International Immunology, Vol 3, No. 6, pp. 571 -577
© 1991 Oxford University Press 0953-8178/91 $3.00
IL-1 up-regulates the expression of cytokine receptors on a factor-dependent human hemopoietic cell line, TF-1 Toshio Kitamura1'2, Fumimaro Takaku2, and Atsushi Miyajlma1 'DNAX Research Institute of Molecular and Cellular Biology, 901 California Avenue, Palo Alto, CA 94304, USA 2 The Third Department of Internal Medicine, Faculty of Medicine, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo Key words. Epo receptor, GM-CSF receptor, IL-3 receptor, IL-5 receptor, factor-dependent cell line
A factor-dependent human hemopoietic cell line, TF-1, requires interleukln 3 (IL-3) or granulocyte/macrophage colony-stimulating factor (GM-CSF) for its long-term growth. We have found that IL-4, IL-5, and IL-6 also support the growth of TF-1 and that IL-1 enhances the prollferative effect of these cytoklnes. Augmentation by IL-1 is associated with up-regulatlon of the receptors for IL-3, IL-5, GM-CSF, and erythropoietln (Epo). IL-1 increased the number of binding sites for IL-3 and Epo without changing their affinities. In contrast, IL-1 Increased the number of high affinity binding sites for GM-CSF and IL-5, whereas the total number of binding sites was unchanged. Chemical crossllnklng experiments Indicated that the receptors for IL-3, IL-5, and GM-CSF were composed of two components and that the molecular masses of the larger components of these cytokine receptors were quite similar (120 kd). The enhanced expression of the larger components of the IL-3, IL-5, and GM-CSF receptors by IL-1 may be responsible for IL-1-Induced up-regulation of these receptors. These observations are consistent with the model that the receptors for IL-3 and GM-CSF share a common component. Introduction Hemopoiesis is regulated by a number of cytokines and stromal cells, lnterleukin-3 (IL-3) and granulocyte/macrophage colonystimulating factor (GM-CSF) act on early hemopoietic progenitors and affect the development of various cell lineages. On the other hand, IL-5, granulocyte-colony stimulating factor (G-CSF), macrophage-colony stimulating factor (M-CSF) and erythropoietin (Epo) are more lineage specific. IL-1, IL-4, and IL-6 show pleiotropic functions on a wide variety of cells (1). In particular, IL-1, produced by different types of cells, acts on early hemopoietic precursors and enhances the effects of other cytokines such as IL-3 and GM-CSF (2), and is also known as hemopoietin-1 (3). There are two structurally distinct IL-1 molecules, IL-1 a and IL-10, encoded by two distinct genes. Despite their structural difference, both IL-1a and IL-1/3 bind to the same receptor and exhibit similar biological activities (4). Cell lines which respond to multiple cytokines are quite useful for the study of cytokine functions. Recently we have established a unique human cell line, TF-1, from a patient with erythroleukemia, which requires IL-3, GM-CSF, or Epo for its growth (5). Here we describe further characterization of this cell line and expand the list of the cytokines that stimulate the
Correspondence to: A. Miyajima Transmitting editor: M. Miyasaka
proliferation of TF-1. We also describe how IL-1 stimulates the proliferation of TF-1 in a synergistic fashion with other cytokines. As cytokines manifest their biological activities through specific receptors and signal transduction pathways, IL-1 may affect components of various cytokine receptors and augment their function. Molecular cloning and characterization of the cytokine receptor genes have revealed that high affinity functional receptors for cytokines such as IL-2 (6), IL-3 (7), IL-5 (8), IL-6 (9,10) and GM-CSF (11,12) are composed of multiple subunits. Recently high affinity receptors for IL-2 (6), GM-CSF (12) and IL-6 (13) have been reconstituted with molecularly cloned receptor components. In these cases, at least two different components are required for high affinity binding of ligands. Whereas the cloned IL-3 (7) and IL-5 receptors (8) bind their ligands with low affinity, high affinity receptors are present on cells which respond to these factors (14,15). Clearly an additional molecule(s) is also required for high affinity ligand binding in these cytokine receptor systems. We have recently published data showing that TF-1 cells express functional receptors for Epo (16), IL-3 (17), and GM-CSF (18). Here we present the enhancing action of IL-1 with IL-3, GMCSF, IL-5, and Epo using TF-1 cells as a model and demonstrate
Received 27 January 1991, accepted 15 March 1991
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Abstract
572
Up-regulation of cytokine receptors by IL-1
that IL-1 up-regulates the high-affinity receptors for these cytokines. Methods TF-1 cell line and cytokines
Proliferation assays
Results Enhancing effect of IL-1 with other cytokines TF-1 cells proliferate in response to IL-3, GM-CSF, and Epo and deprivation of these factors results in cell death within 2 - 3 days (5). We examined the effect of other cytokines including IL-4, IL-5, and IL-6 on the proliferation of TF-1 and found that IL-4, IL-5, and IL-6 sustained the growth of TF-1 in short-term proliferation assays using MTT and thymidine uptake (Fig. 1). In particular,
Cell proliferation was examined mainly by the colorimetric assay (MTT assay) developed by Mossman (19) with minor modifications (5). In brief, after culturing cells using 96-well plates for 48 h in the presence or absence of various concentrations of cytokines, 3-{4,5-dimethylthiazol-2-yl)-2, 5-diphenyitetrazolium bromide (MTT, Sigma) was added (final concentration: 0.5 mg/ml) and the incubation was continued for 6 h. The insoluble purple reaction product produced by MTT reduction was then dissolved in isopropanol/0.04 N HCI, and the optical density (OD: 570-630 nm) was measured with an ELISA reader. The amount of MTT reduction is proportional to the number of the viable cells (19). In some of the experiments, tritiated thymidine (PHJTdR) uptake was also measured. TF-1 cells (1 x 104 cells/well) cultured in the absence of GM-CSF for 20 h were incubated with 0.1 /id of [3H]TdR (Amersham) for 4 h in the presence of various concentrations of cytokines. The cells were harvested onto an absorbent glass paper and counted for radioactivity associated with the cells by a scintillation counter. Binding assays Recombinant human GM-CSF, IL-3, and IL-5 were iodinated using the Bolton-Hunter reagent as described previously (18), and erythropoietin was iodinated by the Chloramine-T method (20) with minor modifications (21). Binding assays were performed as follows: cells were incubated with iodinated cytokines at 4°C for 2 h for GM-CSF, IL-5, and Epo and 8 h for IL-3 to obtain equilibrium binding. Cell-bound radioactivity was separated from free ligand by centrtfugation through n-buthyl phthalate The equilibrium binding data was analysed by the Ligand program (22). Chemical crosslinking Cells incubated with 2 nM of iodinated cytokines at 4°C for 2 h for GM-CSF, IL-5, and Epo and 8 h for IL-3 were collected by centrifugation and resuspended in PBS containing 200 pM
irr 5
irr 4
10-3
irr 2
COS supernatant dilution (IL-4) Hi- ll.1
1
10
100
ng/m/ (IL-5. IL-6) Fig. 1. Response of TF-1 cells to IL-4, IL-5, and IL-6. Growth response of TF-1 cefls to IL-4 (open circle), IL-5 (dosed circle), and IL-6 (open box) were assessed by MTT assay (upper panel) and [3H]thymidirte uptake Cower panel). Results are shown as the mean ± SD of triplicates.
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TF-1 cells (5) are maintained in RPM11640 containing 10% fetal calf serum and 5 ng/ml recombinant GM-CSF. For the IL-1 treatment, the cells were collected, washed twice with phosphatebuffered saline (PBS), and incubated for 20 h in RPMI 1640 medium containing 10 ng/ml IL-1 a. Recombinant human GM-CSF produced in Eschenchia coli, recombinant human IL-3 produced in yeast, and recombinant human IL-5 produced in Chinese hamster ovary cells were provided by Schering-Plough. Recombinant human IL-1 a and 0 produced in E.coli were kind gifts from Ohtsuka Pharmaceutical Co. (Tokushima, Japan). Recombinant IL-4 was produced in COS7 cells transiently transfected with the IL-4 cDNA. Recombinant human IL-6 produced in E.coli was generously provided by Genetics Institute. Recombinant human Epo was a kind gift from Kirin Brewery Co. (Gunma, Japan).
disuccinimidyl suberate (DSS; Pierce Chemical Co., Rockford). After 15 min incubation at 4°C, the cross-linking reaction was stopped by adding 1 ml of ice-cold quenching buffer (50 mM Tns- HCI, pH 7.4,150 mM NaCI, 1 mM EDTA). The cells pelleted by centrifugation were solubilized in 1 % Triton X-100, 50 mM HEPES (pH 7.4), 1 mM phenyimethylsulphonyl fluoride, and 500 U/ml aprotinin. After 1 min incubation on ice, soluble fraction was obtained by centrifugation at 15,000 g for 3 min. Solubilized proteins were mixed with 1/4 volume of 5-fold concentrated Laemmh's sample buffer and analyzed on an SDS/8% polyacrylamide gel. Fixed, stained and dried gels were subjected to autoradiography for 3 - 7 days using Kodak X-Omat AR film with Dupont Lightning Plus intensifying screens.
