinomycin D (1 &ml). After incubation for 18-24 h, cells were stained with 0.02% neutral red, and the stained cells were solubilized in 30% ethanol with 0.01 N-HCl. Percent cytotoxicity was calculated on the basis of absorbance at 490 nm. Detection of TGF-j3 activity. MvlLu cells (1 X lo4 per wells) were cultured with diluted samples in RPM1 1640 medium containing 5% FCS in 96-well microplates for 24 h. Cells were pulse-labeled with 3Hthymidine for the final 4 h, and the incorporated radioactivity was measured. The specificity of TGF-P was ascertained by neutralizing with anti-TGF-B antibody (Becton Dickinson Labware). IFN-y activity was measured with a mouse interferon-ELISA kit (Genzyme Co. MA, USA). Gel filtration of PLM Three hundred microliters of concentrated PLM was applied to a TSK-gel G3000SWXL column (Tosoh, Japan) that was equilibrated and eluted with phosphate buffered saline (PBS, pH 7.2). The column was run at a flow rate of 12 ml/h and 0.3-ml fractions were collected. Individual fractions were tested for growth inhibition of myeloma cells.
Isoelectronic focusing (IEF) Samples were dialyzed against 1% glycine for 24 h and diluted to a final volume of 60 ml containing 0.8% BioLyte ampholytes, pH range 3-10. This solution was loaded into the Rotofor cell (Bio-Rad). Focusing was carried out at 12 Watts constant power for 4 h at 4°C. After the electrofocusing, 20 fractions were harvested and their pH values were measured. Each fraction was dialyzed against PBS for 24 h prior to being tested for growth inhibition activity.
Measurement of cellproliferation Single cell suspensions of myeloma cells were seeded, at a density of 5 X lo2 cells per well, on 96-well microtiter plates, and cultured in medium containing an appropriate amount of PLM, human recombinant TGF-Pl (King Brewing Co., Japan), mouse IFN-ap (see reference 12) or mouse IFN-), (supernatant of ConA-stimulated spleen cell culture). After 5-6 days of incubation, the cells were pulse-labeled with 0.5 &i/well methyl-3Hthymidine for 4-6 h, and were then harvested in an automated collection device (Flow Laboratories, Rockville, MD, USA). Calorimetric determination of cell growth was determined by MTT methods (14). For the assay, 50 pug of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma) was added to cell culture wells and the plates were incubated at 37°C for 4 h. Acidisopropanol was added to all wells and mixed thoroughly to dissolve the dark blue crystals, and optical density was measured at 595 nm.
Effect of PLM on growth of myeloma cells PLM induced a dose-dependent inhibition of 3H-thymidine incorporation by the three strains of myeloma cells. This inhibitory effect actually reflected an inhibition of proliferation as demonstrated by calorimetric measurement of growth. Strains PX-63 and SP-2 were highly susceptible to PLMI at a low concentration, while strain NS-1 was resistant to the same concentration (Fig. 1).
Measurement of cytokine activity in PLM Detection of TNF-a activity. L929 cells suspended in RPM1 1640 medium containing 10% FCS were seeded at a density of 6X lo4 cells per well in 96-well tissue cul-
Growth inhibition pattern of TGF-@, TNF-a, IFN-ap, IFN-y, in myeloma cell lines TNF-a had no effect on the proliferation of any of the three myeloma cell lines. TGF-/3 exhibited a moderate
226
Results
Hepatocyte derived growth inhibitor
dose-dependent inhibitory effect on NS-1 cells; however, it failed to inhibit the proliferation of PX-63 and SP-2 cells. IFN-a/? and IFN-1, had similar dose-de-
pendent inhibitory lines (Fig. 2). Cytokine
activity
effects in all three myeloma cell
in PLM
6 ‘Z a ._
60
TNF-a and IFN-y activity were not detected in PLM. IFN-c@ activity was present in PLM at a concentration of less than 10 units/ml (15). PLM contained TGF-j3 in the range of 1000 of 3000 &ml, but acid treatment was necessary before the assay detected TGF-P, indicating that TGF-j? in PLM was present in a latent form (Table 1).
2 @.
40
Partial characterization
3H-thymldine
uptake
80
20
0
0
3.2
6.3
12.5
Concentration
25
O--/c1 3.2
50
of PLM (a)
6.3
12.5
Concentrsiion
25
50
of PLM (96)
Fig. 1. Effects of the conditioned medium of mouse parenchymal liver cells (PLM) on the growth of three strains of myeloma cells. Myeloma cells (5X102), NS-1 (a), PX-63 (A), and SP-2 (0) were exposed to the indicated concentrations of PLM and incubated at 37°C for 54 days. The number of cells was determined by the MTT or 3H-thymidine uptake method.
