inhibitory effect of interleukin 4 on production of ... · seattle, wa). a cloned il-6/bsf-2...

[CANCER RESEARCH 53, 4643~t647, October 1, 1993]

Inhibitory Effect of Interleukin 4 on Production of Interleukin 6 by Adult T-Cell

Leukemia Cells

Naoki Mori, 2 Fumihiko Shirakawa, Shuichi Murakami, Susumu Oda, and Sumiya Eto

The First Department of Internal Medicine, University of Occupational and Environmental Health, School of Medicine, 1-1 Iseigaoka, Yahatanishiku, Kitakyushu 807, Japan

A B S T R A C T

Freshly isolated leukemic cells from patients with adult T-cell leukemia (ATL) produce high levels of interleukin 6 (IL-6), which is suggested to play an important role in thrombocytosis, elevation of C-reactive protein, and hypercalcemia in ATL. In this study, we investigated the effects of T-cell growth factors such as interleukin 2 (IL-2) and interleukin 4 (IL-4) on IL-6 production by ATL cells in vitro. Although IL-2 and/or IL-4 enhanced the cell proliferation of freshly isolated ATL cells from seven of nine patients, IL-2 did not affect the IL-6 release in most cases. In contrast, another T-cell tropic factor, IL-4 markedly inhibited the release of IL-6 in the conditioned medium in all cases. This IL-4-mediated inhibition of IL-6 release was completely abrogated by the addition of anti-IL.4 monocional antibody. Time course experiments demonstrated that IL-4 reduced the secretion of IL-6 for a prolonged period of time (more than 72 h). By Northern analysis, IL-4 reduced the transcription level of IL-6 mRNA. Furthermore, by flow cytofluorometry with the use of anti-human IL-4 receptor monoclonal antibody, ATL cells showed the significant level of IL-4 receptor on their cell surfaces without any stimulation. These data suggest that IL-4 may play an important regulatory role in the production of IL-6 in ATL.

I N T R O D U C T I O N

IL-4, 3 a product of act ivated T-cells, was originally descr ibed as

B-cell g rowth and differentiat ion factor. IL-4 has been shown to

possess a broader spec t rum of biological activities affecting, a m o n g

others, T-cells, monocy tes , mast cells, and hematopoie t ic progeni tor

cells (1-4). Fur thermore , it has been recently reported that IL-4 exerts suppress ive effects on the spontaneous growth of C M M o L cells (5)

and m y e l o m a cells (6) through the inhibit ion of IL-6 product ion,

which acts as an autocrine or paracrine growth factor of these cells.

IL-6 was purif ied and its gene was c loned f rom HTLV-I- infected

T-cell lines (7). ATL is causally associated with HTLV-I infect ion and ATL cells f requent ly p roduce cytokines such as IL-1 (8, 9), IL-6 (10),

and IL-2 in some cases (11); also, ATL cells proliferate in response to

IL-2 (11) and IL-4 (12, 13). In the pre l iminary exper iments we also

conf i rmed that se rum IL-6 levels in patients wi th ATL increased

significantly, as compared with those of either a symptomat ic carriers or controls. These observat ions led us to invest igate whe ther IL-4

affects the product ion of IL-6 by ATL cells. In this report, we show

that IL-4 marked ly inhibits IL-6 product ion by ATL cells.

M A T E R I A L S A N D M E T H O D S

Patients. Nine patients with ATL in acute phase were studied. The age of the patients, five males and four females, ranged from 45 to 76 years old. The

Received 3/22/93; accepted 7/27/93. The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

i This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan, and research award from the Arthritis Foun- dation, Atlanta, GA.

2 To whom requests for reprints should be addressed. 3 The abbreviations used are: IL-4, interleukin 4; ATL, adult T-cell leukemia; IL-6,

interleukin 6; IL-2, interleukin 2; IL-4R, IL-4 receptor; HTLV-I, human T-cell leukemia virus type I; PBMC, peripheral blood mononuclear cells; MoAb, monoclonal antibody; FCS, fetal calf serum; [3H]TdR, tritiated thymidine; PHA, phytohemagglutinin; CMMoL, chronic myelomonocytic leukemia; PBA, phosphate-buffered saline supplemented with 0.1% bovine serum albumin and 0.1% sodium azide.

diagnosis of ATL was based on clinical features, morphological characteristics, cell surface phenotypes of leukemic cells, serum antibodies against HTLV-I- associated antigens, and the HTLV-I proviral genome in leukemic cells. WBC ranged from 9,800 to 242,900//xl. More than 85% of the peripheral WBCs were leukemic cells, as morphologically determined by Wright-Giemsa staining. CD4-positive (ATL) cells were more than 90% of the PBMC, as evaluated by flow cytofluorometry.