Up-regulation of cytokine receptors by IL-1 573 themselves. However, both IL-1a and IL-1/3 strongly augmented the proliferate effect of IL-3, IL-4, IL-5, IL-6, Epo, and GM-CSF in a dose-dependent manner (Fig. 3). To eliminate the possibility that IL-1 might induce the cytokine production by TF-1 cells, we examined the prohferative activity against TF-1 in the culture supernatant of IL-1 treated TF-1 cells. No such prdiferative activity was detectable from the conditioned media of IL-1 treated TF-1 cells (data not shown).
IL-5 stimulated proliferation and increased cell number, whereas IL-4 and IL-6 only prolonged cell survival (Fig. 2). In contrast to IL-3 and GM-CSF, IL-5 was not able to support the growth of TF-1 cells for more than one week. IL-1a and IL-1/3 had a small effect on cell survival of TF-1 by
0.4 • PI
Up-regulation of cytokine receptors by IL-1
ID
•§ o.i |
Fig. 2. Growth of TF-1 cells in the presence or absence of IL-4, IL-5, or IL-6 Cells were seeded in a 96-well microplate at lO/Vwell in the presence of a 10~ 3 concentration of COS supernatant including IL-4 (open circle), 10 ng/ml IL-5 (dosed circle), or 10 ng/ml IL-6 (open box), or in the absence of these cytokines (closed box). Cell growth was assessed by the MTT assay on days 1,2,4, and 6. Results shown are the mean ± SD of triplicates.
0.1 ng/ml
IL-3
0.3 U/m!
Epo
0.1 ng/ml
GM-CSF
10~J 10"'
10
1 ng/ml
100
IL-5
IL-4
10 ng/ml
IL-6
None
B
¥
0 5-
• ZH • •
g l 0.4-
0.-10 0>m mi H--I0 W ml L-l/5 IOn(( ml O--I0 100ml "">
I S 0.3-|
o.i 0.1 ng/m)
0.3U/ml
0.1 ng/ml
IL-3
Epo
GM-CSF
10
1 ng/ml
IL-5
100
10ng/ml
IL-6
None
Fig. 3. Enhancing effects of IL-1a and IL-10 on the factor-dependent growth of TF-1 cells. Cells were seeded in 96-well microplates at ICH/well in the presence of various cytokines at the indicated concentrations and 0 - 1 0 0 ng/ml of either IL-1a or IL-1/3. Two days later, ceil growth was assessed by the MTT assay. Results shown are the mean ± SD of triplicates.
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As cytokines function through their receptors, the augmentation of the proliferative effects by IL-1 may be due to changes in the receptors and/or their signal transduction systems. We therefore examined the effect of IL-1 on expression of cytokine receptors. Since TF-1 requires either IL-3 or GM-CSF for continuous growth, we usually maintained the cells in medium containing 5 ng/ml GM-CSF; this may have some effect on the expression of cytokine receptors. It is difficult to determine the basal levels of receptor expression in factor-dependent cell lines. We have defined the basal expression level of the receptors for IL-3, IL-5, and Epo in the TF-1 cells cultured in the presence of 5 ng/ml GM-CSF. In this condition, more than half of GM-CSF receptors on TF-1 cells are down-modulated by GM-CSF (data not shown). The basal expression level of GM-CSF receptors was determined in cells cultured in the presence of 10 ng/ml IL-3. To examine the effect of IL-1 on the expression of cytokine receptors, TF-1 cells
574
Up-regulation of cytokine receptors by IL-1
cultured in medium containing either GM-CSF or IL-3 were washed and incubated in the presence or absence of IL-1, and cytokine binding was measured after the incubation. Binding of IL-3, IL-5, GM-CSF, and Epo rapidly increased by culturing cells in the presence of 10 ng/ml IL-1 and reached a plateau level within 6 - 20 h (data not shown). Up-regulation of these receptors by IL-1 was dependent on IL-1 concentration and the maximum effect was obtained at 1 ng/ml of IL-1 (data not shown). In the following experiments, cells were incubated with IL-1 for 20 h at a concentration of 10 ng/ml. We then performed Scatchard analysis using cells that had been incubated for 20 h in the presence or absence of 10 ng/ml IL-1 and compared the expression levels of cytokine receptors (Fig. 4 and Table 1). Binding of IL-3 to TF-1 cells showed a single class of high affinity binding (420 pM). Incubation of cells with IL-1 resulted in increase of receptor number by 5-fold without
a significant change in the dissociation constant. The Epo binding showed a similar binding profile as that of IL-3: a single class of binding site (640 pM) was detected and it was also increased 2.5-fdd by IL-1. This increase was not associated with a significant change in the dissociation constant. The reason for the slight increases in the number of the IL-3 receptor and the Epo receptor in the factor-depletion condition compared to the basal level is not clear, but it is possible that GM-CSF may downregulate the expression of these receptors. In contrast to the IL-3 and Epo receptors, IL-1 increased the high affinity binding sites for GMCSF by 5.6-fold, whereas it did not significantly change the total number of binding sites (-10,000/ceH). IL-5 binding also showed a similar binding profile as that of GM-CSF: high (120 pM) and low (2 nM) affinities were present and the high affinity sites increased by 4.6-fold after IL-1 treatment while the total binding sites increased only 1.3-fold.
0.3-1
IL-3 Receptor
GM-CSF Receptor
0.2 0.10 1000 Sites/Cell
01-1
2000
4000
8000 12000 16000 Sites/Cell
Epo Receptor
IL-5 Receptor 0.1-
1000 2000 3000 Sites/Cell
4000
400 Sites/Cell
800
Fig. 4. Scatchard analysis of cytokine receptors. Open boxes indicate the basal levels of receptor expression- the cells which had been maintained with 5 ng/ml hGM-CSF were used for the analysis of the expression of the receptors for IL-3, IL-5, and Epo, and the cells which had been maintained with 5 ng/ml hlL-3 were used for the analysis of the expression of the GM-CSF receptor. Closed circles indicate the receptors on ceils incubated with 10 ng/ml IL-1a for 20 h. The open circles shown in the left two panels are the Scatchard plot of the TF-1 cells cultured in the absence of any cytokines for 20 h (factor depletion) For GM-CSF and IL-5 receptor, factor depletion also had similar small effects. These figures are representative of two to four independent experiments which gave essentially identical results.
[12Sl]cytokines
To examine which component of the cytokine receptors was increased by the IL-1 addition, we performed cross-linking experiments using 125l-labeled cytokines (Fig. 5). Cross-linking of GM-CSF to TF-1 cells usually showed three bands of about 95, 135, and 215 kd which represent the a subunit (80 kd), the /3 subunit (120 kd) and the complex of the two subunits, respectively after the reduction of the molecular weight of GMCSF (15 kd). IL-1 increased the intensity of those three bands. In particular, the band at 135 kd increased more than the other bands. Cross-linking of IL-3 also showed three proteins of about 85, 135, and 205 kd and IL-1 increased these three proteins equally. Cross-linking of IL-5 revealed two bands of 75 and 140 kd and IL-1 clearly increased the 140 kd band, but not the 75 kd band. Cross-linking of Epo showed also two bands of 125 and 140 kd, and IL-1 increased the intensity of the both bands equally.