100
100-
TGF-f3
:I s
80
m-
MI
3 60P
f
I
TNF-a
20
0
0.1
16
40010000
The inhibitory effect was completely abolished when PLM was heated to 85°C for 30 min, and the effect was markedly reduced by trypsin treatment (Table 2). On gel filtration chromatography, the growth inhibitory activity toward myeloma cells was eluted in two peaks, one with an approximate molecular weight of 18 kDa and the second one at below 10 kDa. The second peak of growth inhibitory activity was observed in the control culture medium, and therefore the molecular weight of PLMI was estimated to be about 18 kDa (Fig. 3). On isoelectronic focusing, the growth inhibitory activity was focused in four peaks, at p1 values of 3.84.0, 6.1-6.3, 6.6, and 7.2-7.4. The growth inhibitory activity of the three peaks with p1 values of 6.16.3, 6.6, and 7.2-7.4 was found in the control medium, and so the p1 of PLMI was estimated to be 3.84.0 (Fig. 3).
Discussion
_
ipa.
Z”
of PLMI
63
125 250 500 10002000
rTGF-P (pg/ml)
The results of these experiments indicated potent PLMI growth inhibitory activity in myeloma cells. In the myeloma cell strain, NS-1, proliferation was
rTNF-a (p@nU
TABLE
EN-a!3
1
Detection
of cytokine
TNF-a
IFN-a/3
ND’
2.7-7.5
activity
in mouse parenchymal
units/ml
liver cells
IFN-y
TGF-/l
ND
102&3192
ng/ml*
’ Not detected. * Detected after acidification. TNF=tumor necrosis factor; IFN=interferon. 0
0.060.25
1
3.8
15
60
J.FN-up (units/ml)
0
0.060.25
1 EN-y
3.8
IS
60
(lmm/d)
Fig. 2. Effect of transforming growth factor (TGF)-fi, interferon (IFN)-ap, IFN-y, and tumor necrosis factor (TNF-a) on the growth of three strains of myeloma cells. Myeloma cells (5x102), NS-I (O), PX-63 (A), and SP2 (O), were exposed to the indicated concentration of TGFp, IFN-a/3, IFN-y, and TNF-a and incubated at 37°C for 54 days. The number of cells was determined by the MTT method.
TABLE
2
Effect of heat and trypsin parenchymal liver cells
treatment
on the inhibitory
Treatment
Percent inhibition
no WC, 30 min 85”C, 30 min Trypsin
58-78 3661 o-22 22-l 1
effect of mouse
(%)
227
M. Takahashi and K. Yokomuro
suppressed by a high concentration of PLM, while this cell line was fairly resistant to the growth inhibitory activity of PLM at a low concentration, compared with the effects in the other two strains. To clarify the characteristics of the growth inhibitory factor in PLM, we compared the inhibition patterns of PLM in the three strains of myeloma cells with those of other wellknown factors: TGF-/I, IFN-o/I, IFN-y, and TNF-(r. TGF-P is a potential negative regulator of proliferation in many cell types (16). It was recently shown that TGF-/I enhanced IgA production by LPS-stimulated splenic B cells (17). We have observed that PLM, which was added to culture of LPS-stimulated B cells, enhanced the production of IgA (unpublished data). Therefore we regarded TGF-/I candidate as an inhibitory factor in the proliferation of myeloma cells, and we assayed the activity of TGF-fi in PLM. We found that a high concentration of TGF-/I (more than 1000 rig/ml) was produced in PLM, but that this TGF-j3 activity could not be detected unless PLM was treated with acid, indicating that the TGF-p was produced as a latent form. In the strain, NS-1, which was resistant to PLM, proliferation was slightly suppressed by rTGF-/3 at a high concentration; however, in the PX63 and SP-2 strains, which were susceptible to PLM, the proliferation was not suppressed at all by rTGF-13 at the same concentration. Besides we have found no effect of anti-TGF-j?l antibody in reducing the inhibitory effect of PLM (data not shown). These findings suggested that growth inhibitor for myeloma cells in PLM was not TGF-j?. None of the three strains of myeloma cells showed suppression of proliferation on treatment with rTNF-ol, nor was TNF-(r activity detected in PLM. Growth in three strains was suppressed by IFN-ccp and IFN-y, depending on their concen-
(A ) Gel Chromatography
PX-63
(B)JEF
Fig. 3. Gel-filtration chromatography (A) and isoelectronic focusing (B) of mouse parenchymal liver cell growth derived inhibitor. Individual fractions were tested for growth inhibitory activity. SP-2 cells (5~ 102) were incubated with each fraction for 5 days. The number of cells was determined by the MTT method.
