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ANTITUMOR EFFECTS OF INTERFERON: MECHANISMS OF THE LONG TERM SUPPRESSION OF TUMORS IN MICE

Objective


DBA/2 mice were injected intravenously with interferon (IFN) alpha and beta resistant 3Cl8 Friend erythroleukaemia cells (FLC). IFN alpha and beta treatment of tumour injected mice increased their survival time and these mice developed a resistance to a second challenge with FLC. The efficacy of IFN alpha and beta in increasing the survival time was compared between normal immunocompetent and immunodeficient mice. The antitumour action of IFN was markedly reduced or abolished in newborn DBA/2 mice, in adult athymic nu/nu and beige DBA/2 mice, and in BALB/c scid/scid mice.
To determine the phenotype of the effector cells involved, FLC injected DBA/2 mice were treated with antibodies to asialo GM1, CD4 or CD8, or with cyclosporin A or silica. IFN alpha and beta treatment proved much less effective in these mice indicating that a variety of effector cell types participated in the IFN induced suppression of visceral metastases. Thus, an intact immune system appeared to be essential to obtain optimal therapeutic effects of IFN alpha and beta in this experimental model.

To investigate the possible mechanisms of Friend erythroleukaemia cell (FLC) tumour inhibition in the liver of interferon treated mice, an in vitro model was developed consisting of a coculture of interferon (IFN) alpha and beta resistant 3Cl8 FLC and syngeneic mouse hepatocytes. Whereas IFN alpha and beta did not inhibit the multiplication of 3Cl8 FLC cultivated alone, it effectively inhibited the multiplication of 3Cl8 FLC in coculture with hepatocytes. The inhibitory effect was directly proportional to the amount of IFN alpha and beta added to the cocultures and more than 90% inhibition of FLC multiplication was noted with 1.6E5 IU/ml IFN alpha and beta on day 3 of coculture. When FLC were separated from the monolayer of hepatocytes by a pored membrane (0.4 um), the inhibitory effect on FLC proliferation was unchanged, indicating that a soluble factor (or factors) released from IFN treated hepatocytes was most important in the inhibition of FLC multiplication. An inhibitory activity of FLC multiplication was detected only in the conditioned medium of IFN treated hepatocytes but not in the conditioned medium of control hepatocytes nor in extracts of IFN treated or control hepatocytes. The inhibitory factor(s) in the conditioned medium of IFN treated hepatocytes was retained by an ultrafiltration membrane (molecular weight cut off 10 000) and its activity was completely abrogated by trypsin digestion. Its stability to treatment with 1 molar acetic acid, as well as a lack of correlation between the antiproliferative effect and the amount of L-arginine in the medium, distinguished this factor(s) from from liver arginase which was also found to be a potent inhibitor of FLC multiplication in vitro. The inhibitory factor(s) was also distinguishable in its biological activity from IFN gamma interleukin 1 alpha and beta, and transforming growth factor beta 1 and beta 2. These results suggest the possibility that the inhibitory effect of IFN alpha and beta on the developme nt of 3Cl8 FLC in the livers of IFN treated mice may be mediated by an IFN induced inhibitor of FLC multiplication.

Human interferon (IFN) alpha or IFN beta has been shown to induce a 17 kD membrane protein in human cells which, when eluted from sodium dodecyl sulphate (SDS) gels, inhibited the multiplication of cells of different human cell lines. It has now been shown that mouse IFN alpha and beta induces a 16 kD membrane protein in L1210 and Friend erythroleukaemia cells (FLC) sensitive to IFN alpha and beta, (but not in the derived IFN alpha and beta resistant cell lines) as well as in primary and monolayer cultures of mouse embryonic fibroblasts and adult mouse hepatocytes, and in suspensions of spleen cells. In addition, IFN alpha and beta enhanced the expression of an 11 kD membrane protein which could be shown by immunoprecipitation to be beta 2-microglobulin.

DBA/2 mice were injected intravenously with 2 x 106 3Cl8 Friend erythroleukaemia cells (FLC), a cell line resistant to interferon (IFN) alpha and beta. Although daily administration of mouse IFN alpha and beta markedly increased the mean survival time, most IFN treated mice continued to harbour FLC in different organs. To investigate the mechanisms responsible for this persistent suppression of FLC growth in IFN treated mice, a series of adoptive transfer experiments were undertaken with sera and spleen cells. Sera from FLC injected , IFN treated mice were very effective in conferring protection on DBA/2 mice even when injected systematically (intravenously) 18 to 24 hours prior to intravenous challenge with FLC. These sera also exhibited antitumour activity when injected subcutaneously or intraperitoneally together with FLC.
The protective factor in serum was shown to be an immunoglobulin. FLC injected, IFN treated mice developed antibodies to FLC demonstrable by radioimmune assay and complement dependent cytotoxicity. Sera from these mice recognised a specific 65 kD FLC membrane antigen(s) not detectable on membrane extracts from RBL-5 or ESb tumour cells or on normal spleen cells.
FLC injected, IFN treated mice also developed a specific cellular response demonstrable by transfer of protection with spleen cells injected intravenously of subcutaneously. Analysis of the responsible spleen cell populations indicated that the effector cells were neither T nor B cells. These results, demonstrating the importance of host humoral and cellular immune mechanisms in the persistent suppression of FLC in IFN treated mice, may be relevant to the use of IFN alpha and beta in patients in whom tumours may regress and tumour cells may then remain latent for extended periods of time.