Discussion IL-1, also known as hemopoietin-1, has hemopoietic potentiating activities and acts on early hemopoietic progenitors synergistically with hemopoietic growth factors such as IL-3 and GM-CSF (2). IL-1 is known to stimulate the growth of hemopoietic cells by inducing the production of hemopoietic growth factors of accessory cells (23,24). Although some reports suggested that
Table 1. IL-1 up-regulation of cytokine receptors expressed on TF-1 cells Factor depletion11
Basal leveP
Site/cell IL-3R EpoR GM-CSFR IL-5R
340 1300 1700 9460 130 340
± ± ± ± ± ±
*d(plM) 120 260
300 1220 60 120
420 ± 90 640 ± 210
60 2100 120 2100
± ± ± ±
30 450 50 540
IL-1 treatment0
Site/cell
Kd(pM)
Site/cell
M l pM)
550 ± 130 1680 ± 190 4800 ± 1720
450 ± 120 680 ± 170 350 ± 160
1820 ± 320 3260 ± 680 9490 ± 3530
320 ± 100 770 ± 140
380 ± 120
310 ± 100
600 ± 80
Results shown are the average ± standard deviation of at least three independent experiments. The basal levels of the receptor expression were examined as described in the legend to Fig. 4. b The cells were washed twice and incubated for 20 h in the absence of any growth factor. c The cells were washed twice and incubated for 20 h in the presence of 10 ng/ml IL-1 a. a
200 ± 70 280 ± 80
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Cross-linking of
Up-regulation of cytokine receptors by IL-1 575
Interestingly, whereas IL-1 up-regulates the IL-3 and Epo receptors without affinity change, IL-1 increases the number of the high affinity receptors for GM-CSF and IL-5 but decreases the low affinity binding sites for GM-CSF and IL-5. Thus, the total binding sites for GM-CSF and IL-5 are not substantially changed by IL-1 (Fig. 4). This observation is quite interesting in view of the molecular composition of the cytokine receptors, because the high affinity receptors for those cytokines are composed of multiple subunits. Although the dissociation constants for the high affinity receptors for GM-CSF and IL-5 increased slightly by the
GM-CSF.R
KDal 205-
IL-3'R
O =d £ o d S
IL-1 treatment, it is unlikely that a third class (intermediate affinity) receptors emerge from the IL-1 treatment, because the sizes of the cross-linked molecule are not changed at all by the IL-1 treatment (Fig. 5). The reason for the slight increase of the dissociation constants caused by IL-1 treatment is currently unknown. The Epo receptor cDNA, which was cloned from a murine erythroleukemic (MEL) cell line subclone 745, expresses two classes of binding sites (Kd 30 pM and 210 pM) on COS7 cells, whereas the MEL subclone 745 shows a single class of binding sites (Kd 240 pM). This result suggests that a cell type specific protein(s) may affect the affinity of the Epo receptor (29). The affinity of the Epo receptor present on TF-1 cells is similar to that of MEL cells subclone 745 and the binding is up-regulated by IL-1 without changing the affinity. Molecular composition of the Epo receptor is still unclear. However, if multiple subunits are involved in binding of Epo, all the components may be upregulated by IL-1. The mouse IL-3 receptor cDNA encodes a mature protein of 120 kd which binds IL-3 with low affinity and additional component(s) should be required for high affinity binding (7). In contrast to the mouse system, there is no evidence indicating the presence of a low affinity IL-3 receptor in human. Although the human IL-3 receptor gene has not yet been isolated, crosslinking experiments suggest the presence of multiple components for the human IL-3 receptor (Fig. 5) (17). Since IL-1 increases the number of binding sites as well as the amount of all the crosslinked proteins, IL-1 may enhance the expression of all those binding components. An alternative possibility is discussed below. In contrast to receptors for Epo and IL-3, TF-1 cells express both high- and low-affinity receptors for GM-CSF and IL-5 as was shown in other cell lines (14,18,30). Recent cloning of the gene
EpcR
KDal
o
IL-5«R
KDal
— 205-
77-
116.577-
43-
43
Fig. 5. Chemical cross-linking of iodinated cytokines to TF-1 cells. The chemical cross-linking experiments were performed as described in Methods on the cells before and after the IL-1 treatment or factor depletion. Cells grown in the presence (IL-1) or absence (FD) of IL-1 are compared with cells (C) grown in normal medium containing either GM-CSF (for IL-3 and IL-5 binding) or IL-3 (for GM-CSF binding).
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colony formation of hemopoietic progenitor cells was directly enhanced by IL-1 (3,25), it was difficult to eliminate the effects of accessory cells completely and the mechanism of this direct synergism has been unclear. Our present data, however, demonstrates that IL-1 enhances the factor-dependent growth of TF-1 cells directly. Thus, a human hemopoietic cell line, TF-1, is particularly useful to study the combined action of IL-1 with other cytokines because the proliferation of TF-1 is strictly dependent on multiple cytokines including IL-3, IL-5, GM-CSF, and Epo, and IL-1 augments the prohferative effects of these cytokines (Fig. 2) (5). In the IL-2 receptor system, it has been reported that IL-1 upregulated the expression of IL-2 receptor (26). We have demonstrated that IL-1 up-regulates the high affinity receptors for IL-3, IL-5, GM-CSF, and Epo. The increased number of receptors may help the oligomerization of the receptor, which seems to be important for signaling in receptor systems such as epidermal growth factor (27) or platelet-derived growth factor (28). Alternatively, it is possible that a minor population of cells bearing a large number of high affinity receptors is present in normal culture conditions and this population is increased by IL-1 treatment. Interrelation between the number of the high-affinity receptors and proliferation potential is not clear.
576
Up-regulation of cytokine receptors by IL-1
Low Affinity
High Affinity
Wgh Affinity
GM-CSF-R
IL-3-R
Low Affinity
IL-5-R
Fig. 6. A possible mechanism of the upregulation of the receptors for human GM-CSF, IL-3, and IL-5
cells have only the high affinity binding site for IL-3, another component of the IL-3 receptor (70 kd) may bind IL-3 with extremely low affinity or may not bind by itself or the abundance of this component may be low. Up-regulation of the 120 kd protein by IL-1 explains the increase of both IL-3 and GM-CSF binding. The observation (34) that IL-5 binding was inhibited by either IL-3 or GM-CSF in basophils leads us to speculate that the /3 subunit of the GM-CSF receptor may also be involved in the formation of the high affinity IL-5 receptor. Molecular cloning of the other subunits of the IL-3 and IL-5 receptors and reconstitution of the high affinity receptors will answer these questions.
Acknowledgements
Similarly, the high affinity IL-5 receptor is also composed of at least two components (55 and 120 kd). The 55 kd subunit binds IL-5 with low affinity and the 120 kd protein was reported to have a relationship with the high affinity binding of IL-5 (14). In the absence of IL-1, TF-1 cells show two classes of IL-5 binding sites. The low affinity binding is probably mediated by the 55 kd subunit and the high affinity site should be composed of the 55 and 120 kd subunits. Again IL-1 treatment increases the number of high affinity sites without increasing the total binding sites. This can be also explained by the selective enhancement of the expression of the 120 kd subunit (Fig. 6). We identified the /3 subunit of the GM-CSF receptor by molecular cloning. Initially we isolated a human cDNA based on homology with the mouse IL-3 binding protein (12). Interestingly, however, the protein encoded by the cloned human cDNA does not bind IL-3 but it conferred a high affinity binding to GM-CSF when it is co-expressed with the a subunrt of the GM-CSF receptor. Although we extensively searched for a cDNA encoding a human IL-3 binding protein using the mouse IL-3 receptor cDNA as a probe, we have not obtained such a clone. It is most likely that, in human, the ft subunit of the GM-CSF receptor is shared by both IL-3 and GM-CSF receptors. In this case, the /3 subunit of the GM-CSF receptor forms a high affinity receptor for IL-3 with an additional component, possibly the 70 kd protein which can be cross-linked with IL-3. This explains the observation that IL-3 competes for GM-CSF binding to the high affinity site and GM-CSF competes for IL-3 binding (31 -33). Since TF-1
We are grateful to Drs T. Yokota, and K. Arai for helpful discussion and for providing IL-4, and to Miss Chie Misawa for her technical assistance We also thank to Drs S. Tindall (Schering Plough), M. Takahashi and Y. Masui (Ohtsuka Pharmaceutical), S. Clark (Genetics Institute), and T. Suzuki (Kirin Brewery) for providing cytokines. This work was supported in part by a Research Grant for Intractable Diseases from the Ministry of Health and Welfare of Japan and by grants-in-aids from the Ministry of Education, Science and Culture of Japan. DNAX Research Institute of Molecular and Cellular Biology is supported by the Schering-Plough Corporation.
Abbreviations cDNA DSS Epo GM-CSF IL MEL MTT
complementary DNA disuccinimidyl suberate erythropoietin granulocyte/macrophage colony-stimulating factor interleukin murine erythroleukemia 3-{4,5-dimethytthiazol-2-yf)-2, 5-diphenyttetrazolium bromide
References 1 Arai, K., Lee, F., Miyajima, A., Miyatake, S., Arai, N., and Yokota, T. 1990. Cytokines: coordinators of immune and inflammatory responses. Annu. Rev. Biochem. 59:783. 2 Moore, M. A. S. 1989. Role of interteukin-1 in hematopoiesis. Immunol. Rev. 8:165. 3 Mochizuki, D. Y., Eisenman, J. R., Cordon, P. J., Larsen, A. D., and Tushinski, R. J. 1987. lnterleukin-1 regulates hematopoietic activity, a rote previously ascribed to nemopcxetin-1. Proc. Natl Acad. Set. USA 84:5267. 4 Oppenheim, J. J. and Kovacs, E. J. 1986. There is more than one interleukin-1. Immunol. Today 7:45. 5 Kitamura, T., Tange, T., Terasawa, T., Chiba, S., Kuwaki, T., Miyagawa, K , Piao, Y.-F., Miyazono, K., Urabe, A., and Takaku F 1989. Establishment and characterization of a unique human cell line that proliferates dependently on GM-CSF, IL-3, or erythropoietin J. Cell Physhl. 140:323. 6 Hatakeyama, M., Tsudo, M., Minamoto, S., Kono, T., Doi, T., Miyata, T , Miyasaka, M., and Taniguchi, T. 1989. lnterleukin-2 receptor p chain gene: generation of three receptor forms by cloned human a and 0 chain cDNAs. Science 244:551. 7 Itoh, N., Yonehara, S., Schreurs, J., Gorman, D. M., Maruyama, K., Ishii, A., Yahara, I., Arai, K., and Miyapma, A. 1990. Cloning of an interleukin-3 receptor: a member of a distinct receptor gene family. Science 247:324. 8 Takaki, S., Tominaga, A., Hitoshi, Y., Mita, S., Sonoda, E., Yamaguchi, N., and Takatsu, K. 1990. Molecular cloning and expression of the murine interleukin-5 receptor. EMBO J. 94367.