Reagents. Recombinant human IL-2 and human IL-6 were provided by Shionogi Pharmaceutical Co., Ltd., Osaka, Japan, and Ajinomoto Central Re- search Laboratories Co., Inc., Yokohama, Japan, respectively. Recombinant human IL-4 and mouse MoAb against recombinant human IL-4 were supplied by Ono Pharmaceutical Co., Ltd., Osaka, Japan. Mouse anti-human IL-4R MoAb M57 (IgG1 isotype) was kindly provided by Dr. S. Gillis (Immunex, Seattle, WA). A cloned IL-6/BSF-2 complementary DNA (pBSF2.38) was provided by Dr. T. Taga (Osaka University, Osaka, Japan) (7).

Cell Separation and Cell Culture. PBMC were separated from heparin- ized blood of nine ATL patients by centrifugation on lymphocyte separation medium (LSM, Litton Bionetics, Kensington, MD). Cells were further incubated at 37~ for 2 h in plastic culture dishes (Falcon 3002; Falcon Plastics, Oxnard, CA) to remove adherent cells. Furthermore, more enriched ATL cell preparations were obtained by CD4-conjugated immunomagnetic beads (Dy- nabeads M-450; Dynal A.S., Oslo, Norway). After purification, the cells in suspension were used for the experiments described below. The percentage of contaminating monocytes was determined by flow cytofluorometry; it was less than 2% of the obtained cells in all patients. Purity of T-cells as evaluated by flow cytofluorometry of anti-CD4-stained cells was more than 95%.

ATL cells were cultured in 24-well culture plates (2 ml/well) at a concen- tration of 5 x 106 cells/ml in RPMI 1640 medium (Nissui Seiyaku Co., Tokyo, Japan) containing 10% FCS (Grand Island Biological Co., Grand Island, NY) with or without various concentrations of IL-2 or IL-4 in the presence or absence of mouse MoAb against recombinant human IL-4. After culture for the indicated periods, culture supernatants were collected by centrifugation to measure the levels of released IL-6. The viability of the cells determined by trypan blue exclusion always exceeded 90%.

As a positive control for IL-6 mRNA expression, SALT-3 cells (HTLV-I- infected cell line, a gift from Dr. K. Sagawa, Kurume University, Kurume, Japan) were cultured in RPMI 1640 medium containing 10% FCS at 37~

Proliferative Response. ATL cells (1 x 105) from patients were incubated in 96-well flat bottomed microtiter plates in the presence of various concentrations of IL-2 or IL-4 in 200/xl of RPMI 1640 medium containing 10% FCS in triplicate for 72 h. The cells were pulsed with 0.5/xCi of [3H]TdR for the last 24 h. [3H]TdR incorporation was measured by liquid scintillation counting after harvesting the cells with a cell harvester (Abekagaku Co., Funabashi, Japan).

Detection of IL-6 in Culture Supernatant. IL-6 in the culture supernatant of ATL cells was measured by using the human IL-6 enzyme-linked immunosorbent assay kit (Toray-Fuji Bionix Inc., Tokyo, Japan). The sensitivity of this assay is 10 pg/ml of IL-6 and there is no cross-reactivity with other cytokines.

Northern Blot Analysis for IL-6 Gene Expression. Total cellular RNA was isolated from the cultured cells as previously described by using the single-step isolation procedure (14). Twenty-/xg RNA samples were subjected to gel electrophoresis by using 1% agarose containing 6% formaldehyde and were blotted onto nylon membrane filters (Hybond-N, Amersham Japan, Tokyo, Japan). TaqI-BanII fragment of pBSF2.38 complementary DNA was labeled with [32p]dCTP by using the multiprime DNA labeling kit (Amersham Japan).

