The immunoglobulin production of human peripheral B lymphocytes induced by phorbol myristate acetate

CELLULAR

IMMUNOLOGY

72, 88-96 (1982)

The lmmunoglobulin Production of Human Peripheral B Lymphocytes Induced by Phorbol Myristate Acetate ISAMU SUGAWAKA Department of Immunobiology, Karolinska Institute, Wallenberg Laboratory, Lilla Frescativiigen 7, S-104 05, Stockholm, Sweden Received February 2. 1982; accepted May 31, 1982 The mechanism of immunoglobulin production of human peripheral B lymphocytes induced by the croton oil-derived tumor-promoting agent, phorbol my&ate acetate (PMA), was investigated. The following results were obtained. (i) PMA at a concentration of 1 or 5 &ml induced polyclonal IgM production but the level of IgM synthesis was lower than that induced by PWM, when mononuclear cells (MNC) were cultured with PMA for 6 days. (ii) When T and B lymphocytes were cultured with PMA for 6 days, PMA could induce significant IgM production. However, addition of human monocytes potentiated Ig production by PMA markedly. (iii) When purified B or non-T lymphocytes were cultured with PMA, there was no significant IgM production. (iv) When OKT4+ cells or OKT8+ cells were added to the nonT cells and cultured with PMA. OKT4+ cell addition enhanced PMAdriven IgM production remarkably, while OKT8+ cell addition did not have any effect on it, suggestingthat the target cells of suppression of B-cell differentiation by PMA are OKT4+ cells. It was concluded that PMA required monocytes for the optimal immunoglobulin production of B cells and that OKT4+ cells were playing a major role in PMA-induced immunoglobulin production of human B cells. INTRODUCTION

The croton oil-derived tumor-promoting agent (l), phorbol my&ate acetate enhances the action of a carcinogen in inducing epidermal tumors of mouse skin (2), is mitogenic for lymphocytes (3, 4) or chicken embryo fibroblasts (5), potentiates lectin-induced T-cell activation (6), restores the capacity of macrophagedepleted T-cell populations to respond to Con A (7), enhances the production of Interleukin 2 (8), and directly stimulates macrophages from P388Dl tumor line to the synthesis of Interleukin 1 (9). PMA inhibits the terminal differentiation of various committed embryonic cells ( lo), and mouse Friend erythroleukemia ( 11) or myeloid leukemia cells ( 12). PMA can induce Ig secretion in human B-cell lines ( 13), differentiation of CLL cells in vitro (14, 15), a terminal cell differentiation in some murine (16) and human myeloid leukemia (17), and histiocytic lymphoma cells (18). (PMA),’

’ Abbreviations used: PMA, phorbol my&ate acetate; PWM, pokeweed mitogen; MNC, mononuclear cells; SRBC, sheep erythrocyte; FCS, fetal calf serum; Con A, concanavalin A; BSS, balanced salt solution; PFC, plaque-forming cell; DMSO, dimethyl sulfoxide; CLL, chronic lymphoid leukemia; Ig, immunoglobulin. 88 0008-8749/82/ 130088-09$02.00/0 Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved

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In our previous report2 we reported that PMA was a monocyte-independent human B-cell mitogen and monocyte-dependent human T-cell mitogen and that PMA did not interact with the activation receptors used by Con A or PHA. Therefore, I investigated the effect of PMA on immunoglobulin production of human peripheral B lymphocytes in different cell populations further, using a protein A-SRBC hemolytic plaque assay (19). We report here first that PMA requires monocyte participation for the optimal immunoglobulin production of B cells and that OKT4+ cells are playing a major role in PMA-driven immunoglobulin production of human B cells. MATERIALS AND METHODS MNC and T- and B-cell preparation. Human peripheral blood from healthy adult volunteers was collected in heparin. It was then diluted l/2 in physiological saline and layered on Lymphoprep (Nyegaard, Oslo, Norway) (20). After centrifugation at 1500 rpm for 30 min, the cells at the interface were collected, pooled, washed twice in balanced salt solution (BSS), and resuspended in RPM1 1640 (GIBCO, Europe, Glasgow, Scotland) containing 10% human AB serum. To deplete monocytes contained in human peripheral blood, one part of silica suspension (KAC-2, Nippon Kotai Kenkyujo, Gunma, Japan) was added to nine parts of human peripheral blood and the blood was incubated at 37°C for 90 min (according to an instruction sheet made by Nippon Kotai Kenkyujo, Japan). The blood was then diluted l/2 in 0.9% NaCl and layered on Lymphoprep. After centrifugation, the cells at the interface were collected and washed. Phagocytic cells were included in the pellet. The washed cells contained less than 2% cells phagocytosing silica particles. To deplete monocytes out of T- and B-cell population, the cells were further incubated on FCS-coated petri dishes (Intermed, Denmark) for 60 min (2 1). Thereafter, the supematants were collected and washed twice. The cells contained less than 0.1% silica-phagocytosing cells and are referred to as T and B cells. T- and B-cell separation. In IO-ml plastic tubes (Falcon plastics, 2057) 4 X lo6 T and B cells/ml were mixed with 200 X lo6 fresh sheep red blood cells (SRBC) in a final volume of 5 ml of RPM1 1640 containing 10% fetal calf serum (FCS) (previously absorbed with sheep and human erythrocytes and heat inactivated) (22). The cell mixture was incubated for 30 min at 37°C centrifuged for 5 min, at 1000 rpm, and then stored overnight at 4°C before being carefully resuspended and layered on Lymphoprep. After centrifugation at 1500 rpm for 30 min at 4’C, the cells at the interface were collected, pooled, and washed in BSS and are referred to as B cells. The pelleted cells are referred to as T cells after removal of SRBC by osmotic shock. To obtain highly purified T and B cells, the T cells or B cells were rerosetted with SRBC. The purity of the T cells and B cells were about 98 and about 96%, respectively, by complement-mediated cell lysis (23). Separation of purified monocytes. The 10’ MNC were incubated on FCS-coated petri dishes (Intermed, Denmark) for 2 hr. Thereafer, the supematants were discarded and the petri dishes were washed in warm media gently three times with Pasteur pipets (21). The bottoms were scraped carefully with a rubber policeman ’ I. Sugawara and S. Ishizaka, The degree of monocyte participation by phorbol myristate acetate, submitted for publication.

in human B- and T-cell activation

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and the cells were washed twice in BSS. The purity of the monocytes was about 99%, as judged by Latex particle ingestion test and the phagocytic cells were irradiated (2000 Rad) when used. Isolation of pur$ed non-T cells and T-cell subsets by complement-mediated cell lysis utilizing the monoclonal antibodies, OKT3, OKT4, OKT8, and OkNl. The OKT3 antibody reacts with 100% of peripheral T cells, OKT4 antibody reacts with 50-60% of peripheral T cells, OKT8 antibody reacts with 35% of peripheral T cells, and OKM 1 antibody reacts with 78% of adherent mononuclear cells and 18% of null cells and does not react with peripheral B and T cells. In order to obtain purified non-T cells, OKT4+ and OKTS+ cells, 5 X lo6 of the cells at the interface after the SRBC rosetting procedures, or 5 X lo6 T cells were resuspended in 1 ml of OKT3,OKT8, and OKMl or OKT4 and OKMl antibody (Ortho Diagnostics, Raritan, N.J.) diluted l/200 in RPM1 1640 containing 5% FCS and incubated for 1 hr at 4°C. After incubation, fresh rabbit serum as a source of complement was added at a final dilution of 1:5 and incubation was further carried out for 1 hr at 37°C in a humidified atmosphere. Complement-mediated cell lysis (23) showed that OKT3-treated cells were more than 97% B cells, less than 0.2% Latex phagocytosing cells, and less than 2% T cells: OKT8- and OKM 1-treated cells contained about 90% OKT4+ cells and less than 0.1% phagocytic cells and OKT4and OKMl-treated cells contained about 88% OKT8+ cells and less than 0.1% phagocytic cells. OKT4+ cells signify a T-cell population remaining after treatment with OKT8, OKM 1 plus complement, and OKT8+ cells signify a T-cell population remaining after treatment with OKT4, OKMl plus complement. Stimulants. PMA was purchased from Sigma Chemical Company, St. Louis, Missouri, and was dissolved in dimethyl sulfoxide (DMSO) and diluted to the optimal concentration with culture media. The content of DMSO was less than 0.2% of the total volume, which did not influence the cells used. PWM was obtained from Serva Feinchemica, Heidelberg, West Germany, and P-L Biochemicals Inc., Milwaukee, Wisconsin. Plaque-forming cell (PFC) responses to PMA. The 5 X IO6 MNC or 3-4 X lo6 T and B cells or 2.5-3 X lo6 non-T cells or 3 X lo6 B cells were incubated for 6 days with PMA ( 1 and 5 pg/ml) or PWM (20 &ml), using Marbrook culture bottles (No. 7, Takahashi Giken Co., Tokyo, Japan). In some experiments a certain number of monocytes was added to the cell population to evaluate monocyte requirement in immunoglobulin production of B cells. The inner tube with a dialyzable membrane contained 1 ml of cell suspension and the mitogen. The outer bottle contained 10 ml of RPM1 1640 containing 10% human AB serum. Since PMA can pass through the dialyzable membrane, it was also placed in the other bottle. After 6 days of culture, the cell suspensions were collected and washed in BSS at 1200 rpm for 10 min. The viable cells were counted in a Btirker hemocytometer. The number of immunoglobulin-secreting cells was determined by a hemolytic plaque assay in Staphylococcus aureus protein A-coated SRBC (19). Of the cell suspension, 25 ~1 was added to 0.2 ml of a 0.5% liquid agar supplemented with 1.6 ml/dl agar of 3% DEAE dextran containing 25 ~1of a 1:8 dilution of protein A-coupled SRBC. Then, 25 ~1 of rabbit anti-human IgM (diluted 1:60, DAK0 Immunoglobulins, Copenhagen, Denmark) and 25 ~1 of SRBC absorbed guinea pig complement (diluted 1:8, Flow Laboratory) were added to the cells in Ellerman tubes. The above mixture was placed on petri dishes and covered with coverglassesand incubated at 37°C for 4