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for a second component of the GM-CSF receptor has revealed that the high affinity GM-CSF receptor is composed of a (80 kd) and /3 (120 kd) subunits (12). The a subunit binds GM-CSF with low affinity (11), whereas the 0 subunit does not bind GM-CSF by itself. Co-expression of the a and /3 subunits forms a high affinity receptor for GM-CSF (12). This is consistent with the observation that the larger band detected by cross-linking is correlated with the high affinity binding of GM-CSF (18). If there is an excess amount of the a subunit in TF-1 cells, TF-1 cells should have high affinity receptors (a//S) and low affinity receptors (a). Since IL-1 increases the number of the high affinity sites without changing the total binding sites (Fig. 4) and preferentially increases the larger cross-linked band (Fig. 5), IL-1 may enhance the expression of the 0 subunit selectively. This is consistent with the results that the mRNA for the /3 chain of the GM-CSF receptor increased greatly with IL-1 treatment (unpublished results). This also explains the loss of low affinity sites by IL-1 because the increased level of the /3 subunrt reduces the free a subunit (Fig. 6).
Up-regulation of cytokine receptors by IL-1 577 23 Fibbe, W. E., Van Damme, J., BflBau, A., Gosefink, H. M., Voogt, P. J., Van Eeden, G., Ralph, P., Altonic, B. W., and Falkenberg, J. H. F. 1988. lnterfeukin-1 induces human marrow stromal cells in long-term culture to produce graruilocyte colony-stimulating factor and macrophage colony-stimulating factor. Blood 71:430. 24 Zsebo, K. M., Yuschenkoff, V. N., Schiffer, S., Chang, D., McCaU, E., Dinarrello, C. A., Brown, M. A., Altorock, B., and Bagley, G. C. J. 1988. Vascular endothelial cells and granulopoiesis. interteukin-1 stimulates release of G-CSF and GM-CSF. Blood 71:99. 25 Williams, D. E. and Broxmeyer, H E. 1988. lnterleukin-1 a enhances the in vitro survival of purified murine granulocyte-macrophage progenitor ceDs in the absence of colony-stimulating factor. Proc. Nati Acad. Sci. USA 84:3871. 26 Kaye, J , Gillis, S., Mizel. S B., Shevach, E. M., Malex, T. R., Dinarello, C. A., Lachman, L B., and Janeway, C. A. J. 1984. Growth of a cloned helper T cell line induced by a monoclonal antibody specific for the antigen receptor: interleukin 1 is required for the expression of receptors for interleukin 2. J. Immunol 133:1339. 27 Cochet, C , Kashles, O., Chambaz, E. M., Borrello, I., King, C. R., and Schlessinger, J. 1988. Demonstration of epidermal growth factor-induced receptor dimerization in living cells using a chemical covalent cross-linking agent. J Biol. Chem. 2633290. 28 Heldm, C. H., Ernlund, A., Rorsman, C , and Ronnstrand, L. 1989. Dimerization of B-type platelet-derived growth factor receptors occurs after ligand binding and is closely associated with receptor kinase activation. J. Bio!. Chem. 264:8905. 29 D'Andrea, A. D., Lodish, H. F., and Wong, G. G. 1989. Expression cloning of the munne erythropoietin receptor. Cell 57:277. 30 Walker, F. and Burgess, A. W. 1985 Specific binding of radioiodinated granulocyte-macrophage colony-stimulaling factor to hemopoietic cells. EMBO J. 4.933. 31 Park, L. S., Friend, D, Price, V., Anderson, D., Singer, J., Pnckett, K S., and Urdal, D. L. 1989. Heterogeneity in human interleukin-3 receptors. J. Bid Chem. 264:5420 32 Budel, L. M., Elbaz, O , Hoogerbrugge, H., Delwel, R., Mahmoud, L. A., Lowenberg, B., and Touw, I. P. 1990. Common binding structure for granulccyte macrophage colony-stimulating factor and interleukin-3 on human acute myeloid leukemia cells and monocytes. Blood 75:1439. 33 Onetto-Pothier, N., Aumont, N., Haman, A., Park, L , Clark, S. C , De Lean, A., and Hoang, T. 1990. IL-3 inhibits the binding of GMCSF to AML blasts, but the cytokines act synergistically in supporting blast proliferation. Leukemia 4:329. 34 Lopez, A. F , Eglinton, J. M., Lyons, A. B., Tapley, P. M., To, L. B., Park, L S., Clarke, S. C, and Vadas, M. A. 1990. Human interleukin-3 inhibits the binding of granulocyte-macrophage colony-stimulating factor and interleukin-5 to basophils and strongly enhances their functional activity. J. Cell. Physiol. 145:69.
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9 YamasaW, K., Taga, T., Hirata. Y., Yawata, H., Kawanishi, Y., Seed, B., Taniguchi, T., Hirarto, T. and Kishimoto, T. 1988. Cloning and expression of the human interieukin-6 (BSF-2/IFN02) receptor. Science 241:825. 10 Taga, T., Hibi, M., Hirata, Y., Yamasaki, K.. Yasukawa, K , Matsuda, T., Hirano, T., and Kishimoto, T. 1989. lnterteukin-6 triggers the association of its receptor with a possible signal transducer, gp130. Cell 58:573. 11 Gearing, D. P., King, J. A , Gough, N. M., and Nicola, N. A. 1989. Expression cloning of a receptor for human granulocyte-macrophage colony-stimulating factor. EMBO J. 8:3667. 12 Hayashida, K., Kitamura, T., Gorman, D. M., Arai, K , Yokota, T., and MiyajmaA 1990. Molecular cloning of a second subunit of the human GM-CSF receptor: reconstitution of a high affinity GM-CSF receptor. Proc. Nati Acad. Sci. USA 87:9655. 13 Hibi, M., Murakami, M., Sarto, M., Hirano, T., Taga, T., and Kjshimoto, T. 1990. Molecular cloning and expression of an IL-6 signal transducer, gp130. Cell 63:1149 14 Mita, S., Tominaga, A., Hitoshi, Y , Sakamoto, K., Honjo, T., Akagi, M., Kikuchi, Y., Yamaguchi, N., and Takatsu, K. 1989. Characterization of high-affinity receptors for mterieukin 5 on interleukin 5-dependent cell lines. Proc. Nati Acad. Sci USA 86:2311. 15 Schreurs, J., Aral, K , and Miyajima, A. 1990. Evidence for a lowaffinity interleukin-3 receptor. Growth Factors 2:221 16 Kitamura, T , Tojo, A , Kuwaki, T., Chiba, S., Miyazono, K , Urabe, A., and Takaku, F. 1989. Identification and analysis of human erythropoietin receptors on a factor-dependent cell line, TF-1. Blood 73:375. 17 Kuwaki, T , Krtamura, T., Tojo, A., Matsuki, S., Tamai, Y., Miyazono, K., and Takaku, F. 1989. Characterization of human interleukin-3 receptors on a mult-factor-dependent cell line Biochem. Biophys. Res Commun. 161:16. 18 Chiba, S., Tojo, A., Kitamura, T., Urabe, A., Miyazono, K., and Takaku, F. 1990. Characterization and molecular features of the cell surface receptor for human granulocyte-macrophage colonystimulating factor. Leukemia 4:22. 19 Mosmann, T 1983 Rapid colorimetric assays for cellular growth and survival, application to proliferation and cytotoxicity assays. J. Immunol. Methods 65:55. 20 Greenwood, F. C , Hunter, W. H., and Glover, J. S. 1963. The preparation of '31I-Iabeled human growth hormone of high-specific activity. Biochem. J. 89.114. 21 Fukamachi, H., Top, A , Sarto, T., Krtamura, T., Nakata, M., Urabe, A., and Takaku, F. 1987. Internalization of radioiodinated erythropoietin and the ligand-induced modulation of its receptor in murine erythroleukemic cells. Int. J. Cell Cloning 5.209. 22 Munson, P. J. 1983. LIGAND: a computerized analysis of ligand binding data. Methods Enzymot. 92:543.