Cytofluorometric Measurements of IL-4R Expression. Cells (1 x 106) were washed and resuspended in 100 /xl PBA. For determination of IL-4R expression, cells were incubated for 30 min at 4~ with 1 /xg mouse anti- human IL-4R MoAb M57, washed twice with 1 ml PBA, and further incubated for 30 min with 50/xl fluorescein isothiocyanate-conjugated goat anti-mouse

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EFFECT OF IL-4 ON 1L-6 PRODUCTION BY ATL CELLS

Fig. 1. Proliferative response of ATL cells to IL-2 and IL-4. [3H]TdR incorporation of peripheral blood leukemic cells cultured with IL-2 or IL-4 was examined. Points, mean of triplicate cultures; bars, SE.

Table 1 Grouping of ATL cases by abilities to respond to IL-2 and IL-4

Group Patient no. Response to IL-2 Response to IL-4

1 1, 3, 5, 6 + + 2 2 , 4 + - 3 8 - + 4 7, 9 - -

IgG F(ab')e (Immunotech, Marseille, France) diluted 1:50 in phosphate-buffered saline. After the second incubation, the cells were washed twice with 1 ml PBA and resuspended in 600 txl PBA. Cytofluorometric analysis was performed with FACScan flow cytometry (Becton Dickinson Immunocytometry Systems, Inc., Mountain View, CA). For controls, an isotype control antibody was used.

As a positive control for IL-4R expression, PBMC from normal donors were activated with PHA-P (Difco Laboratories, Detroit, MI) at 0.1% for 72 h.

RESULTS

Cell Proliferation of ATL Cells in Response to IL-2 and IL-4. As shown in Fig. 1 and Table 1, ATL cells from peripheral blood of 4 (patients 1, 3, 5, and 6) of 9 patients proliferated in response to both IL-2 and IL-4 in a dose-dependent manner. ATL cells from 2 patients (patients 2 and 4) responded to IL-2, but did not respond to IL-4. In contrast, ATL cells from 1 patient (patient 8) proliferated in response to IL-4, but did not respond to IL-2. In 2 patients (patients 7 and 9), ATL cells did not proliferate in response to either IL-2 or IL-4. The base-line values of [3H]TdR incorporation varied from 398 to 8343 cpm. The levels of the spontaneous proliferation seem to bear some relationship to the clinical phase of the disease, as reported previously (13). The maximal response was obtained in most patients in the range of 1 to 10 ng/ml of either IL-2 or IL-4. As shown in Table 1, ATL cells from 6 of 9 patients responded to IL-2 and from 5 of 9 patients responded to IL-4. In agreement with a recent report by Sawada et al.

(10), IL-6 itself did not show any effects on [3H]TdR incorporation of ATL cells at all (data not shown).

Release of IL-6 into Medium. As shown in Fig. 2, culture supernatants of ATL cells sampled from nine patients and cultured for 72 h displayed quite high levels of IL-6 without any stimulation (83.5 to 20,600 pg/ml). Normal T-cells from healthy donors did not secret IL-6 without stimulation (less than 10 pg/ml). Apart from one sample (.patient 2), which was markedly enhanced for IL-6 release by IL-2, IL-2 did not show any effects on IL-6 secretion (not statistically significant) (Fig. 2). In contrast, when ATL cells from nine patients were incubated with IL-4 for 72 h, the levels of IL-6 secretion were

markedly decreased in all patients (P < 0.005; Wilcoxon test). This effect of IL-4 was observed in a dose-dependent manner. Three representative cases were shown in Fig. 3; other cases showed similar results (data not shown). Furthermore, the effect of IL-4 on IL-6 release was almost completely antagonized by anti-IL-4 MoAb. The representative case, No. 3, was shown in Fig. 4. The inhibition of IL-6 release by IL-4 was almost completely (95%) abrogated by the addition of anti-IL-4 MoAb. In other cases, anti-IL-4 MoAb also markedly reversed the inhibitory effect of IL-4 on IL-6 release (data not shown). These results suggest that IL-4 may markedly suppress the production and/or secretion of IL-6 by ATL cells.