Ig PRODUCTION

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Exp.1

MNC

MNC MNC MN P;M PiA PiA (20)(l) (5)

FIG. 1. PFC responses Marbrook culture bottles performed. All the values PMA (I), PMA (1 &ml);

NT

91

BY PMA

NT

NT

NT

”

MNC MNC

P+WMPM; A? (20)(l) (5)

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MNC MNC PAA &A (1) (5)

of purified non-T cells to PMA. 5 X were cultured for 6 days and protein show mean of the duplicate cultures. PMA (5), PMA (5 rg/ml); NT, non-T

NT

NT

NT

P;M PM; (20)(l)

NT P;IA 151

lo6 MNC or 3 X IO6 non-T cells in A-SRBC hemolytic plaque assay was PWM (20), PWM (Serva, 20 &ml); cells.

hr. The results were expressed as PFC per lo6 viable cells. All the experiments were done in the duplicate cultures. RESULTS PMA-Induced Plaque-Forming Cell (PFC) Responses Since we found that PMA is a monocyte-independent human B-cell mitogen and a monocyte-dependent human T-cell mitogen,’ I investigated whether PMA can induce Ig production in purified B or non-T cell populations. As shown in Fig. 1, PMA failed to induce significant IgM production even in the presence of monocytes. This finding was further confirmed by the data that there was also no significant polyclonal Ig production when monocytes were added to purified B-cell population (Fig. 2). Next, we tested whether PMA can induce polyclonal Ig secretion in T- and B-cell populations, since PMA can stimulate T cells significantly in the absence of monocytes.’ As depicted in Fig. 3, significant IgM production was recognized, but its degree was always lower than that by PWM. It seems that helper T cells play an important role in PMA-driven Ig production. When monocytes were added to Tand B-cell populations, addition of monocytes potentiated polyclonal immunoglobulin production induced by PMA (Fig. 4). Monocytes also seem to be an important factor in PMA-induced Ig production. To evaluate the role of T cells in detail, T cells were separated into OKT4+ cells and OKT8+ cells, utilizing OKT4, OKT8, and rabbit complement. When OKT4+

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cells were placed into a non-T-cell population, addition of OKT4+ cells enhanced polyclonal Ig induction by PMA (Fig. 5). On the contrary, OKT8+ cells did not have any effect on PMA-driven Ig production (Fig. 6). Since OKT4+ cells and OKT8+ cells mean inducer/helper T-cell subclass and human suppressor/cytotoxic T-cell subclass, respectively, it seemsthat helper T cells are playing a major role in significant immunoglobulin production induced by PMA. There was slight IgM production at the addition of OKT4+ cells and OKT8+ cells. This suggeststhat OKT8+ cells act on B cells directly or on OKT4+ cells. DISCUSSION I showed in this paper that PMA required monocyte participation for the optimal immunoglobulin production of peripheral B cells and that OKT4+ cells (inducer/ helper T-cell subclass) were playing a primary role in PMA-induced Ig production. As PMA can stimulate B cells directly, there is a possibility that PMA can induce Ig production of B cells directly. In the human B-cell line ( 13), CLL cells ( 14, 15), and lymphoma cell lines ( 18), it is possible. The reason for this is that certain dividing B cells (cell lines) may be at a differentiation state close to the Ig-producing plasmacytes and can be easily triggered to mature by PMA. However, as already shown in Figs. 1 and 2, this is not the case in normal human B lymphocytes. Judging from the data that there is significant Ig production in T and B populations without monocyte participation, helper T-cell and B-cell interactions may be very