228
tration, but there was no difference in the degree of suppression among these three strains. IFN-?/ activity was not detected in PLM, although IFN-c$ activity was detected at less than 10 units/ml. However, this concentration produced no difference among strains. On the basis of our findings, regarding the difference in suppression of proliferation among the three strains of myeloma cells, the activity of each factor in PLM, and molecular size and isoelectronic point of the active fraction, we believe that the growth inhibitory substance in PLM is not TGF-/I, TNF-a, IFN-a/3 or IFNy. Several investigators have demonstrated the existence of the cell growth modulator in the liver. Chapekar et al. reported the presence of a factor in rat liver homogenate that had exerted negative and positive effects on cell proliferation (18). Woodman et al. reported that after partial hepatectomy nonparenchymal cells secreted hepatocyte proliferation growth inhibitor (19). They showed that their factors were distinct from TGF-/I or any other previously described inhibitor of cell proliferation. Why the myeloma strains showed different susceptibility to the inhibitory factor in PLM is not known, As one of the noticeable different features between the susceptible and the resistant strains, it was shown that the quantity of 3H-thymidine uptake differs remarkably in these strains. The number of myeloma cells was calculated every 24 h by both the MTT and 3H-thymidine uptake methods. The proliferation of culture cells assayed by the MTT method was increased by approximately the same amount in all three strains, while the quantity of 3H-thymidine uptake in DNA was l/5-1/7 lower in the two susceptible strains compared with the resistant strain (Fig. 4). However, the total quantity of DNA was about the same in all three strains, sug-
SP-2
Days
Fig. oma The (0)
4. Growth rate of three strains of myeloma cells. Myelcells (5 X 102) were seeded in 100 ,ul medium per well. number of cells was determined every 24 h by the MTT or 3H-thymidine uptake (0) method.
Hepatocyte derived growth inhibitor
gesting that there may be a difference in the synthesizing process of pyrimidine nucleotide. It is not clear whether this is related to the difference in the susceptibility of myeloma cells to PLM. However, it appears to be important in understanding the suppressive mechanism that NS-1, a resistant clone, has characteristics similar to those of PX-63, a sensitive clone, since these two clones were derived from the same source.
References 1. Richman LK, Klingenstein RJ, Richman JA, Strober W, Berzofsky JA. The murine Kupffer cell: I. Characterization of the cell serving an accessory function in antigen-specific T cell proliferation. J Immunol 1979; 123: 2602-9. 2. Thomas HC, Ryan CJ, Benjamin IS, Blumgart LH, MacSween RNM. The immune response in cirrhotic rats: the induction of tolerance to orally administered protein antigens. Gastroenterology 1976; 71: 114-7. 3. Brown WR, Kloppel TM. The liver and IgA: immunological, cell biological and clinical implications. Hepatology 1989; 9: 763-84. 4. Manning RJ, Walker PG, Carter L, Barrington PJ, Jackson GDE Studies on the origins of biliary immunoglobulins in rats. Gastroenterology 1984; 87: 173-9. of 5 Takahashi M, Yokomuro K. Phenotypic characterization antibody-secreting cells in the liver. Cell Immunol 1993; 152: 220-5. 6 Kutteh WH, Rainey WE, Carr BR. Regulation of interleukin-6 production in human fetal Kupffer cells. Stand J Immunol 1991; 33: 607-13. I Gaillard T, Mulsch A, Busse R, Klein H, Decker K. Regulation of nitric oxide production by stimulated rat Kupffer cells. Pathobiology 1991; 59: 280-3. 8. Mabuchi A, Komuro T, Saizawa T, Sakamoto T, Watari E, Yokomuro K. The liver and the hematolymphoid system: I. The regulation of nylon-passed spleen cell proliferation by active factors released from syngeneic nonparenchymal liver cells. J Leukocyte Biol 1991; 50: 402-l 1. 9. Decker K. Biologically active products of stimulated liver macrophages (Kupffer cells). Eur J Biochem 1990; 192: 24561. I
.