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© 1991 Oxford University Press 0953-8178/91 $3.00
IL-1 up-regulates the expression of cytokine receptors on a factor-dependent human hemopoietic cell line, TF-1 Toshio Kitamura1'2, Fumimaro Takaku2, and Atsushi Miyajlma1 'DNAX Research Institute of Molecular and Cellular Biology, 901 California Avenue, Palo Alto, CA 94304, USA 2 The Third Department of Internal Medicine, Faculty of Medicine, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo Key words. Epo receptor, GM-CSF receptor, IL-3 receptor, IL-5 receptor, factor-dependent cell line
A factor-dependent human hemopoietic cell line, TF-1, requires interleukln 3 (IL-3) or granulocyte/macrophage colony-stimulating factor (GM-CSF) for its long-term growth. We have found that IL-4, IL-5, and IL-6 also support the growth of TF-1 and that IL-1 enhances the prollferative effect of these cytoklnes. Augmentation by IL-1 is associated with up-regulatlon of the receptors for IL-3, IL-5, GM-CSF, and erythropoietln (Epo). IL-1 increased the number of binding sites for IL-3 and Epo without changing their affinities. In contrast, IL-1 Increased the number of high affinity binding sites for GM-CSF and IL-5, whereas the total number of binding sites was unchanged. Chemical crossllnklng experiments Indicated that the receptors for IL-3, IL-5, and GM-CSF were composed of two components and that the molecular masses of the larger components of these cytokine receptors were quite similar (120 kd). The enhanced expression of the larger components of the IL-3, IL-5, and GM-CSF receptors by IL-1 may be responsible for IL-1-Induced up-regulation of these receptors. These observations are consistent with the model that the receptors for IL-3 and GM-CSF share a common component. Introduction Hemopoiesis is regulated by a number of cytokines and stromal cells, lnterleukin-3 (IL-3) and granulocyte/macrophage colonystimulating factor (GM-CSF) act on early hemopoietic progenitors and affect the development of various cell lineages. On the other hand, IL-5, granulocyte-colony stimulating factor (G-CSF), macrophage-colony stimulating factor (M-CSF) and erythropoietin (Epo) are more lineage specific. IL-1, IL-4, and IL-6 show pleiotropic functions on a wide variety of cells (1). In particular, IL-1, produced by different types of cells, acts on early hemopoietic precursors and enhances the effects of other cytokines such as IL-3 and GM-CSF (2), and is also known as hemopoietin-1 (3). There are two structurally distinct IL-1 molecules, IL-1 a and IL-10, encoded by two distinct genes. Despite their structural difference, both IL-1a and IL-1/3 bind to the same receptor and exhibit similar biological activities (4). Cell lines which respond to multiple cytokines are quite useful for the study of cytokine functions. Recently we have established a unique human cell line, TF-1, from a patient with erythroleukemia, which requires IL-3, GM-CSF, or Epo for its growth (5). Here we describe further characterization of this cell line and expand the list of the cytokines that stimulate the
Correspondence to: A. Miyajima Transmitting editor: M. Miyasaka
proliferation of TF-1. We also describe how IL-1 stimulates the proliferation of TF-1 in a synergistic fashion with other cytokines. As cytokines manifest their biological activities through specific receptors and signal transduction pathways, IL-1 may affect components of various cytokine receptors and augment their function. Molecular cloning and characterization of the cytokine receptor genes have revealed that high affinity functional receptors for cytokines such as IL-2 (6), IL-3 (7), IL-5 (8), IL-6 (9,10) and GM-CSF (11,12) are composed of multiple subunits. Recently high affinity receptors for IL-2 (6), GM-CSF (12) and IL-6 (13) have been reconstituted with molecularly cloned receptor components. In these cases, at least two different components are required for high affinity binding of ligands. Whereas the cloned IL-3 (7) and IL-5 receptors (8) bind their ligands with low affinity, high affinity receptors are present on cells which respond to these factors (14,15). Clearly an additional molecule(s) is also required for high affinity ligand binding in these cytokine receptor systems. We have recently published data showing that TF-1 cells express functional receptors for Epo (16), IL-3 (17), and GM-CSF (18). Here we present the enhancing action of IL-1 with IL-3, GMCSF, IL-5, and Epo using TF-1 cells as a model and demonstrate
Received 27 January 1991, accepted 15 March 1991
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Abstract
572
Up-regulation of cytokine receptors by IL-1
that IL-1 up-regulates the high-affinity receptors for these cytokines. Methods TF-1 cell line and cytokines
Proliferation assays
Results Enhancing effect of IL-1 with other cytokines TF-1 cells proliferate in response to IL-3, GM-CSF, and Epo and deprivation of these factors results in cell death within 2 - 3 days (5). We examined the effect of other cytokines including IL-4, IL-5, and IL-6 on the proliferation of TF-1 and found that IL-4, IL-5, and IL-6 sustained the growth of TF-1 in short-term proliferation assays using MTT and thymidine uptake (Fig. 1). In particular,
Cell proliferation was examined mainly by the colorimetric assay (MTT assay) developed by Mossman (19) with minor modifications (5). In brief, after culturing cells using 96-well plates for 48 h in the presence or absence of various concentrations of cytokines, 3-{4,5-dimethylthiazol-2-yl)-2, 5-diphenyitetrazolium bromide (MTT, Sigma) was added (final concentration: 0.5 mg/ml) and the incubation was continued for 6 h. The insoluble purple reaction product produced by MTT reduction was then dissolved in isopropanol/0.04 N HCI, and the optical density (OD: 570-630 nm) was measured with an ELISA reader. The amount of MTT reduction is proportional to the number of the viable cells (19). In some of the experiments, tritiated thymidine (PHJTdR) uptake was also measured. TF-1 cells (1 x 104 cells/well) cultured in the absence of GM-CSF for 20 h were incubated with 0.1 /id of [3H]TdR (Amersham) for 4 h in the presence of various concentrations of cytokines. The cells were harvested onto an absorbent glass paper and counted for radioactivity associated with the cells by a scintillation counter. Binding assays Recombinant human GM-CSF, IL-3, and IL-5 were iodinated using the Bolton-Hunter reagent as described previously (18), and erythropoietin was iodinated by the Chloramine-T method (20) with minor modifications (21). Binding assays were performed as follows: cells were incubated with iodinated cytokines at 4°C for 2 h for GM-CSF, IL-5, and Epo and 8 h for IL-3 to obtain equilibrium binding. Cell-bound radioactivity was separated from free ligand by centrtfugation through n-buthyl phthalate The equilibrium binding data was analysed by the Ligand program (22). Chemical crosslinking Cells incubated with 2 nM of iodinated cytokines at 4°C for 2 h for GM-CSF, IL-5, and Epo and 8 h for IL-3 were collected by centrifugation and resuspended in PBS containing 200 pM
irr 5
irr 4
10-3
irr 2
COS supernatant dilution (IL-4) Hi- ll.1
1
10
100
ng/m/ (IL-5. IL-6) Fig. 1. Response of TF-1 cells to IL-4, IL-5, and IL-6. Growth response of TF-1 cefls to IL-4 (open circle), IL-5 (dosed circle), and IL-6 (open box) were assessed by MTT assay (upper panel) and [3H]thymidirte uptake Cower panel). Results are shown as the mean ± SD of triplicates.
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TF-1 cells (5) are maintained in RPM11640 containing 10% fetal calf serum and 5 ng/ml recombinant GM-CSF. For the IL-1 treatment, the cells were collected, washed twice with phosphatebuffered saline (PBS), and incubated for 20 h in RPMI 1640 medium containing 10 ng/ml IL-1 a. Recombinant human GM-CSF produced in Eschenchia coli, recombinant human IL-3 produced in yeast, and recombinant human IL-5 produced in Chinese hamster ovary cells were provided by Schering-Plough. Recombinant human IL-1 a and 0 produced in E.coli were kind gifts from Ohtsuka Pharmaceutical Co. (Tokushima, Japan). Recombinant IL-4 was produced in COS7 cells transiently transfected with the IL-4 cDNA. Recombinant human IL-6 produced in E.coli was generously provided by Genetics Institute. Recombinant human Epo was a kind gift from Kirin Brewery Co. (Gunma, Japan).
disuccinimidyl suberate (DSS; Pierce Chemical Co., Rockford). After 15 min incubation at 4°C, the cross-linking reaction was stopped by adding 1 ml of ice-cold quenching buffer (50 mM Tns- HCI, pH 7.4,150 mM NaCI, 1 mM EDTA). The cells pelleted by centrifugation were solubilized in 1 % Triton X-100, 50 mM HEPES (pH 7.4), 1 mM phenyimethylsulphonyl fluoride, and 500 U/ml aprotinin. After 1 min incubation on ice, soluble fraction was obtained by centrifugation at 15,000 g for 3 min. Solubilized proteins were mixed with 1/4 volume of 5-fold concentrated Laemmh's sample buffer and analyzed on an SDS/8% polyacrylamide gel. Fixed, stained and dried gels were subjected to autoradiography for 3 - 7 days using Kodak X-Omat AR film with Dupont Lightning Plus intensifying screens.
