Immunology 1990 70 473-477

Cytokine secretion effected by synergism of the immunomodulator AS101 and the protein kinase C inducer bryostatin B. SREDNI, Y. KALECHMAN, M. ALBECK,* 0. GROSS, D. AURBACH,* P. SHARON,* S. N. SEHGALt M. J. GURWITHt & H. MICHLIN CAIR Institute, Department of Life Sciences and *Department of Chemistry, Bar Ilan University, Ramat Gan, Israel and t Wyeth Ayerst Research, Philadelphia, U.S.A.

Acceptedfor publication 9 April 1990

SUMMARY AS 101, a synthetic organotellurium compound, was found to have immunomodulating properties by initiation of cytokine production in vitro and in vivo. Phase I/IT clinical trials currently in progress on AIDS and cancer patients treated with AS101 show significant increases in various immunological parameters, with minimal toxicity. Recently, AS1O1 and the protein kinase C (PKC) inducer, phorbol myristate acetate (PMA), were shown to synergize in the secretion of interleukin-2 (IL-2) and colonystimulating factor (CSF) in vitro, by human and mouse lymphoid cells. The bryostatins, a group of natural macrocyclic lactones isolated from marine invertebrates (Bugula neritina) have been reported to be potent PKC activators with no tumour promoting activity. In this study, we investigated the synergistic effect of AS 101 and a partially purified preparation of bryostatin on the production of several cytokines. Our data confirm the presence of synergism, which greatly enhances cell proliferation, IL-2, tumour necrosis factor (TNF) and interferon-gamma (IFN-y) secretion by human mononuclear cells (MNC) and the production of IL-2 and TNF by mouse cells. The absence of tumour-promoting activity of the bryostatins makes them particularly good candidates, in combination with AS 101, for immunomodulation in vivo in clinically immunosuppressed conditions. Treatment of AIDS patients with AS 101 also shows promising results (Ruiz-Palacios et al., 1988). It is now well known that antigen- and mitogen-mediated lymphocyte activation is correlated with the induction of Ca2+ influx and the activation of protein kinase C (PKC) (Berridge, 1984; Trunch, Goldstein & Schmidt-Vershult, 1985; Nishizuka, 1986). The mechanism by which AS 101 stimulates lymphocytes to secrete IL-2 was found to involve the triggering of Ca2+ influx (Sredni et al., 1987). In a recent publication it was demonstrated that IL-2 secretion and cell proliferation of both human and mouse lymphocytes, as well as the production of CSF by mouse splenocytes, were significantly enhanced by the synergistic effect of ASlOl and the PKC activator phorbol myristate acetate (PMA) (Sredni et al., 1990a). It was recently reported that the bryostatins, a group of natural compounds extracted from the marine invertebrate Bugula neritina from the phylum Bryozoa, are non-tumour promoting activators of PKC. It was demonstrated that bryostatins together with Ca2+ ionophore stimulate human lymphocytes to secrete IL-2 and IFN-y as effectively as PMA and Ca2+ ionophore (Mohr, Pettit & Plessing-Menze, 1987). In view of the absence of tumour-promoting activity of the bryostatins in contrast to the phorbol esters, and their PKC activation potential, we hypothesized that they could be useful in combination with ASIOl in augmenting cytokine secretion. We therefore investigated the in vito synergism between AS101

INTRODUCTION AS 101, a synthetic organotelluric compound developed in the laboratory at Bar Ilan University, was found to possess immunomodulating and anti-viral properties with minimal toxicity (Sredni et al., 1987, 1988; Albeck, Tamari & Sredni., 1989; Nyska et al., 1989; Shani et al., 1990; Sredni et al, 1990b). AS 101 was found to induce the production of various cytokines by human and mouse haemopoietic cells in vitro, and to enhance the production of interleukin-2 (IL-2), colony-stimulating factor (CSF) and IL-2 receptor expression by spleen cells of AS 101injected mice (Sredni et al., 1987, 1988). Systemic administration of AS 101 to mice transplanted with primary tumours appeared to decrease the tumour volume and to increase the survival rate of the tumour-bearing mice (Sredni et al., 1987, 1988). AS101, which is presently in extensive Phase I/II clinical trials in patients with advanced malignancies, shows enhancement of IL-2, tumour necrosis factor (TNF) and interferongamma (IFN-y) production and IL-2 receptor levels without significant toxic effects (Shani et al., 1990; Sredni et al., 1990b). Abbreviations: IFN-y, interferon-gamma; IL-2, interleukin-2; MNC, mononuclear cells; PKC, protein kinase C; PMA, phorbol myristate acetate; TNF, tumour necrosis factor. Correspondence: Professor B. Sredni, Dept. of Life Sciences, Bar Ilan University, Ramat Gan, 52900 Israel.