10. Rosenbaum J, Philippe M, Preaux AM, Lesce MC, Dhumeaux D. Mouse hepatic endothelial cells in culture secrete a growth inhibitor for hepatic lipocytes and Balb/c3T3 fibroblasts. J Hepatol 1989; 9: 2955300. 11. Sakamoto T, Mabuchi A, Kuriya SI, Sudo T, Aida T, Asano G, Shoji T, Yokomuro K. Production of granulocyte-macrophage colony-stimulating factor by adult murine parenchymal liver cells (hepatocytes). Reg Immunol 1991; 3: 26& 7. 12. Tsukui T, Kikuchi K, Mabuchi A, Sudo T, Sakamoto T, Yokomuro K, Sato N, Tsuneoka K, Shikita M, Aida T, Asano G, Watari E. Production of macrophage colony-stimulating factor by adult murine parenchymal liver cells (hepatocytes). J Leukocyte Biol 1992; 52: 383-389. 13. Tsukui T, Kikuchi K, Mabuchi A, Sudo T, Sakamoto T, Yokomuro K. Production of interleukin-1 by primary cultured parenchymal liver cell (hepatocytes). Exp Cell Res 1994; 210: 172-6. 14. Mosmann T. Rapid calorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Meth 1983; 65: 55-63. 15. Mabuchi A, Watari E, Ikeda M, Watanabe Y, Yokomuro K. Growth control of primary culture hepatocytes by nonparenchymal liver cells: role of interferon produced by liver sinusoidal cells. J Nippon Med Sch 1994; 61: 120-8. 16. McMahon J, Richards WL, Campo AA, Song MKH, Thorgeirsson SS. Differential effects of transforming growth factor-p on proliferation of normal and malignant rat liver epithelial cells in culture. Cancer Res 1986; 46: 4665-71. 17. Sonoda E, Hitoshi Y, Yamaguchi N, Ishii T, Tominaga A, Takatsu K. Differential regulation of IgA production by TGF-/J induces surface &A-positive cells bearing IL-5 receptor, whereas IL-5 promotes their survival and maturation into IgA-secreting cells. Cell Immunol 1992; 140: 158872. 18. Chapekar MS, Huggett AC, Thorgeirsson SS. Growth modulatory effects of a liver-derived growth inhibitor, transforming growth factor /?l, and recombinant tumor necrosis factor (x, in normal and neoplastic cells. Exp Cell Res 1989; 185: 247757. 19. Woodman AC, Selden CA, Hodgson HJF. Partial purification and characterization of an inhibitor of hepatocyte proliferation derived from nonparenchymal cells after partial hepatectomy. J Cell Physiol 1992; 151: 405-14.
229
Copyright 0 European Association for the Sludy of the Liver 1996 Journal of Hepatology ISSN 0168-8278
Mouse parenchymal liver cells in culture secrete a growth inhibitor for myeloma cells Megumi Takahashi
and Kozo Yokomuro
Nippon Medical School, Department of Microbiology and Immunology, Tokyo, Japan
Methods: Growth inhibitory activity in the conditioned medium of mouse parenchymal liver cells was examined in three strains of myeloma cells. Results: ‘Ikvostrains of myeloma cells were highly sensitive to a low concentration of mouse parenchymal liver cell derived growth inhibitor, whereas one strain was resistant to the same concentration. Interferon-@ and transforming growth factor-j? activity were detected in mouse parenchymal liver cells, while interferon-y and tumor necrosis factor-a were not. The growth suppression exerted by mouse parenchymal liver cell derived growth inhibitor in the three myeloma strains was distinct from that exerted by transforming growth factor-j?, tumor necrosis
factor-a, interferon-up and interferon-y. The mouse parenchymal liver cell derived growth inhibitor was eluted with a peak activity in the 18 kDa range and focused into p1 values of 3.840, and it was lost when mouse parenchymal liver cells were treated with heat or trypsin. Conclusion: These results indicate that mouse parenchymal liver cell derived growth inhibitor differs from the well-characterized growth inhibitors, transforming growth factor-/?, tumor necrosis factor-a, interferon-a/? and interferon-y.