Transcription of IL-6 mRNA. Next, we studied the effects of IL-2 and IL-4 on the transcription level of IL-6. As shown in Fig. 5, an HTLV-I-infected cell line, SALT-3, and freshly isolated ATL cells (,patients 1, 3, and 6) showed high levels of IL-6 message without any stimulation. IL-4 also markedly inhibited the transcription levels of

Fig. 2. Effects of IL-2 and IL-4 on IL-6 release from ATL cells. Cells were cultured for 72 h at 5 x 106 cells/ml in RPMI 1640 medium containing 10% FCS. IL-2 and IL-4 (10 ng/ml) were added at the initiation of culture. IL-6 levels in the culture supernatant were measured by using an enzyme-linked immunosorbent assay. Points, means; bars, SE. N.S., not significant.

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EFFECT OF IL-4 ON IL-6 PRODUCTION BY ATL CELLS

positive cells were markedly increased up to 17% (Fig. 7B). ATL cells from patients showed surprisingly high levels of IL-4R expression without any stimulation. Two representative cases were shown in Fig. 7, C and D, and their IL-4R expression was 78 and 42%, respectively. Other cases also showed high IL-4R expression when compared with normal healthy donors (data not shown). These results suggest that ATL cells show quite high expression of IL-4R without stimulation.

DISCUSSION

IL-6 is one of the important and multifunctional cytokines that regulate host defense responses. For example, it is one of the major physiological stimulants for the hepatic production of acute phase

Fig. 3. Dose-response curves of the inhibitory effect of IL-4 on IL-6 release from ATL cells. Cells were cultured for 72 h with different concentrations of IL-4. Points, means of the percentage of the control cultured without IL-4; bars, SE.

Fig. 4. Abrogation of inhibitory effect of IL-4 on IL-6 release by anti-IL-4 MoAb. Addition of increasing concentrations of anti-IL-4 MoAb to ATL cells from patient 3 cultured in the presence of 10 ng/ml of IL-4 for 72 h completely restores IL-6 levels produced in the culture medium. Columns, means of IL-6 release; bars, SE.

Fig. 5. Effects of IL-2 and IL-4 on IL-6 mRNA expression in ATL cells. ATL cells from patients 3, 1, and 6 were cultured without (Lanes 2, 5, and 8), with (Lanes 3 and 6) IL-2 (10 ng/ml), and with (Lanes 4, 7, and 9) IL-4 (10 ng/ml) for 2 h. The control Lane 1 contains RNA from an HTLV-I -infected cell line, SALT-3 cells. Top, Northern blot analysis, using a probe for IL-6. Bottom, ethidium bromide staining pattern of gel. Approximately equal amounts (20/xg) of RNA were electrophoresed in each lane.

IL-6 mRNA in Northern analysis. In contrast, IL-2 did not show any effects. These results suggest that IL-4 may decrease the transcription level of IL-6 message by ATL cells.

Kinetics of IL-6 Secretion. We performed time course experiments to characterize the kinetics of the effect of IL-4 on IL-6 secretion. As shown in Fig. 6, the release of IL-6 from ATL cells in the absence of IL-4 was markedly increased after 6 h in culture. However, it was markedly reduced by the addition of 10 ng/ml IL-4 at from 6 h to more than 72 h.

Expression of IL-4R on ATL Cells. Finally, we studied whether ATL cells have IL-4R without any stimulation. As shown in Fig. 7, the expression of IL-4R was examined by flow cytofluorometry by using a MoAb M57 directed against IL-4R. This antibody inhibited IL-4- dependent cell proliferation by PHA-activated human PBMC and could not bind to IL-4R when it was saturated with IL-4 (data not shown). Normal mononuclear cells from a healthy donor expressed only a very low level or almost none of IL-4R on their surface (2% positive in Fig. 7A). However, when cultured with PHA, IL-4R-

4645

Fig. 6. Time course of IL-6 release from ATL cells cultured with or without IL-4. ATL cells from patient 3 were cultured with (Q) or without (O) 10 ng/ml of IL-4. IL-6 levels were measured by using an enzyme-linked immunosorbent assay. Points, means of triplicate cultures; bars, SE.