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and a partially purified preparation of bryostatin. The evidence we found for a synergistic effect of the two compounds on the proliferation rate and the secretion of various cytokines by human and mouse cells strongly suggests that a combined treatment might prove useful in clinically immunosuppressed conditions.

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Reagents PMA (Sigma Chemical Co., St Louis, MO) was dissolved in ethanol and stored at -20°. Before being added to cells, PMA was diluted in culture medium to the appropriate concentration. The final concentration of ethanol was less than 0-1 %. Actinomycin D (Dactinomycin, Merck, NJ) was dissolved in RPMI and stored at -200, protected from light. AS101 [ammonium trichloro(dioxoethylene-0,0'-)tellurate, a low molecular weight synthetic compound, synthesized at Bar Ilan University] was dissolved in PBS at a concentration of 50 yg/ml and stored at 40. Bryostatin, a macrocyclic lactone extracted from the marine invertebrate B. neritina of the phylum Bryozoa, was collected in the Mediterranean Sea and partially purified according to Pettit et al. (1982). Seven fractions obtained after extraction and Sephadex chromatography were evaporated and dissolved in dimethylsulphoxide (DMSO), at concentrations of 20 jug/ml, and stored at - 20°. One of the fractions was found to be the most potent and was used in the present study. Before adding to cell culture, the bryostatin was dissolved in culture medium. DMSO concentration in the culture was always less than 1%. Cell and culture preparation Human mononuclear cells (MNC), separated from fresh heparinized blood of healthy donors by Ficoll-Hypaque density gradient centrifugation (Pharmacia, Fine Chemicals, Uppsala, Sweden), were washed twice and suspended in enriched RPMI1640 medium at a concentration of 15 x 106 cells/ml, as described previously (Sredni et al., 1990a). Mouse splenocytes were obtained from 2-3-month-old male BALB/c mice. After hypotonic lysis to remove erythrocytes, the cells were suspended in enriched RPMI-1640 medium at a concentration of 5 x 106 cells/ml. Mouse peritoneal macrophages (PEC) were isolated from BALB/c mice (from Tel-Aviv University, Israel) injected 72 hr before with 2 ml of 3% thioglycolate (Difco Lab., Detroit, MI). The cells were washed twice and suspended in enriched RPMI1640 medium at a concentration of 1 x 106 cells/ml. The cultures were incubated at 370 in a humidified incubator containing 7 5 CO2 in air. T-cell proliferation assay Freshly isolated human MNC were placed in 96-well microtitre plates in 200 p1 of enriched RPMI-1640 medium at a concentration of 2 x 105 cells/well. The various compounds to be tested for their proliferative potential were added in the appropriate concentrations. Cultures were harvested after 3 days. For the last 18-24 hr before harvesting the cultures were pulsed with 1 uCi/well of [3H]thymidine (Nuclear Research Center, Negev, Israel) and counted by liquid scintillation.

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Figure 1. Synergism between ASlOI and bryostatin in stimulating IL-2 production by mouse splenocytes. Mouse spleen cells were incubated with 0-100 ng/ml bryostatin (-) or brysostatin in the presence of 0 1 Pg/ ml AS 101 (5). Supernatants were collected after 24 hr and assayed for IL-2 activity on mouse IL-2-dependent CTLL cells (the results represent mean + SME of five experiments).