I
period of embryonic life in mammals, the liver plays a major role in the development of the hematopoietic system. In adults, the liver regulates not only the intermediary metabolism but also the participation of both the immune and hematopoietic systems in the host defense reaction. In recent years, there has been increased direct and indirect evidence of the immunological function of the liver; this includes phagocytosis and the processing of antigens passing through the intestine (l), the inducement of oral tolerance (2), the transport of polymeric immunoglobulin A into bile (3), the presence of antibody-forming cells (4,5), and cytokine production (6-8). It is well known that Kupffer cells are potent producers of inflammatory cytokines such as TNF-cc, IL- 1, and IL-6 (9). Hepatic sinusoidal endothelial cells and Ito cells are also able to secrete certain cytokines (10). Recently, it has been revealed that not only hepatic sinusoidal cells but also hepatocytes produce various cytokines. Sakamoto
et al. and Tsukui et al. have reported that the conditioned medium of mouse parenchymal liver cells (PLM) contains IL-l, IL-6, GM-CSF, and M-CSF (11-13). These cytokines found in PLM act as positive growth regulators, predominantly promoting cell proliferation and differentiation. In this study, we have examined the inhibitory effect of PLM on the proliferation of various established cell clones. PLM had no inhibitory effect on the proliferation of mouse lymphoma and L cell clones at a high concentration. However, we found that, among mouse myeloma cells, two strains were highly susceptible but one was resistant at a low concentration of PLM. To elucidate the characteristics of the growth inhibitor in PLM, we compared its active factor with well-characterized regulators of cell growth in the manner of suppression of myeloma cell growth. The factor was partially purified gel filtration and isoelectronic focusing.
Received 19 October 1994; revised 30 January; accepted 23 February
Materials and Methods Cell lines Non-secreting mouse myeloma P3/NS l/ 1-Ag4- 1, P3X63-Ag8.6563, and Sp2/0-Ag14 were routinely pass-
N THE EARLY
1995
Correspondence: M. Takahashi, Nippon Medical School, Department of Microbiology and Immunology, l-l-5, Sedagi, Bunkyo-ku, Tokyo 113, Japan.
Key words: Cytokines; Growth inhibitor; Myeloma cells; Parenchymal liver cells.
225
M. Takahashi and K. Yokomuro
aged in RPM1 1640 medium supplement with 10% fetal calf serum (FCS) and 50 PM 2-ME in our laboratory. Preparation of parenchymal liver cell conditioned medium (PLM) The liver cells were isolated from normal BALB/C mice according to the collagenase digestion method described by Sakamoto et al. (11). Briefly, the livers were perfused in situ with 0.05% collagenase (150-300 units/mg Wako, Tokyo, Japan). Parenchymal liver cells were separated from whole liver cells suspension by low-speed centrifugation and 5 ml of the cell suspension was seeded into lo-cm diameter culture dishes, at a concentration of 1X lo6 cells/ml, in RPM1 1640 medium containing 10% FCS. After 3 h, nonadherent cells were removed by washing, and only the adherent cells were cultured. Twenty-four hours later, PLM was centrifuged at 500 g for 10 min to remove dead cells and cell debris. According to morphological observation, cultured parenchymal liver cells consisted of 99% typical parenchymal liver cells. To investigate the characteristics of the active factors, we heated PLM to 55,65,75, and 85°C for 30 min and subjected the preparation to trypsin treatment; PLM was incubated with 1650 units/ml of trypsin (Mochida, Japan) for 3 h at 37°C. Trypsin digestion was stopped by adding 1 mg/ml of soybean trypsin inhibitor (Sigma, St. Louis, MO, USA).
ture plates. After 18-24 h of incubation, L929 cultures were replaced with serial dilutions of the test samples and medium containing Actinomycin D (1 &ml). After incubation for 18-24 h, cells were stained with 0.02% neutral red, and the stained cells were solubilized in 30% ethanol with 0.01 N-HCl. Percent cytotoxicity was calculated on the basis of absorbance at 490 nm. Detection of TGF-j3 activity. MvlLu cells (1 X lo4 per wells) were cultured with diluted samples in RPM1 1640 medium containing 5% FCS in 96-well microplates for 24 h. Cells were pulse-labeled with 3Hthymidine for the final 4 h, and the incorporated radioactivity was measured. The specificity of TGF-P was ascertained by neutralizing with anti-TGF-B antibody (Becton Dickinson Labware). IFN-y activity was measured with a mouse interferon-ELISA kit (Genzyme Co. MA, USA). Gel filtration of PLM Three hundred microliters of concentrated PLM was applied to a TSK-gel G3000SWXL column (Tosoh, Japan) that was equilibrated and eluted with phosphate buffered saline (PBS, pH 7.2). The column was run at a flow rate of 12 ml/h and 0.3-ml fractions were collected. Individual fractions were tested for growth inhibition of myeloma cells.