Friend erythroleukaemia cells (FLC) passaged in mice are highly tumorigenic and multiply extensively in the livers of suckling DBA/2 mice without differentiating. In contrast, in vitro passaged FLC injected intravenously were of low tumorigenicity, multiplied to a limited extent in the livers of suckling mice and underwent differentiation from the proerythroblast to the orthochromatic erythroblast stage in the liver. The presence of characteristic C-type virions budding from the cell surface in various stages of erythroid differentiation served as a marker of the injected FLC. When the same in vitro passaged FLC, which differentiated in the liver, were injected subcutaneously in suckling mice they formed large subcutaneous tumours consisting of sheets of undifferentiated tumour cells. It is concluded that the tumorigenicity of FLC depended on the site of tumour growth and that there was an inverse correlation between the tumorigenic capacity and the capacity to differentiate.

Friend erythroleukaemia cells (FLC) (H-2d) injected intravenously multiply extensively in the livers of syngeneic DBA/2 mice and not at all in the livers of allogeneic C57Bl/6 mice. Results indicate that interferon alpha (IFN alpha) is an important factor in the resistance of allogeneic mice to the multiplication of FLC in the liver.
After intravenous inoculation of FLC there was an inverse correlation between the presence of IFN alpha in the serum and the capacity of FLC to multiply in the liver. Thus, all 44 FLC injected adult C57Bl/6 mice had circulating IFN alpha and FLC did not multiply in the liver of any of the mice. Interferon was not detected in the serum of 83% of 41 FLC injected DBA/2 mice (and was found only at a low titer in 17% of the mice) and FLC multiplied in the liver of all mice.
FLC did multiply in the livers of newborn C57Bl/6 mice and in the livers of irradiated adult C57Bl/6 mice, and IFN alpha was not detected in their sera. In contrast, after intravenous inoculation of FLC, IFN alpha was detected in the sera of 3 week old and athymic nu/nu adult C57Bl/6 mice, while FLC failed to multiply in the liver.
FLC also induced IFN alpha in congenic B10.D2 (H-2d) mice and FLC did not multiply in the liver.
It is suggested that, depending on the site of tumour implantation, different host mechanisms have various degrees of importance in controlling the growth and/or rejection of allogeneic tumour cells, and that IFN alpha is particularly important when FLC are injected intravenously.

Peritumoral injection of relatively low doses of either mouse interferon (IFN) alpha and beta (10 000-20 000 units/injection) or of recombinant human interleukin-1 (IL-1) beta (125-250 ng/injection) in mice transplanted subcutaneously with Friend erythroleukaemia cells (FLC) resulted in some inhibition of primary tumour growth, inhibition of liver and splenic metastases and increased survival time. A synergistic antitumour effect was observed in mice injected with both IL-1 and IFN alpha and beta. Highly purified mouse IFN beta also exerted a synergistic antitumour effect when combined with IL-1 beta in mice injected with FLC. The antitumour action of IL-1 and IFN was markedly reduced in mice treated with antibodies to CD4 antigens. Antibodies to asialo GM1 also diminished the antitumour effect by the combined cytokine treatment. The combined IL-1 and IFN therapy was effective in natural killer (NK) deficient bg/bg mice, although the extent of the antitumour response in these mice was less than that observed in bg+ mice.

Peritumoral injection of recombinant human interleukin 1 beta (IL-1 beta) in mice transplanted subcutaneously with Friend erythroleukaemia cells (FLC) resulted in a marked increase in survival time and inhibition of metastatic tumour growth in liver and spleen. In contrast, interleukin 2 (IL-2) treatment alone did not significantly inhibit the development of FLC metastases. A synergistic antitumour effect was observed after combined IL-1 and IL-2 therapy of these mice.
The antitumour action of IL-1 and IL-2 treatment was abolished, or markedly reduced, in mice treated with antibodies to CD4 or CD8 antigens, whereas antibodies to asialo GM1 were ineffective. A clear cut increase in the percentage of CD4+ cells was observed in the spleens of cytokine treated mice on days 17 and 23. On day 23 of cytokine therapy, CD8+ cells were increased in both spleens and lymph nodes. On day 17, infiltrates of host reactive cells (ie lymphocytes, granulocytes and monocytes) were observed in both spleen and liver from FLC injected mice treated with IL-1 and IL-2, in association with tumour cells. On days 17 and 23, spleen cells and cells recovered from mesenteric lymph nodes of IL-1 and IL-2 treated mice exerted a potent antitumour effect as determined by Winn assay experiments. This antitumour activity was abolished by preincubation of spleen cells with antiCD8 antibody, but not by treatment with antibodies to asialo GM1 (antibodies to CD4 exerted only a slight effect).
Combined IL-1 and IL-2 therapy was more effective on established FLC tumours than on early tumour transplanted mice. IL-1 and IL-2 treatment was also highly effective in increasing the survival time of mice from which the subcutaneous primary tumours were excised 7 days after FLC injection.
These data indicate that in mice injected with FLC, the antitumour effects of IL-1 and IL-2 are mediated by CD4+ and CD8+ cells (but not natural killer (NK) cells), and suggest that this combined cytokine treatment may be effec tive against established metastatic tumours.
NEANT.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

Centre National de la Recherche Scientifique (CNRS)
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ISTITUTO SUPERIORE DI SANITA
Italy
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