E F F E C T O F I L - 4 O N I L - 6 P R O D U C T I O N B Y A I ' L C E L L S

E ,d z m

O

i l d 0 ' ' 11~

. . . . . ; > . . . . . . . , , , , , , , , , ' , , , , , , , , , , , , , ,

D

Fluorescence Intensity Fig. 7. Assay of IL-4R on cell surfaces by flow cytometry. Cells were analyzed by

cytofluorometry with MoAb M57 against human IL-4R. Fluorescein isothiocyanate conjugated goat anti-mouse IgG F(ab')2 was used as a secondary antibody to stain the cells. Histograms of anti-IL-4R-stained cells ( ) are superimposed over histograms of control cells ( ..... ). A, unstimulated PBMC; B, PHA-activated PBMC; C, ATL cells from patient 1; D, ATL cells from patient 3.

proteins including C-reactive protein (15). Also, IL-6 promotes the maturation of megakaryocyte (16) and it induces bone resorption (17). In ATL, several characteristic clinical features are frequently observed as following: high levels of C-reactive protein, accelerated erythrocyte sedimentation rate in the absence of anemia, thrombocytosis, hypercalcemia (more than 70% cases), generalized bone resorption, and markedly increased osteoclasts in bone. These clinical observations lead us to an idea that IL-6 may play an important role in ATL. In the preliminary experiments, we confirmed that the sera from ATL patients showed quite high levels of IL-6, and that ATL cells produce high levels of IL-6 into the culture medium.

In this report, we examined the effects of T-cell growth factors on IL-6 production by ATL cells. IL-4, in contrast to IL-2, markedly inhibited the production of IL-6 by ATL cells in all 9 patients. IL-4 also markedly inhibited the transcription levels of IL-6. These effects were completely abrogated by the addition of the anti-IL-4 MoAb. Furthermore, ATL cells showed quite high levels of IL-4R expression without any stimulation, in contrast to normal mononuclear cells.

IL-4 acts on many cell types by displaying either agonistic or antagonistic effects. IL-4 exhibits different effects on cells of a single lineage at different stages of differentiation. IL-4 either induces or inhibits expression of certain cytokines by various cell types. It has been recently demonstrated that IL-4 down-regulates clonogenic growth of CMMoL cells (5) and myeloma cells (6) by antagonizing the growth stimulatory effects of endogenous IL-6. However in ATL, IL-4 enhanced the cell proliferation of ATL cells from 5 of 9 patients examined. In addition, IL-6 did not show any effect on proliferation of ATL cells.

One may argue about the contamination of monocytes in ATL cell preparations because IL-4 also markedly inhibits the production of IL-1, tumor necrosis factor, and IL-6 by monocytes (18). Whereas monocytes cannot produce these cytokines without any stimulation, ATL cells and HTLV-I-infected cell lines spontaneously produce quite high levels of IL-6 (7, 10). Considering the high purity of ATL cells used in this study and the undetectable levels of IL-6 in the culture supernatants sampled from normal donors, the large amount of IL-6 detected in the culture supernatants sampled from ATL patients

seemed to be produced by ATL cells themselves. On the other hand, IL-loz is known to be undetectable in the culture medium of normal human monocytes. Our preliminary studies indicated that IL-4 also inhibits the production of IL-lc~ in the ATL culture medium collected in this study. This information strongly suggests that IL-4 may inhibit IL-6 production by ATL cells.

The mechanism of action of IL-4 that interferes with IL-6 expression is not yet clear. Our studies indicate that IL-6 steady-state mRNA levels were reduced in ATL cells cultured in the presence of IL-4, but further analyses are required to assess the relative contribution of changing rates of transcription initiation and/or mRNA stability to determine the influence of IL-4 on ATL cells steady-state mRNA levels.

In this study, we also found high expression of IL-4R on ATL cells without any stimulation. IL-4 mediates its action through the cell surface receptor of a single class of high affinity receptors for IL-4, as has been detected on hematopoietic cells (19). Indeed, the levels and magnitude of the effects of IL-4 on cell proliferation and IL-6 production were different among ATL cells. High responders in cell proliferation and IL-6 production such as in patients 1 and 3 expressed quite high levels of IL-4R on their surface (78 and 42% of IL-4R- positive cells, respectively). In addition, non- or low responders in cell proliferation or IL-6 production such as in patients 2 and 9 expressed lower levels of IL-4R (8.3 and 9.8%, respectively) than that of high responders. However, their (patients 2 and 9) IL-4R expression was still significantly high when compared with normal mononuclear cells (2% positive cells). In preliminary experiments, there seems to be clear correlation between levels of IL-4R expression and cell proliferation in response to IL-4.