IL-2 production and quantification Human MNC, 15 x 106/ml, or mouse splenocytes, 5 x 106/ml, suspended in enriched RPMI- 1640 culture medium, were seeded in 24-well cluster wells in the presence of various concentrations of the stimulants. The cultures were incubated for 48 and 24 hr, respectively. Supernatants were assessed for IL-2 activity by their ability to stimulate the proliferation of the IL-2-responsive murine cell line, CTLL, as described by Gillis et al. (1978). TNF secretion and bioassay Human MNC, 1-5 x 106/ml, or mouse PEC, I x 106/ml, cells in enriched culture medium were incubated for 96 hr and 24 hr respectively, in the presence of the stimulators tested. Supernatants obtained were tested for TNF levels by their cytotoxicity for the murine target cells LM (a derivative of cell line L929), obtained from the ATCC (no. CCL 23), as described previously (Flick & Gifford, 1984). TNF levels were defined as the reciprocal of the dilution resulting in 50%, cell cytotoxicity.

IFN-y secretion and bioassay IFN-y levels were tested in supernatants prepared by the same method as that for human TNF. IFN-y activity of supernatant fluids was titrated by their ability to inhibit the cytopathic effect of the vesicular stomatitis virus (VSV) on the monolayers of HeP2 cells (ATCC, CCL-12) (Havell & Vilcek, 1972). IFN-y levels were calculated in reference to a standard containing 480 U/ml IFN-y. RESULTS Effect of AS101 and bryostatin on IL-2 production by mouse lymphoid cell In order to investigate the combined effect of AS101 and bryostatin on IL-2 production, mouse splenocytes were stimulated with 0-1 pg/ml AS101 and increasing concentrations of bryostatin (0-100 ng/ml) (Fig. 1). Under these experimental conditions, splenocytes stimulated with either AS101 or bryostatin did not show significant IL-2 production but at the same concentrations a strong synergistic effect was found between the two compounds, with an optimal activity at 50 ng/ml bryostatin.

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Cytokine secretion by ASJOJ and bryostatin

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Figure 3. Induction of IFN-y secretion by human MNC stimulated with 0 5 Mg/ml ASlOI and 0-200 ng/ml bryostatin (0) or ASI01 with 10 ng/ml PMA (U) or bryostatin alone (-) (the results represent mean + SME of four experiments).

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produced by ASI01 and PMA, while PMA alone did not stimulate secretion of IFN-y (data not shown).

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Figure 2. Synergism between AS101 and bryostatin in stimulating human cell proliferation and IL-2 production. (a) Proliferation of MNC incubated with 01 yg/ml AS101 and 0-100 ng/ml bryostatin (0) or bryostatin alone (U) (the results represent mean + SME of five experiments). (b) IL-2 production by human MNC stimulated with 0-1 ,pg/ml AS101 and 0-100 ng/ml bryostatin (E) or bryostatin alone (-) (the results represent mean + SME of five experiments).

Combined effect of AS101 and bryostatin on human lymphoid cell proliferation and IL-2 secretion In these experiments, cell proliferation (Fig. 2a) and IL-2 production (Fig. 2b) of human MNC stimulated by 0-1 Mg/ml ASI01 and 0-100 ng/ml bryostatin were tested. Figure 2a shows that addition of bryostatin and 0-1 g/ml AS 10 1 to MNC led to a strong enhancement of the proliferation rate. Maximal proliferation was obtained by adding 50 ng/ml bryostatin. Figure 2b demonstrates IL-2 secretion by MNC stimulated with 0-1 pg/ml AS10, in conjunction with bryostatin, with an optimal effect achieved at 25 ng/ml. Secretion of IFN-y by human MNC as the result of the synergistic effect of AS101 and bryostatin After demonstrating the synergism of ASIO1 and bryostatin on IL-2 production and cell proliferation, the combined effect of these compounds was tested on the secretion of other lymphokines. AS101, at a concentration of 0-5 Mg/ml, and increasing quantities of bryostatin (0-200 ng/ml) or PMA (10 ng/ml) were added to human MNC and incubated for 96 hr. Figure 3 summarizes the IFN-y levels, tested in the supernatants. AS10, 0-5 yg/ml, in conjunction with bryostatin, stimulated enhanced amounts of IFN-y, with maximal activity of 210 + 30 U/ml obtained at 25 ng/ml (compared to 37-5 + 7-5 U/ml obtained when 0-5 pg/ml AS101 was added alone). As can be seen in Fig. 3, IFN-y levels produced by stimulation of ASO1 and bryostatin were 1-75 times those