Exp.1

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ICE

B

B

B

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BB

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FIG. 2. Effect of 0.5 X lo6 monocyte addition on PFC responses of purified B cells to PMA. 5 X lo6 MNC or 3 X lo6 B cells in Marbrook culture bottles were incubated for 6 days and protein A-SRBC hemolytic plaque assay was performed. Each value represents mean of the duplicate cultures. PWM (20), PWM (Serva, 20 &ml); PMA (1), PMA (1 &ml); PMA (5), PMA (5 &ml); B, B lymphocytes; M, 0.5 X lo6 monocytes.

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8 7 6

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MNC

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TB

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(20)(l)

(5)

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lln L

MNC MNC TB

B

TB

TB

+ + +

PWM PMA PMA (20)(l) (5)

P;IA PiiA (1) (5)

FIG. 3. Plaque-forming cell (PFC) responses of TB cells to PMA. 5 X lo6 MNC or 3 X IO6 TB cells in Marbrook culture bottles were cultured for 6 days and protein A-SRBC hemolytic plaque assay was performed. All the values represent mean of the duplicate cultures. PWM (20), PWM (Serva, 20 &ml); PMA (l), PMA (1 &ml); PMA (5), PMA (5 &ml); TB, T and B cells,

Exp.2

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TB

TB

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I

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1, r= TB

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< TB

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A

;

(1) (5) 1201

P;M (20)

PtlA PtiA P;M (1) (5) (20)

FIG. 4. Effect of lo6 monocyte addition on PFC responses of Marbrook culture bottles were cultured for 6 days and protein performed. All the values show mean of the duplicate cultures. i &ml); PMA( l), PMA (1 &ml); PMA(S), PMA (5 &ml); TB, 93

TB cells to PMA. 4 X lo6 TB cells in A-SRBC hemolytic plaque assay was WM(20), PWM (P-L Biochemicals, 20 T and B cells; M, IO6 monocytes.

ISAMU SUGAWARA

Exp.2

NT

NT

NT

p+NA 0th (5)

NT

NT

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NT PiA (5)

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(5)

FIG. 5. Effect of 106OKT4+ cell addition on PFC responsesof non-T cells to OMA. 2.5 X lo6 nonT cells in Marbrook culture bottles were cultured for 6 days and protein A-SRBC hemolytic plaque assay was performed. Each value shows mean of the duplicate cultures. PWM(ZO), PWM (P-L Biochemicals, 20 &ml); PMA( l), PMA (1 pg/ml); PMA(S), PMA (5 &ml); NT, non-T cells, OKT4+ cells; OKT8, OKT8+ cells.

important in polyclonal Ig production by PMA. At the same time, addition of monocytes potentiated PMA-induced Ig production. This suggeststhat monocytes and/or soluble factor(s) released by monocytes are important in inducing Ig production. OKT4+ cells are required to generate helper factor(s) capable of polyclonahy activating B cells independent of additional T-cell help (24). The helper factor(s) may enhance Ig production by PMA. Thus, studies about the possible role of soluble factor(s) released from monocytes and helper factor(s) by OKT4+ cells will be necessary to elucidate the precise mechanism of PMAdriven Ig production. My data also show that OKT8+ cells did not have any effect on Ig production induced by PMA (Fig. 6). Furthermore, there was slight IgM production at the addition of OKT4+ and OKT8+ cells. Taken together, it may be concluded that OKT8+ cells act on OKT4+ cells directly rather than B cells. The interaction between OKT4+ cells and OKT8+ cells is of importance in considering Ig production induced by PMA. Thomas et al. (24) demonstrated that the targets of suppression of B-cell differentiation induced by ahoantigen-triggered helper factor or PWM are OKT4+ &Is. This may be the case in PMA-driven Ig production, since their data are consistent with my data. Soluble factor(s) from OKT8+ cells may also be important and there is a possibility that the soluble factor(s) may suppress a helper function of B-cell differentiation induced bv OKT4+ cells. Further study will be necessary.

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Ig PRODUCTION INDUCED BY PMA

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r

i NT

7 PiA (5) N

1 NT + OKT8

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(5)

NT +

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NT +

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NT I

OKT8

OKT8

OKTB

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PiA (1)

PiA

(5)

FIG. 6. Effect of lo6 OKT8+ cell addition on PFC responses of non-T cells to PMA. 2.5 X lo6 nonT cells in Marbrook culture bottles were cultured for 6 days and protein A-SRBC hemolytic plaque assay was performed. All the values represent mean of the duplicate cultures. PWM(20), PWM (P-L Biochemicals, 20 pg/ml); PMA( l), PMA (1 &ml); PMA(S), PMA (5 &ml); NT, non-T cells; OKT8, lo6 OKT8+ cells.