Isoelectronic focusing (IEF) Samples were dialyzed against 1% glycine for 24 h and diluted to a final volume of 60 ml containing 0.8% BioLyte ampholytes, pH range 3-10. This solution was loaded into the Rotofor cell (Bio-Rad). Focusing was carried out at 12 Watts constant power for 4 h at 4°C. After the electrofocusing, 20 fractions were harvested and their pH values were measured. Each fraction was dialyzed against PBS for 24 h prior to being tested for growth inhibition activity.
Measurement of cellproliferation Single cell suspensions of myeloma cells were seeded, at a density of 5 X lo2 cells per well, on 96-well microtiter plates, and cultured in medium containing an appropriate amount of PLM, human recombinant TGF-Pl (King Brewing Co., Japan), mouse IFN-ap (see reference 12) or mouse IFN-), (supernatant of ConA-stimulated spleen cell culture). After 5-6 days of incubation, the cells were pulse-labeled with 0.5 &i/well methyl-3Hthymidine for 4-6 h, and were then harvested in an automated collection device (Flow Laboratories, Rockville, MD, USA). Calorimetric determination of cell growth was determined by MTT methods (14). For the assay, 50 pug of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma) was added to cell culture wells and the plates were incubated at 37°C for 4 h. Acidisopropanol was added to all wells and mixed thoroughly to dissolve the dark blue crystals, and optical density was measured at 595 nm.
Effect of PLM on growth of myeloma cells PLM induced a dose-dependent inhibition of 3H-thymidine incorporation by the three strains of myeloma cells. This inhibitory effect actually reflected an inhibition of proliferation as demonstrated by calorimetric measurement of growth. Strains PX-63 and SP-2 were highly susceptible to PLMI at a low concentration, while strain NS-1 was resistant to the same concentration (Fig. 1).
Measurement of cytokine activity in PLM Detection of TNF-a activity. L929 cells suspended in RPM1 1640 medium containing 10% FCS were seeded at a density of 6X lo4 cells per well in 96-well tissue cul-
Growth inhibition pattern of TGF-@, TNF-a, IFN-ap, IFN-y, in myeloma cell lines TNF-a had no effect on the proliferation of any of the three myeloma cell lines. TGF-/3 exhibited a moderate
226
Results
Hepatocyte derived growth inhibitor
dose-dependent inhibitory effect on NS-1 cells; however, it failed to inhibit the proliferation of PX-63 and SP-2 cells. IFN-a/? and IFN-1, had similar dose-de-
pendent inhibitory lines (Fig. 2). Cytokine
activity
effects in all three myeloma cell
in PLM
6 ‘Z a ._
60
TNF-a and IFN-y activity were not detected in PLM. IFN-c@ activity was present in PLM at a concentration of less than 10 units/ml (15). PLM contained TGF-j3 in the range of 1000 of 3000 &ml, but acid treatment was necessary before the assay detected TGF-P, indicating that TGF-j? in PLM was present in a latent form (Table 1).
2 @.
40
Partial characterization
3H-thymldine
uptake
80
20
0
0
3.2
6.3
12.5
Concentration
25
O--/c1 3.2
50
of PLM (a)
6.3
12.5
Concentrsiion
25
50
of PLM (96)
Fig. 1. Effects of the conditioned medium of mouse parenchymal liver cells (PLM) on the growth of three strains of myeloma cells. Myeloma cells (5X102), NS-1 (a), PX-63 (A), and SP-2 (0) were exposed to the indicated concentrations of PLM and incubated at 37°C for 54 days. The number of cells was determined by the MTT or 3H-thymidine uptake method.
100
100-
TGF-f3
:I s
80
m-
MI
3 60P
f
I
TNF-a
20
0
0.1
16
40010000
The inhibitory effect was completely abolished when PLM was heated to 85°C for 30 min, and the effect was markedly reduced by trypsin treatment (Table 2). On gel filtration chromatography, the growth inhibitory activity toward myeloma cells was eluted in two peaks, one with an approximate molecular weight of 18 kDa and the second one at below 10 kDa. The second peak of growth inhibitory activity was observed in the control culture medium, and therefore the molecular weight of PLMI was estimated to be about 18 kDa (Fig. 3). On isoelectronic focusing, the growth inhibitory activity was focused in four peaks, at p1 values of 3.84.0, 6.1-6.3, 6.6, and 7.2-7.4. The growth inhibitory activity of the three peaks with p1 values of 6.16.3, 6.6, and 7.2-7.4 was found in the control medium, and so the p1 of PLMI was estimated to be 3.84.0 (Fig. 3).