Secretion of TNF by human MNC and mouse macrophages stimulated by AS101 and bryostatin In these experiments we examined whether the synergistic effect of AS101 and bryostatin is more general and also affects macrophages. Human MNC and mouse PEC cells, isolated by peritoneal puncture 72 hr after i.p. thioglycolate injection, were incubated with AS101 and bryostatin for 96 hr and 24 hr, respectively. Similar cultures were incubated with AS101 and PMA. PMA was added at a concentration at which no TNF was secreted. Figure 4a shows that 01 g/ml AS I 0 1 together with bryostatin (0-100 ng/ml) led to remarkable levels of TNF production, with a maximal effect being reached at 50 ng/ml bryostatin. In these experiments the combined effect of AS 101 and PMA (10 ng/ml) was also tested and was found to be lower than the optimal effect of AS101 and bryostatin (48+16 compared to 88 + 24 U/ml). Low levels of TNF production were noticed when ASIOI was added alone. Figure 4b shows the production of TNF by mouse PEC cells induced by 0 5 pg/ml AS101 and increasing amounts of bryostatin (0-100 ng/ml) or with PMA (20 ng/ml). Bryostatin at a concentration of 100 ng/ml with ASI01 induced maximal TNF production and was found to be higher than TNF levels secreted by AS101 and PMA (18-7 U/ml versus 10-7 U/ml). Bryostatin at a concentration of 100 ng/ml induced maximal TNF production and was found to be higher than TNF levels secreted by ASlOI and PMA (18.7 U/ml versus 10-7 U/ml). DISCUSSION In the present study we describe the ability of the immunomodulator ASIOI to synergize with the PKC activator bryostatin in inducing the secretion ofseveral cytokines. We demonstrate that ASO1 in combination with bryostatin stimulates human and mouse lymphoid cells to proliferate and secrete IL-2 (Figs 1 and 2). Since IL-2 is known to increase the number and activity of cytotoxic, natural killer (NK) and lymphokine-activated killer (LAK) cells capable of anti-tumour activity, the potentiation of

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1987; Lee et al., 1989). Moreover, the pharmacokinetics of IL-2 indicate a short half-life in vivo (Thompson et al., 1987). The present study shows a strong synergism between AS 101 and bryostatin in cytokine secretion. Since AS101 has been shown to possess multipotential stimulatory activities and to exhibit low toxicity (Sredni et al., 1987, 1988; Shani et al., 1990), and bryostatin, in contrast to the phorbol ester PMA, has been shown to have no tumour-promoting activity (Pettit et al., 1982), we believe that a combined treatment with the two agents might increase the endogenous production of cytokines, thus improving the restoration of immune responses in conditions involving immune suppression. These conditions are known to exist in advanced malignancies, viral infections and immunodeficiency disorders including AIDS, as well as immunosuppressive conditions brought about by effects of aggressive therapies such as chemotherapy and radiotherapy.

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REFERENCES

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Figure 4. Induction of TNF production by human MNC and mouse PEC cells by the synergistic effect of ASlOl and bryostatin. (a) TNF production by human MNC incubated for 96 hr with 0 1 pg/ml ASIO1 and 0-100 ng/ml bryostatin (0) or 10 ng/ml PMA (U) or bryostatin alone (U) (the results represent mean+SME of four experiments). (b) TNF production by mouse PEC cells obtained after thioglycolate injection, incubated for 24 hr with 0-5 Pg/ml ASIOI and 0-100 ng/ml bryostatin (Li) or 10 ng/ml PMA (0) or bryostatin alone (-) (the results represent mean + SME of three experiments).