Up-regulation of cytokine receptors by IL-1 573 themselves. However, both IL-1a and IL-1/3 strongly augmented the proliferate effect of IL-3, IL-4, IL-5, IL-6, Epo, and GM-CSF in a dose-dependent manner (Fig. 3). To eliminate the possibility that IL-1 might induce the cytokine production by TF-1 cells, we examined the prohferative activity against TF-1 in the culture supernatant of IL-1 treated TF-1 cells. No such prdiferative activity was detectable from the conditioned media of IL-1 treated TF-1 cells (data not shown).
IL-5 stimulated proliferation and increased cell number, whereas IL-4 and IL-6 only prolonged cell survival (Fig. 2). In contrast to IL-3 and GM-CSF, IL-5 was not able to support the growth of TF-1 cells for more than one week. IL-1a and IL-1/3 had a small effect on cell survival of TF-1 by
0.4 • PI
Up-regulation of cytokine receptors by IL-1
ID
•§ o.i |
Fig. 2. Growth of TF-1 cells in the presence or absence of IL-4, IL-5, or IL-6 Cells were seeded in a 96-well microplate at lO/Vwell in the presence of a 10~ 3 concentration of COS supernatant including IL-4 (open circle), 10 ng/ml IL-5 (dosed circle), or 10 ng/ml IL-6 (open box), or in the absence of these cytokines (closed box). Cell growth was assessed by the MTT assay on days 1,2,4, and 6. Results shown are the mean ± SD of triplicates.
0.1 ng/ml
IL-3
0.3 U/m!
Epo
0.1 ng/ml
GM-CSF
10~J 10"'
10
1 ng/ml
100
IL-5
IL-4
10 ng/ml
IL-6
None
B
¥
0 5-
• ZH • •
g l 0.4-
0.-10 0>m mi H--I0 W ml L-l/5 IOn(( ml O--I0 100ml "">
I S 0.3-|
o.i 0.1 ng/m)
0.3U/ml
0.1 ng/ml
IL-3
Epo
GM-CSF
10
1 ng/ml
IL-5
100
10ng/ml
IL-6
None
Fig. 3. Enhancing effects of IL-1a and IL-10 on the factor-dependent growth of TF-1 cells. Cells were seeded in 96-well microplates at ICH/well in the presence of various cytokines at the indicated concentrations and 0 - 1 0 0 ng/ml of either IL-1a or IL-1/3. Two days later, ceil growth was assessed by the MTT assay. Results shown are the mean ± SD of triplicates.
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As cytokines function through their receptors, the augmentation of the proliferative effects by IL-1 may be due to changes in the receptors and/or their signal transduction systems. We therefore examined the effect of IL-1 on expression of cytokine receptors. Since TF-1 requires either IL-3 or GM-CSF for continuous growth, we usually maintained the cells in medium containing 5 ng/ml GM-CSF; this may have some effect on the expression of cytokine receptors. It is difficult to determine the basal levels of receptor expression in factor-dependent cell lines. We have defined the basal expression level of the receptors for IL-3, IL-5, and Epo in the TF-1 cells cultured in the presence of 5 ng/ml GM-CSF. In this condition, more than half of GM-CSF receptors on TF-1 cells are down-modulated by GM-CSF (data not shown). The basal expression level of GM-CSF receptors was determined in cells cultured in the presence of 10 ng/ml IL-3. To examine the effect of IL-1 on the expression of cytokine receptors, TF-1 cells
574
Up-regulation of cytokine receptors by IL-1
cultured in medium containing either GM-CSF or IL-3 were washed and incubated in the presence or absence of IL-1, and cytokine binding was measured after the incubation. Binding of IL-3, IL-5, GM-CSF, and Epo rapidly increased by culturing cells in the presence of 10 ng/ml IL-1 and reached a plateau level within 6 - 20 h (data not shown). Up-regulation of these receptors by IL-1 was dependent on IL-1 concentration and the maximum effect was obtained at 1 ng/ml of IL-1 (data not shown). In the following experiments, cells were incubated with IL-1 for 20 h at a concentration of 10 ng/ml. We then performed Scatchard analysis using cells that had been incubated for 20 h in the presence or absence of 10 ng/ml IL-1 and compared the expression levels of cytokine receptors (Fig. 4 and Table 1). Binding of IL-3 to TF-1 cells showed a single class of high affinity binding (420 pM). Incubation of cells with IL-1 resulted in increase of receptor number by 5-fold without
a significant change in the dissociation constant. The Epo binding showed a similar binding profile as that of IL-3: a single class of binding site (640 pM) was detected and it was also increased 2.5-fdd by IL-1. This increase was not associated with a significant change in the dissociation constant. The reason for the slight increases in the number of the IL-3 receptor and the Epo receptor in the factor-depletion condition compared to the basal level is not clear, but it is possible that GM-CSF may downregulate the expression of these receptors. In contrast to the IL-3 and Epo receptors, IL-1 increased the high affinity binding sites for GMCSF by 5.6-fold, whereas it did not significantly change the total number of binding sites (-10,000/ceH). IL-5 binding also showed a similar binding profile as that of GM-CSF: high (120 pM) and low (2 nM) affinities were present and the high affinity sites increased by 4.6-fold after IL-1 treatment while the total binding sites increased only 1.3-fold.
0.3-1
IL-3 Receptor
GM-CSF Receptor
0.2 0.10 1000 Sites/Cell
01-1
2000
4000
8000 12000 16000 Sites/Cell
Epo Receptor
IL-5 Receptor 0.1-
1000 2000 3000 Sites/Cell
4000
400 Sites/Cell
800
Fig. 4. Scatchard analysis of cytokine receptors. Open boxes indicate the basal levels of receptor expression- the cells which had been maintained with 5 ng/ml hGM-CSF were used for the analysis of the expression of the receptors for IL-3, IL-5, and Epo, and the cells which had been maintained with 5 ng/ml hlL-3 were used for the analysis of the expression of the GM-CSF receptor. Closed circles indicate the receptors on ceils incubated with 10 ng/ml IL-1a for 20 h. The open circles shown in the left two panels are the Scatchard plot of the TF-1 cells cultured in the absence of any cytokines for 20 h (factor depletion) For GM-CSF and IL-5 receptor, factor depletion also had similar small effects. These figures are representative of two to four independent experiments which gave essentially identical results.
[12Sl]cytokines
To examine which component of the cytokine receptors was increased by the IL-1 addition, we performed cross-linking experiments using 125l-labeled cytokines (Fig. 5). Cross-linking of GM-CSF to TF-1 cells usually showed three bands of about 95, 135, and 215 kd which represent the a subunit (80 kd), the /3 subunit (120 kd) and the complex of the two subunits, respectively after the reduction of the molecular weight of GMCSF (15 kd). IL-1 increased the intensity of those three bands. In particular, the band at 135 kd increased more than the other bands. Cross-linking of IL-3 also showed three proteins of about 85, 135, and 205 kd and IL-1 increased these three proteins equally. Cross-linking of IL-5 revealed two bands of 75 and 140 kd and IL-1 clearly increased the 140 kd band, but not the 75 kd band. Cross-linking of Epo showed also two bands of 125 and 140 kd, and IL-1 increased the intensity of the both bands equally.