Discussion
_
ipa.
Z”
of PLMI
63
125 250 500 10002000
rTGF-P (pg/ml)
The results of these experiments indicated potent PLMI growth inhibitory activity in myeloma cells. In the myeloma cell strain, NS-1, proliferation was
rTNF-a (p@nU
TABLE
EN-a!3
1
Detection
of cytokine
TNF-a
IFN-a/3
ND’
2.7-7.5
activity
in mouse parenchymal
units/ml
liver cells
IFN-y
TGF-/l
ND
102&3192
ng/ml*
’ Not detected. * Detected after acidification. TNF=tumor necrosis factor; IFN=interferon. 0
0.060.25
1
3.8
15
60
J.FN-up (units/ml)
0
0.060.25
1 EN-y
3.8
IS
60
(lmm/d)
Fig. 2. Effect of transforming growth factor (TGF)-fi, interferon (IFN)-ap, IFN-y, and tumor necrosis factor (TNF-a) on the growth of three strains of myeloma cells. Myeloma cells (5x102), NS-I (O), PX-63 (A), and SP2 (O), were exposed to the indicated concentration of TGFp, IFN-a/3, IFN-y, and TNF-a and incubated at 37°C for 54 days. The number of cells was determined by the MTT method.
TABLE
2
Effect of heat and trypsin parenchymal liver cells
treatment
on the inhibitory
Treatment
Percent inhibition
no WC, 30 min 85”C, 30 min Trypsin
58-78 3661 o-22 22-l 1
effect of mouse
(%)
227
M. Takahashi and K. Yokomuro
suppressed by a high concentration of PLM, while this cell line was fairly resistant to the growth inhibitory activity of PLM at a low concentration, compared with the effects in the other two strains. To clarify the characteristics of the growth inhibitory factor in PLM, we compared the inhibition patterns of PLM in the three strains of myeloma cells with those of other wellknown factors: TGF-/I, IFN-o/I, IFN-y, and TNF-(r. TGF-P is a potential negative regulator of proliferation in many cell types (16). It was recently shown that TGF-/I enhanced IgA production by LPS-stimulated splenic B cells (17). We have observed that PLM, which was added to culture of LPS-stimulated B cells, enhanced the production of IgA (unpublished data). Therefore we regarded TGF-/I candidate as an inhibitory factor in the proliferation of myeloma cells, and we assayed the activity of TGF-fi in PLM. We found that a high concentration of TGF-/I (more than 1000 rig/ml) was produced in PLM, but that this TGF-j3 activity could not be detected unless PLM was treated with acid, indicating that the TGF-p was produced as a latent form. In the strain, NS-1, which was resistant to PLM, proliferation was slightly suppressed by rTGF-/3 at a high concentration; however, in the PX63 and SP-2 strains, which were susceptible to PLM, the proliferation was not suppressed at all by rTGF-13 at the same concentration. Besides we have found no effect of anti-TGF-j?l antibody in reducing the inhibitory effect of PLM (data not shown). These findings suggested that growth inhibitor for myeloma cells in PLM was not TGF-j?. None of the three strains of myeloma cells showed suppression of proliferation on treatment with rTNF-ol, nor was TNF-(r activity detected in PLM. Growth in three strains was suppressed by IFN-ccp and IFN-y, depending on their concen-
(A ) Gel Chromatography
PX-63
(B)JEF
Fig. 3. Gel-filtration chromatography (A) and isoelectronic focusing (B) of mouse parenchymal liver cell growth derived inhibitor. Individual fractions were tested for growth inhibitory activity. SP-2 cells (5~ 102) were incubated with each fraction for 5 days. The number of cells was determined by the MTT method.