IL-2 production might be of great significance for enhancing host defence mechanisms against tumours (Hefeneider et al., 1983; Rosenberg, 1985; Lotzova, 1988; Lotzova et al., 1988). Bryostatin was also shown to synergize with ASIOl in the secretion of significant levels of TNF (Fig. 4a, b), a monocyte/ macrophage derived cytokine with cytotoxic activity against tumour cells (Feinman et al., 1987; Urban et al., 1986), and of IFN-y (Fig. 3), which activates macrophage and NK cells (Brunda & Rosembaum, 1984; Saiki & Fidler, 1985). Although there was also a synergistic effect between ASIOI and PMA on the production of the two cytokines, the augmentation with bryostatin was higher. The role of the above-mentioned cytokines in tumour therapy and haematopoiesis has been extensively investigated in recent clinical studies. Treatment with IL-2 alone or in combination with LAK cells has been shown to be therapeutically beneficial in some types of human cancer as well as in murine tumour models (Lafreniere & Rosenberg, 1985; Rosenberg et al., 1987; Kedar, Ben-Aziz & Shiloni, 1988). In recent years a growing number of publications have reported that combinations ofdifferent cytokines have synergistic therapeutic effects in murine tumour models and cancer patients (Rosenberg, Schwarz & Speiss, 1988; Dermetri et al., 1989; Zimmerman et al., 1989). For effective treatment with recombinant cytokines, high doses are required and are associated with serious toxicities, especially when given in combination (Thomspon et al.,

ALBECK M., TAMARI T. & SREDNI B. (1989) A synthesis and properties of ammonium trichloro (dioxoethylene-0,0') tellurate (AS101), a new immunomodulating compound. Synthesis, 8, 635. BERRIDGE M.J. (1984) Inositol triphosphate and diacylglycerol as second messengers. Biochem. J. 220, 345. BRUNDA M.J. & ROSENBAUM D. (1984) Modulation of murine natural killer cell activity in vitro and in vivo by recombinant human interferons. Cancer Res. 44, 597. DERMETRI G.D., SPRIGGS D.R., SHERMAN M.L., ARTHUR K.A., IMAMURA K. & KuFE D.W. (1989) Phase I trial of recombinant human tumor necrosis factor and interferon-gamma: effects of combination cytoline administration in vivo. J. Clin. Oncology, 7, 1545. FEINMAN R., HENRIKsEN-DEsLEFANo D., TsuJIMOTO M. & VILCEK J. (1987) Tumor necrosis factor is an important mediator of tumor cell killing by human monocytes. J. Immunol. 138, 635. FLICK D.A. & GIFFORD G.E. (1984) Comparison of in vitro cell cytotoxic assays for tumor necrosis factor. J. immunol. Meth. 68, 167. GILLIS S., FERM MM., Ou N. & SMITH K.A. (1987) T cell growth factor: parameters of production and quantitative microassay for activity. J. Immunol. 120, 2027. HAVELL E.A. & VILCEK J. (1972) Production of high-titered interferon in cultures of human diploid cells. J. Antimicrob. Agents Chemother. 2, 476. HEFENEIDER S.H., CONLON P.S., HENNEY C.S. & GILLIS S. (1983) In vivo interleukin administration augments the generation of alloreactive cytolytic T lymphocytes and resident natural killer cells. J. Immunol. 130, 222. KEDAR E., BEN-Aziz R. & SHILONI E. (1988) Therapy of advanced solid tumors in mice using chemotherapy in combination with IL-2 with and without lympholine-activated killer cells. Israel J. Med. Sci. 24, 494. LAFRENIERE R. & ROSENBERG S.A. (1985) Successful immunotherapy of murine experimental hepatic metastases with lymphokine-activated killer cells and recombinant interleukin-2. Cancer Res. 45, 3735. LEE R.E., LOTZE M.T., SKIBBER J.M., TUCKER E., BONOw R.O., OGNIBENE F.P. et al. (1989) Cardiorespiratory effects of immunotherapy with interleukin-2. J. Clin. Oncology, 7, 7. LOTZOVA E. (1988) Role of natural killer cells in defense against leukemia: therapeutic considerations. Natl. Immun. Cell Growth Regul. 7, 170. LOTZOVA E., SAVARY C.A., FREEDMAN R.S., EDWARDS C.L. & WHARTON S.T. (1988) Recombinant IL-2 activated NK cells mediate LAK activity against ovarian cancer. Int. J. Cancer, 42, 225. MOHR H., PETTIT G.R. & PLESSING-MENZE A. (1987) Co-induction of