Discussion IL-1, also known as hemopoietin-1, has hemopoietic potentiating activities and acts on early hemopoietic progenitors synergistically with hemopoietic growth factors such as IL-3 and GM-CSF (2). IL-1 is known to stimulate the growth of hemopoietic cells by inducing the production of hemopoietic growth factors of accessory cells (23,24). Although some reports suggested that
Table 1. IL-1 up-regulation of cytokine receptors expressed on TF-1 cells Factor depletion11
Basal leveP
Site/cell IL-3R EpoR GM-CSFR IL-5R
340 1300 1700 9460 130 340
± ± ± ± ± ±
*d(plM) 120 260
300 1220 60 120
420 ± 90 640 ± 210
60 2100 120 2100
± ± ± ±
30 450 50 540
IL-1 treatment0
Site/cell
Kd(pM)
Site/cell
M l pM)
550 ± 130 1680 ± 190 4800 ± 1720
450 ± 120 680 ± 170 350 ± 160
1820 ± 320 3260 ± 680 9490 ± 3530
320 ± 100 770 ± 140
380 ± 120
310 ± 100
600 ± 80
Results shown are the average ± standard deviation of at least three independent experiments. The basal levels of the receptor expression were examined as described in the legend to Fig. 4. b The cells were washed twice and incubated for 20 h in the absence of any growth factor. c The cells were washed twice and incubated for 20 h in the presence of 10 ng/ml IL-1 a. a
200 ± 70 280 ± 80
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Cross-linking of
Up-regulation of cytokine receptors by IL-1 575
Interestingly, whereas IL-1 up-regulates the IL-3 and Epo receptors without affinity change, IL-1 increases the number of the high affinity receptors for GM-CSF and IL-5 but decreases the low affinity binding sites for GM-CSF and IL-5. Thus, the total binding sites for GM-CSF and IL-5 are not substantially changed by IL-1 (Fig. 4). This observation is quite interesting in view of the molecular composition of the cytokine receptors, because the high affinity receptors for those cytokines are composed of multiple subunits. Although the dissociation constants for the high affinity receptors for GM-CSF and IL-5 increased slightly by the
GM-CSF.R
KDal 205-
IL-3'R
O =d £ o d S
IL-1 treatment, it is unlikely that a third class (intermediate affinity) receptors emerge from the IL-1 treatment, because the sizes of the cross-linked molecule are not changed at all by the IL-1 treatment (Fig. 5). The reason for the slight increase of the dissociation constants caused by IL-1 treatment is currently unknown. The Epo receptor cDNA, which was cloned from a murine erythroleukemic (MEL) cell line subclone 745, expresses two classes of binding sites (Kd 30 pM and 210 pM) on COS7 cells, whereas the MEL subclone 745 shows a single class of binding sites (Kd 240 pM). This result suggests that a cell type specific protein(s) may affect the affinity of the Epo receptor (29). The affinity of the Epo receptor present on TF-1 cells is similar to that of MEL cells subclone 745 and the binding is up-regulated by IL-1 without changing the affinity. Molecular composition of the Epo receptor is still unclear. However, if multiple subunits are involved in binding of Epo, all the components may be upregulated by IL-1. The mouse IL-3 receptor cDNA encodes a mature protein of 120 kd which binds IL-3 with low affinity and additional component(s) should be required for high affinity binding (7). In contrast to the mouse system, there is no evidence indicating the presence of a low affinity IL-3 receptor in human. Although the human IL-3 receptor gene has not yet been isolated, crosslinking experiments suggest the presence of multiple components for the human IL-3 receptor (Fig. 5) (17). Since IL-1 increases the number of binding sites as well as the amount of all the crosslinked proteins, IL-1 may enhance the expression of all those binding components. An alternative possibility is discussed below. In contrast to receptors for Epo and IL-3, TF-1 cells express both high- and low-affinity receptors for GM-CSF and IL-5 as was shown in other cell lines (14,18,30). Recent cloning of the gene
EpcR
KDal
o
IL-5«R
KDal
— 205-
77-
116.577-
43-
43
Fig. 5. Chemical cross-linking of iodinated cytokines to TF-1 cells. The chemical cross-linking experiments were performed as described in Methods on the cells before and after the IL-1 treatment or factor depletion. Cells grown in the presence (IL-1) or absence (FD) of IL-1 are compared with cells (C) grown in normal medium containing either GM-CSF (for IL-3 and IL-5 binding) or IL-3 (for GM-CSF binding).
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colony formation of hemopoietic progenitor cells was directly enhanced by IL-1 (3,25), it was difficult to eliminate the effects of accessory cells completely and the mechanism of this direct synergism has been unclear. Our present data, however, demonstrates that IL-1 enhances the factor-dependent growth of TF-1 cells directly. Thus, a human hemopoietic cell line, TF-1, is particularly useful to study the combined action of IL-1 with other cytokines because the proliferation of TF-1 is strictly dependent on multiple cytokines including IL-3, IL-5, GM-CSF, and Epo, and IL-1 augments the prohferative effects of these cytokines (Fig. 2) (5). In the IL-2 receptor system, it has been reported that IL-1 upregulated the expression of IL-2 receptor (26). We have demonstrated that IL-1 up-regulates the high affinity receptors for IL-3, IL-5, GM-CSF, and Epo. The increased number of receptors may help the oligomerization of the receptor, which seems to be important for signaling in receptor systems such as epidermal growth factor (27) or platelet-derived growth factor (28). Alternatively, it is possible that a minor population of cells bearing a large number of high affinity receptors is present in normal culture conditions and this population is increased by IL-1 treatment. Interrelation between the number of the high-affinity receptors and proliferation potential is not clear.
576
Up-regulation of cytokine receptors by IL-1
Low Affinity
High Affinity
Wgh Affinity
GM-CSF-R
IL-3-R
Low Affinity
IL-5-R
Fig. 6. A possible mechanism of the upregulation of the receptors for human GM-CSF, IL-3, and IL-5
cells have only the high affinity binding site for IL-3, another component of the IL-3 receptor (70 kd) may bind IL-3 with extremely low affinity or may not bind by itself or the abundance of this component may be low. Up-regulation of the 120 kd protein by IL-1 explains the increase of both IL-3 and GM-CSF binding. The observation (34) that IL-5 binding was inhibited by either IL-3 or GM-CSF in basophils leads us to speculate that the /3 subunit of the GM-CSF receptor may also be involved in the formation of the high affinity IL-5 receptor. Molecular cloning of the other subunits of the IL-3 and IL-5 receptors and reconstitution of the high affinity receptors will answer these questions.
Acknowledgements
Similarly, the high affinity IL-5 receptor is also composed of at least two components (55 and 120 kd). The 55 kd subunit binds IL-5 with low affinity and the 120 kd protein was reported to have a relationship with the high affinity binding of IL-5 (14). In the absence of IL-1, TF-1 cells show two classes of IL-5 binding sites. The low affinity binding is probably mediated by the 55 kd subunit and the high affinity site should be composed of the 55 and 120 kd subunits. Again IL-1 treatment increases the number of high affinity sites without increasing the total binding sites. This can be also explained by the selective enhancement of the expression of the 120 kd subunit (Fig. 6). We identified the /3 subunit of the GM-CSF receptor by molecular cloning. Initially we isolated a human cDNA based on homology with the mouse IL-3 binding protein (12). Interestingly, however, the protein encoded by the cloned human cDNA does not bind IL-3 but it conferred a high affinity binding to GM-CSF when it is co-expressed with the a subunrt of the GM-CSF receptor. Although we extensively searched for a cDNA encoding a human IL-3 binding protein using the mouse IL-3 receptor cDNA as a probe, we have not obtained such a clone. It is most likely that, in human, the ft subunit of the GM-CSF receptor is shared by both IL-3 and GM-CSF receptors. In this case, the /3 subunit of the GM-CSF receptor forms a high affinity receptor for IL-3 with an additional component, possibly the 70 kd protein which can be cross-linked with IL-3. This explains the observation that IL-3 competes for GM-CSF binding to the high affinity site and GM-CSF competes for IL-3 binding (31 -33). Since TF-1
We are grateful to Drs T. Yokota, and K. Arai for helpful discussion and for providing IL-4, and to Miss Chie Misawa for her technical assistance We also thank to Drs S. Tindall (Schering Plough), M. Takahashi and Y. Masui (Ohtsuka Pharmaceutical), S. Clark (Genetics Institute), and T. Suzuki (Kirin Brewery) for providing cytokines. This work was supported in part by a Research Grant for Intractable Diseases from the Ministry of Health and Welfare of Japan and by grants-in-aids from the Ministry of Education, Science and Culture of Japan. DNAX Research Institute of Molecular and Cellular Biology is supported by the Schering-Plough Corporation.
Abbreviations cDNA DSS Epo GM-CSF IL MEL MTT
complementary DNA disuccinimidyl suberate erythropoietin granulocyte/macrophage colony-stimulating factor interleukin murine erythroleukemia 3-{4,5-dimethytthiazol-2-yf)-2, 5-diphenyttetrazolium bromide
References 1 Arai, K., Lee, F., Miyajima, A., Miyatake, S., Arai, N., and Yokota, T. 1990. Cytokines: coordinators of immune and inflammatory responses. Annu. Rev. Biochem. 59:783. 2 Moore, M. A. S. 1989. Role of interteukin-1 in hematopoiesis. Immunol. Rev. 8:165. 3 Mochizuki, D. Y., Eisenman, J. R., Cordon, P. J., Larsen, A. D., and Tushinski, R. J. 1987. lnterleukin-1 regulates hematopoietic activity, a rote previously ascribed to nemopcxetin-1. Proc. Natl Acad. Set. USA 84:5267. 4 Oppenheim, J. J. and Kovacs, E. J. 1986. There is more than one interleukin-1. Immunol. Today 7:45. 5 Kitamura, T., Tange, T., Terasawa, T., Chiba, S., Kuwaki, T., Miyagawa, K , Piao, Y.-F., Miyazono, K., Urabe, A., and Takaku F 1989. Establishment and characterization of a unique human cell line that proliferates dependently on GM-CSF, IL-3, or erythropoietin J. Cell Physhl. 140:323. 6 Hatakeyama, M., Tsudo, M., Minamoto, S., Kono, T., Doi, T., Miyata, T , Miyasaka, M., and Taniguchi, T. 1989. lnterleukin-2 receptor p chain gene: generation of three receptor forms by cloned human a and 0 chain cDNAs. Science 244:551. 7 Itoh, N., Yonehara, S., Schreurs, J., Gorman, D. M., Maruyama, K., Ishii, A., Yahara, I., Arai, K., and Miyapma, A. 1990. Cloning of an interleukin-3 receptor: a member of a distinct receptor gene family. Science 247:324. 8 Takaki, S., Tominaga, A., Hitoshi, Y., Mita, S., Sonoda, E., Yamaguchi, N., and Takatsu, K. 1990. Molecular cloning and expression of the murine interleukin-5 receptor. EMBO J. 94367.