It has been reported that IL-2 and IL-4 mRNA expression was undetectable in leukemic cells from ATL patients (9). However, it is possible that ATL cells are regulated by IL-2 and IL-4 which are locally produced by activated T-cells present in lymphoid tissues in vivo. In fact, although IL-2 was not detected in cultures of PBMC isolated from one patient (patient 4) by immunoassay, we detected significant levels of IL-2 in cultures of mononuclear cells isolated from a lymph node of the same patient.

Several clinical features of ATL may be explained by up-regulated IL-6 production by ATL cells, such as the frequently observed elevation of C-reactive protein, accelerated erythrocyte sedimentation rate, thrombocytosis, and hypercalcemia (15-17). We showed that IL-4 markedly inhibits IL-6 synthesis by ATL cells at the mRNA and protein levels in a dose-dependent manner, but at the same time it enhances the cell growth of ATL cells. These characteristics are a remarkable contrast with CMMoL cells and myeloma cells. In these cells, IL-4 also inhibits IL-6 production but does inhibit the cell growth of CMMoL cells and myeloma cells (5, 6). This evidence suggests that differences may exist in the signals of IL-4 which induce cell growth and inhibit IL-6 production. Recently we demonstrated that IL-4 does not have an effect on the production of parathyroid hormone-related protein by ATL cells; however, IL-2 enhances cell growth and parathyroid hormone-related protein production in ATL cells (20). Thus, IL-4 seems to be not an enhancing factor in cytokines production in ATL. The details of action and mechanisms of IL-4 signals in cell growth and IL-6 production in ATL are currently un- clear. However, this evidence strongly suggests that IL-4 may inhibit a signal involved in IL-6 gene expression and may activate another signal involved in cell proliferation in ATL. The mechanisms of up- regulated IL-6 production and inhibitory effect of IL-4 in ATL is currently under investigation in our laboratory. In this study, these observations suggest a possibility that cell growth and IL-6 production may not be correlated in ATL, even in vivo, and some mechanisms may exist for the explanation of up-regulated production of IL-6 in

4646



EFFECT OF IL-4 ON IL-6 PRODUCTION BY ATL CELLS

ATL. IL-4 may give us a new clue to study the mechanism of up- regulated IL-6 production in ATL.

REFERENCES

1. Brown, M. A., Pierce, J. H., Watson, C. J., Falco, J., Ihle, J. N., and Paul, W. E. B cell stimulatory factor-1/IL-4 mRNA is expressed by normal and transformed mast cells. Cell, 50: 809---818, 1987.

2. Te Velde, A. A., Klomp, J. P. G., Yard, B. A., De Vries, J. E., and Figdor, C. G. Modulation of phenotypic and functional properties of human peripheral blood monocytes by IL-4. J. Immunol., 140: 1548-1554, 1988.

3. Hu-Li, J., Shevach, E. M., Mizuguchi, J., Ohara, J., Mosmann, T., and Paul, W. E. B cell stimulatory factor-1 (interleukin 4) is a potent costimulant for normal resting T lymphocytes. J. Exp. Med., 165: 157-172, 1987.

4. Broxmeijer, H. E., Lu, L., Cooper, S., Rubin, B. Y., Gillis, S., and Williams, D. E. Synergistic effects of purified recombinant human and murine B-cell growth factor/ IL-4 on colony formation in vitro by hematopoietic progenitor cells. J. Immunol., 141: 3852-3862, 1988.

5. Akashi, K., Shibuya, T., Harada, M., Takamatsu, Y., Uike, N., Eto, T., and Niho, Y. lnterleukin 4 suppresses the spontaneous growth of chronic myelomonocytic leukemia cells. J. Clin. Invest., 88: 223-230, 1991.

6. Herrmann, F., Andreeff, M., Gruss, H. J., Brach, M. A., Lubbert, M., and Mertels- mann, R. lntefleukin-4 inhibits growth of multiple myelomas by suppressing interleukin-6 expression. Blood, 78: 2070-2074, 1991.