228
tration, but there was no difference in the degree of suppression among these three strains. IFN-?/ activity was not detected in PLM, although IFN-c$ activity was detected at less than 10 units/ml. However, this concentration produced no difference among strains. On the basis of our findings, regarding the difference in suppression of proliferation among the three strains of myeloma cells, the activity of each factor in PLM, and molecular size and isoelectronic point of the active fraction, we believe that the growth inhibitory substance in PLM is not TGF-/I, TNF-a, IFN-a/3 or IFNy. Several investigators have demonstrated the existence of the cell growth modulator in the liver. Chapekar et al. reported the presence of a factor in rat liver homogenate that had exerted negative and positive effects on cell proliferation (18). Woodman et al. reported that after partial hepatectomy nonparenchymal cells secreted hepatocyte proliferation growth inhibitor (19). They showed that their factors were distinct from TGF-/I or any other previously described inhibitor of cell proliferation. Why the myeloma strains showed different susceptibility to the inhibitory factor in PLM is not known, As one of the noticeable different features between the susceptible and the resistant strains, it was shown that the quantity of 3H-thymidine uptake differs remarkably in these strains. The number of myeloma cells was calculated every 24 h by both the MTT and 3H-thymidine uptake methods. The proliferation of culture cells assayed by the MTT method was increased by approximately the same amount in all three strains, while the quantity of 3H-thymidine uptake in DNA was l/5-1/7 lower in the two susceptible strains compared with the resistant strain (Fig. 4). However, the total quantity of DNA was about the same in all three strains, sug-
SP-2
Days
Fig. oma The (0)
4. Growth rate of three strains of myeloma cells. Myelcells (5 X 102) were seeded in 100 ,ul medium per well. number of cells was determined every 24 h by the MTT or 3H-thymidine uptake (0) method.
Hepatocyte derived growth inhibitor
gesting that there may be a difference in the synthesizing process of pyrimidine nucleotide. It is not clear whether this is related to the difference in the susceptibility of myeloma cells to PLM. However, it appears to be important in understanding the suppressive mechanism that NS-1, a resistant clone, has characteristics similar to those of PX-63, a sensitive clone, since these two clones were derived from the same source.
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10. Rosenbaum J, Philippe M, Preaux AM, Lesce MC, Dhumeaux D. Mouse hepatic endothelial cells in culture secrete a growth inhibitor for hepatic lipocytes and Balb/c3T3 fibroblasts. J Hepatol 1989; 9: 2955300. 11. Sakamoto T, Mabuchi A, Kuriya SI, Sudo T, Aida T, Asano G, Shoji T, Yokomuro K. Production of granulocyte-macrophage colony-stimulating factor by adult murine parenchymal liver cells (hepatocytes). Reg Immunol 1991; 3: 26& 7. 12. Tsukui T, Kikuchi K, Mabuchi A, Sudo T, Sakamoto T, Yokomuro K, Sato N, Tsuneoka K, Shikita M, Aida T, Asano G, Watari E. Production of macrophage colony-stimulating factor by adult murine parenchymal liver cells (hepatocytes). J Leukocyte Biol 1992; 52: 383-389. 13. Tsukui T, Kikuchi K, Mabuchi A, Sudo T, Sakamoto T, Yokomuro K. Production of interleukin-1 by primary cultured parenchymal liver cell (hepatocytes). Exp Cell Res 1994; 210: 172-6. 14. Mosmann T. Rapid calorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Meth 1983; 65: 55-63. 15. Mabuchi A, Watari E, Ikeda M, Watanabe Y, Yokomuro K. Growth control of primary culture hepatocytes by nonparenchymal liver cells: role of interferon produced by liver sinusoidal cells. J Nippon Med Sch 1994; 61: 120-8. 16. McMahon J, Richards WL, Campo AA, Song MKH, Thorgeirsson SS. Differential effects of transforming growth factor-p on proliferation of normal and malignant rat liver epithelial cells in culture. Cancer Res 1986; 46: 4665-71. 17. Sonoda E, Hitoshi Y, Yamaguchi N, Ishii T, Tominaga A, Takatsu K. Differential regulation of IgA production by TGF-/J induces surface &A-positive cells bearing IL-5 receptor, whereas IL-5 promotes their survival and maturation into IgA-secreting cells. Cell Immunol 1992; 140: 158872. 18. Chapekar MS, Huggett AC, Thorgeirsson SS. Growth modulatory effects of a liver-derived growth inhibitor, transforming growth factor /?l, and recombinant tumor necrosis factor (x, in normal and neoplastic cells. Exp Cell Res 1989; 185: 247757. 19. Woodman AC, Selden CA, Hodgson HJF. Partial purification and characterization of an inhibitor of hepatocyte proliferation derived from nonparenchymal cells after partial hepatectomy. J Cell Physiol 1992; 151: 405-14.
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