Downloaded from http://intimm.oxfordjournals.org/ at Dalhousie University on June 24, 2015
for a second component of the GM-CSF receptor has revealed that the high affinity GM-CSF receptor is composed of a (80 kd) and /3 (120 kd) subunits (12). The a subunit binds GM-CSF with low affinity (11), whereas the 0 subunit does not bind GM-CSF by itself. Co-expression of the a and /3 subunits forms a high affinity receptor for GM-CSF (12). This is consistent with the observation that the larger band detected by cross-linking is correlated with the high affinity binding of GM-CSF (18). If there is an excess amount of the a subunit in TF-1 cells, TF-1 cells should have high affinity receptors (a//S) and low affinity receptors (a). Since IL-1 increases the number of the high affinity sites without changing the total binding sites (Fig. 4) and preferentially increases the larger cross-linked band (Fig. 5), IL-1 may enhance the expression of the 0 subunit selectively. This is consistent with the results that the mRNA for the /3 chain of the GM-CSF receptor increased greatly with IL-1 treatment (unpublished results). This also explains the loss of low affinity sites by IL-1 because the increased level of the /3 subunrt reduces the free a subunit (Fig. 6).
Up-regulation of cytokine receptors by IL-1 577 23 Fibbe, W. E., Van Damme, J., BflBau, A., Gosefink, H. M., Voogt, P. J., Van Eeden, G., Ralph, P., Altonic, B. W., and Falkenberg, J. H. F. 1988. lnterfeukin-1 induces human marrow stromal cells in long-term culture to produce graruilocyte colony-stimulating factor and macrophage colony-stimulating factor. Blood 71:430. 24 Zsebo, K. M., Yuschenkoff, V. N., Schiffer, S., Chang, D., McCaU, E., Dinarrello, C. A., Brown, M. A., Altorock, B., and Bagley, G. C. J. 1988. Vascular endothelial cells and granulopoiesis. interteukin-1 stimulates release of G-CSF and GM-CSF. Blood 71:99. 25 Williams, D. E. and Broxmeyer, H E. 1988. lnterleukin-1 a enhances the in vitro survival of purified murine granulocyte-macrophage progenitor ceDs in the absence of colony-stimulating factor. Proc. Nati Acad. Sci. USA 84:3871. 26 Kaye, J , Gillis, S., Mizel. S B., Shevach, E. M., Malex, T. R., Dinarello, C. A., Lachman, L B., and Janeway, C. A. J. 1984. Growth of a cloned helper T cell line induced by a monoclonal antibody specific for the antigen receptor: interleukin 1 is required for the expression of receptors for interleukin 2. J. Immunol 133:1339. 27 Cochet, C , Kashles, O., Chambaz, E. M., Borrello, I., King, C. R., and Schlessinger, J. 1988. Demonstration of epidermal growth factor-induced receptor dimerization in living cells using a chemical covalent cross-linking agent. J Biol. Chem. 2633290. 28 Heldm, C. H., Ernlund, A., Rorsman, C , and Ronnstrand, L. 1989. Dimerization of B-type platelet-derived growth factor receptors occurs after ligand binding and is closely associated with receptor kinase activation. J. Bio!. Chem. 264:8905. 29 D'Andrea, A. D., Lodish, H. F., and Wong, G. G. 1989. Expression cloning of the munne erythropoietin receptor. Cell 57:277. 30 Walker, F. and Burgess, A. W. 1985 Specific binding of radioiodinated granulocyte-macrophage colony-stimulaling factor to hemopoietic cells. EMBO J. 4.933. 31 Park, L. S., Friend, D, Price, V., Anderson, D., Singer, J., Pnckett, K S., and Urdal, D. L. 1989. Heterogeneity in human interleukin-3 receptors. J. Bid Chem. 264:5420 32 Budel, L. M., Elbaz, O , Hoogerbrugge, H., Delwel, R., Mahmoud, L. A., Lowenberg, B., and Touw, I. P. 1990. Common binding structure for granulccyte macrophage colony-stimulating factor and interleukin-3 on human acute myeloid leukemia cells and monocytes. Blood 75:1439. 33 Onetto-Pothier, N., Aumont, N., Haman, A., Park, L , Clark, S. C , De Lean, A., and Hoang, T. 1990. IL-3 inhibits the binding of GMCSF to AML blasts, but the cytokines act synergistically in supporting blast proliferation. Leukemia 4:329. 34 Lopez, A. F , Eglinton, J. M., Lyons, A. B., Tapley, P. M., To, L. B., Park, L S., Clarke, S. C, and Vadas, M. A. 1990. Human interleukin-3 inhibits the binding of granulocyte-macrophage colony-stimulating factor and interleukin-5 to basophils and strongly enhances their functional activity. J. Cell. Physiol. 145:69.
Downloaded from http://intimm.oxfordjournals.org/ at Dalhousie University on June 24, 2015
9 YamasaW, K., Taga, T., Hirata. Y., Yawata, H., Kawanishi, Y., Seed, B., Taniguchi, T., Hirarto, T. and Kishimoto, T. 1988. Cloning and expression of the human interieukin-6 (BSF-2/IFN02) receptor. Science 241:825. 10 Taga, T., Hibi, M., Hirata, Y., Yamasaki, K.. Yasukawa, K , Matsuda, T., Hirano, T., and Kishimoto, T. 1989. lnterteukin-6 triggers the association of its receptor with a possible signal transducer, gp130. Cell 58:573. 11 Gearing, D. P., King, J. A , Gough, N. M., and Nicola, N. A. 1989. Expression cloning of a receptor for human granulocyte-macrophage colony-stimulating factor. EMBO J. 8:3667. 12 Hayashida, K., Kitamura, T., Gorman, D. M., Arai, K , Yokota, T., and MiyajmaA 1990. Molecular cloning of a second subunit of the human GM-CSF receptor: reconstitution of a high affinity GM-CSF receptor. Proc. Nati Acad. Sci. USA 87:9655. 13 Hibi, M., Murakami, M., Sarto, M., Hirano, T., Taga, T., and Kjshimoto, T. 1990. Molecular cloning and expression of an IL-6 signal transducer, gp130. Cell 63:1149 14 Mita, S., Tominaga, A., Hitoshi, Y , Sakamoto, K., Honjo, T., Akagi, M., Kikuchi, Y., Yamaguchi, N., and Takatsu, K. 1989. Characterization of high-affinity receptors for mterieukin 5 on interleukin 5-dependent cell lines. Proc. Nati Acad. Sci USA 86:2311. 15 Schreurs, J., Aral, K , and Miyajima, A. 1990. Evidence for a lowaffinity interleukin-3 receptor. Growth Factors 2:221 16 Kitamura, T , Tojo, A , Kuwaki, T., Chiba, S., Miyazono, K , Urabe, A., and Takaku, F. 1989. Identification and analysis of human erythropoietin receptors on a factor-dependent cell line, TF-1. Blood 73:375. 17 Kuwaki, T , Krtamura, T., Tojo, A., Matsuki, S., Tamai, Y., Miyazono, K., and Takaku, F. 1989. Characterization of human interleukin-3 receptors on a mult-factor-dependent cell line Biochem. Biophys. Res Commun. 161:16. 18 Chiba, S., Tojo, A., Kitamura, T., Urabe, A., Miyazono, K., and Takaku, F. 1990. Characterization and molecular features of the cell surface receptor for human granulocyte-macrophage colonystimulating factor. Leukemia 4:22. 19 Mosmann, T 1983 Rapid colorimetric assays for cellular growth and survival, application to proliferation and cytotoxicity assays. J. Immunol. Methods 65:55. 20 Greenwood, F. C , Hunter, W. H., and Glover, J. S. 1963. The preparation of '31I-Iabeled human growth hormone of high-specific activity. Biochem. J. 89.114. 21 Fukamachi, H., Top, A , Sarto, T., Krtamura, T., Nakata, M., Urabe, A., and Takaku, F. 1987. Internalization of radioiodinated erythropoietin and the ligand-induced modulation of its receptor in murine erythroleukemic cells. Int. J. Cell Cloning 5.209. 22 Munson, P. J. 1983. LIGAND: a computerized analysis of ligand binding data. Methods Enzymot. 92:543.
Downloaded from http://intimm.oxfordjournals.org/ at Dalhousie University on June 24, 2015