Very recently, it is reported that the OKT4+ cell subsetsof human T cells contain cells that can be activated to differentiate into suppressor cells independent of OKT8+ cells (25). It will be important to study the role of “the suppressor cells” in PMA-driven Ig secretion. In summary, Ig production is induced in human peripheral blood cells by PMA, but the reaction requires the cooperation of T cells (OKT4+ cells) similarly to induction by PWM. PMA will provide a valuable tool in the study of human lymphocyte differentiation in vitro. It is our next step to study how PMA activates peripheral blood cells and renders B cells mature to secrete Ig biochemically and ultra-structurally. ACKNOWLEDGMENTS This work was made possible in part by support from Mr. Mitsuo Sugawara, who is a lawyer and director of Tokyo Marunouchi Law Center, Tokyo, Japan, and by the Swedish Medical Research Council and the Swedish Cancer Society.

REFERENCES 1. Hecker, E., Methods Cancer Rex 6, 439, 1971. 2. Van Duuren, B. L., Prog. Exp. Tumor Rex 11, 3 1, 1969. 3. Touraine, J. L., Hadden, J. W., Touraine, F., Hadden, E. M., Estensen, R., and Good, R. Exp. Med. 145,460, 1977. 4. Abb, J., Bayliss, G. J., and Deinhardt, F., .I Immunol. 122, 1639, 1979. 5. Driedger, P. E., and Blumberg, P. M., Cancer Rex 37, 3257, 1977.

A., J.

96 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

19. 20. 21. 22. 23. 24. 25.

ISAMU SUGAWARA Mastro, A. M., and MUller, G. C., Exp. Cell Rex 88, 40, 1974. Rosenstreich, D. L., and Mizel, S. B., J. Immunol. 123, 1749, 1979. Fuller-Farrar, J., Hilfiker, M. L., Farrar, W. L., and Farrar, J. J., Cell. Zmmunol. 58, 156, 1981. Mizel, S. B., Oppenheim, J. J., and Rosenstreich, D. L., J. Zmmunol. 120, 1497, 1978. Cohen, R., Pacifici, M., Rubinstein, N., Biehl, J., and Holtzer, H., Nature (London) 266, 538, 1977. Rovera, G., O’Brien, T., and Diamond, L., Proc. Nat. Acad. Sci. USA 74, 2894, 1977. Kasukabe, T., Honma, Y., and Hozumi, M., Gann 70, 119, 1979. Ralph, P., and Kishimoto, T., J. Clin. Invest. 68, 1093, 1981. Totterman, T. H., Nilsson, K., and Sundstrom, C., Nature (London) 288, 176, 1980. Totterman, T. H., Nilsson, K., Claesson, L., Simonsson, B., and Aman, P., Hum. LymphcyteDtQf.k 1, 13, 1981. Lotem, J., and Sachs, L., Proc. Nat. Acad. Sci. USA 76, 5158, 1979. Rovera, G., Santoli, D., and Damsky, C., Proc. Nat. Acad. Sci. USA 76, 1779, 1979. Nilsson, K., Andersson, L. C., Gahmberg, C. G., and Forsbeck, K., In “New Trends in Human Immunology and Cancer Immunotherapy” (B. Serrou and C. Rosenfeld, Eds.). Holt-Saunders & Doin, New York, 1981. Gronowicz, E., Coutinho, A., and Melchers, F., Eur. J. Immunol. 6, 588, 1976. Bbyum, A., Stand. J. Clin. Lab. Invest. 21, (Suppl. 97), 77, 1968. Kumagai, K., Itoh, K., Hinuma, S., and Tada, M., J. Immunol. Methods. 29, 17, 1979. Foumier, E., and Charmire, J., Cell. Immunol. 60, 212, 1981. Van Wauwe, J., and Goossens, J., Immunology, 42, 157, 1981. Thomas, Y., Sosman, J., Rogozinski, L., Irigoyen, O., Kung, P. C., Goldstein, G., and Chess, L. J. Immunol. 126, 1948, 1981. Thomas, Y., Rogozinski, L., Irigoyen, 0. H., Friedman, S. M., Kung, P. C., Goldstein, G., and Chess, L., J. Exp. Med. 154, 459, 1981.