7. Hirano, T., Yasukawa, K., Harada, H., Taga, T., Watanabe, Y., Matsuda, T., Kashi- wamura, S., Nakajima, K., Koyama, K., Iwamatsu, A., Tsunasawa, S., Sakiyama, E, Matsui, H., Takahara, Y., Taniguchi, T., and Kishimoto, T. Complementary DNA for a novel human interleukin (BSF-2) that induces B lymphocytes to produce immuno- globulin. Nature (Lond.), 324: 73-76, 1986.

8. Shirakawa, E, Tanaka, Y., Oda, S., Eto, S., and Yamashita, U. Autocrine stimulation of interleukin l a in the growth of adult human T-ceU leukemia cells. Cancer Res., 49: 1143-1147, 1989.

9. Kodaka, T., Uchiyama, T., Umadome, H., and Uchino, H. Expression of cytokine

mRNA in leukemic cells from adult T cell leukemia patients. Jpn. J. Cancer Res., 80: 531-536, 1989.

10. Sawada, T., Tsuda, H., and Takatsuki, K. Spontaneous production of interleukin 6 by adult T-cell leukemia cells. Br. J. Cancer, 62: 923-924, 1990.

11. Arima, N., Daitoku, Y., Yamamoto, Y., Fujimoto, K., Ohgaki, S., Kojima, K., Fuku- mori, J., Matsushita, K., Tanaka, H., and Onoue, K. Heterogeneity in response to interleukin 2 and interleukin 2-producing ability of adult T cell leukemic cells. J. Immunol., 138: 306%3074, 1987.

12. Uchiyama, T., Kamio, M., Kodaka, T., Tamori, S., Fukuhara, S., Amakawa, R., Uchino, H., and Araki, K. Leukemic ceils from adult T-cell leukemia patients proliferate in response to interleukin-4. Blood, 72: 1182-1186, 1988.

13. Mori, N., Yamashita, U., Tanaka, Y., Nakata, K., Oda, S., Morimoto, I., and Eto, S. Interleukin-4 induces proliferation of adult T-ceU leukemia cells. Eur. J. Haematol., 50: 133-140, 1993.

14. Chomczynski, P., and Sacchi, N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162: 156- 159, 1987.

15. Castell, J. V., Gomez-Lechon, M. J., David, M., Hirano, T., Kishimoto, T., and Heinrich, P. C. Recombinant human interleukin-6 (IL-6/BSF/HSF) regulates the synthesis of acute phase proteins in human hepatocytes. FEBS Lett., 232: 347-350, 1988.

16. Ishibashi, T., Kimura, H., Uchida, T., Kariyone, S., Friese, P., and Burstein, S. A. Human interleukin 6 is a direct promoter of maturation of megakaryocyte in vitro. Proc. Natl. Acad. Sci. USA, 86: 5953-5957, 1989.

17. Ishimi, Y., Miyaura, C., Jin, C. H., Akatsu, T., Abe, E., Nakamura, Y., Yamaguchi, A., Yoshiki, S., Matsuda, T., Hirano, T., Kishimoto, T., and Suda, T. IL-6 is produced by osteoblasts and induces bone resorption. J. Immunol., 145: 3297-3303, 1990.

18. Te Velde, A. A., Huijbens, R. J. F., Heije, K., De Vries, J. E., and Figdor, C. G. Interleukin-4 (IL-4) inhibits secretion of IL-1/3, tumor necrosis factor o~, and IL-6 by human monocytes. Blood, 76: 1392-1397, 1990.

19. Ohara, J., and Paul, W. E. Receptors for B-cell stimulatory factor-1 expressed on cells of hematopoietic lineage. Nature (Lond.), 325: 537-540, 1987.

20. Mori, N., Ohsumi, K., Murakami, S., Wake, A., Shirakawa, F., Morimoto, I., Oda, S., and Eto, S. Enhancing effect of interleukin-2 on production of parathyroid hormone- related protein by adult T-cell leukemia cells. Jpn. J. Cancer Res., 84: 425-430, 1993.

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1993;53:4643-4647. Cancer Res Naoki Mori, Fumihiko Shirakawa, Shuichi Murakami, et al. by Adult T-Cell Leukemia CellsInhibitory Effect of Interleukin 4 on Production of Interleukin 6

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