Cytokine secretion by ASJOI and bryostatin lymphokine synthesis by the antineoplastic bryostatins. J. Immunobiology 175, 420. NISHIZUKA Y. (1986) Studies and perspectives of protein kinase C. Science, 233, 305. NYSKA A., WANER T., PIRAK M., ALBECK M. & SREDNI B. (1989) Toxicity study in rats of a tellurium based immunomodulating drug, AS 101: a potential drug for AIDS and cancer patients. Arch. Toxicol. 63, 386. PETTIT G.R., HERALD C. L., DOUBEK D.L. & HERALD D.L. (1982) Isolation and structure of bryostatin. J. Am. Chem. Soc. 104, 6848. ROSENBERG S. (1985) Lymphokine-activated killer cells: a new approach to the immunotherapy of cancer. JNCI 75, 595. ROSENBERG S., LOTZE M.T., MUUL L.M., CHANG A.E., Avis F.P., LEITMAN S. et al. (1987) A progress report on the treatment of 157 patients with advanced cancer using lymphokine-activated killer cells and interleukin-2 or high doses of interleukin alone. N. Engl. J. Med. 316, 889. ROSENBERG S.A., SCHWARZ S.L. & SPIESS P.J. (1988) Combination immunotherapy for cancer: synergistic antitumor interactions of interleukin-2, alfa interferon and tumor-infiltrating lymphocytes. JNCI 80, 1393. RuIZ-PALACIos G.M., PONCE DE LEON S., ALARCON-SEGOVIA D., ALCOCER VARELA J., CRUZ A., NARES F., PONCE DE LEON A., ROJAS G. & SREDNI B. (1988) Tolerance and response to AS-101, a new immunomodulator, in AIDS patients. IV International Conference on AIDS, 3038. Stokholm International Fairs, Stokholm, Sweden. SAIKI I. & FIDLER I.J. (1985) Synergistic activation by recombinant mouse interferon-y and muramyl dipeptide of tumoricidal properties in mouse macrophages. J. Immunol. 135, 684. SHANI A., TICHLER T., CATANE R., GURWITH M., GEZIN A., LEVI E. et al. (1990) The immunologic effects of AS101 in the treatment of cancer patients. Nati. Immun. Cell Growth Regul. 9, 182.

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SREDNI B., CASPI R.R., KLEIN A., KALECHMAN Y., DANZIGER Y., BEN YA'AKOV M., TAMARI T., SHALIT F. & ALBECK M. (1987) A new

immunomodulating compound (AS 101) with potential therapeutic application. Nature (Lond.), 330, 173. SREDNI B., CASPI R.R., LUSTIG S., KLEIN A., KALECHMAN Y., DANZIGER Y., BEN YA'AKOV M., TAMARI T., SHALIT F. & ALBECK M. (1988) The

biological activity and immunotherapeutic properties of AS101, a synthetic organotellurium compound. Nat!. Immun. Cell Growth Regul. 7, 163. SREDNI B., KALECHMAN Y., SHALIT F. & ALBECK M. (1990a) Synergism between ASlOI and PMA in lymphokine production. Immunology, 69, 110. SREDNI B., SHANI A., CATANE R., GEZIN A., LEVI E., SHLESINGER M. et al. (1990b) Recent Advances in Chemotherapy, p. 851.1-851.4. E. Lewin-Epstein Ltd, Tel Aviv. THOMPSON J.A., DOUGLAS J.L., Cox W.W., LINDGREN C.G., COLLINS C. & NERAAS K.A. (1987) Recombinant interleukin-2 toxicity, pharmacokinetics and immunomodulatory effects in a phase I trial. Cancer Res. 47, 4202. TRUNCH A.F., GOLDSTEIN A.P. & SCHMIDT-VERHULST A.M. (1985) Early steps of lymphocytic activation bypassed by synergy between calcium ionophores and phorbol ester. Nature (Lond.), 313, 318. URBAN J.L., SHEPARD H.M., ROTHENSTEIN J.L., SUGARMAN B.J. & SCHREIBER H. (1986) TNF: a potent effector molecule for tumor cell killing by activated macrophages. PNAS 83, 5233. ZIMMERMAN R.S., GAUNY S., CHEN A., LANDRE P., WINKELHAKE J.L. (1989) Sequence dependence of adminstration of human recombinant tumor necrosis factor and interleukin-2 in murine tumor therapy. JNCI 81, 227.