In this study, we analyzed interleukin-2 (IL-2) and IL-2 receptor (IL-2R) expression in murine B16F10 melanoma and studied the effect of recombinant IL-2 (rIL-2) on the proliferation of these cells. Flow cytometry analysis revealed the presence of the IL-2R α subunit in B16F10 melanoma, with a mean posi-tivity rate of 30%. Using confocal microscopy, the expression of this chain could be visualized on the surface of B16F10 cells and in intracellular compartments when the cells were permeabilized with ethanol. In addition to the α subunit, the IL-2R β subunit was also expressed in B16F10 cells as shown by reverse transcription and polymerase chain reaction analysis. The functionality of the IL-2R on B16F10 cells was shown by the fact that cell proliferation increased dose-dependently with the addition of rIL-2 to the culture medium. We also detected expression of the IL-2 gene in B16F10 cells. In Northern blot assays, a typical band of 0.9 kb corresponding to IL·2 mRNA was observed, although supernatants from B16F10 cultures had no detectable IL-2 activity. Furthermore, the addition of neutralizing antibody (anti-IL-2) to cell cultures had no effect on cell proliferation. From these results, we concluded that an IL-2 signalling system is present in murine B16F10 melanoma cells and that IL-2 favors B16F10 cell proliferation, suggesting a role for this cytokine in the tumoral activity of these cells.
To investigate the significance of host immunity in metastasis we have simultaneously evaluated metastatic development and the tumoricidal action of host defenses in an experimental system for liver metastasis which involves the intrasplenic injection of B16F10 melanoma cells in syngeneic mice. In addition, three experimental groups were treated with immunosuppressive doses of cyclosporin A (CsA) during the following periods of the malignant process: 1st-5th days, 1st-12th days and 7th-12th days. Analysis of cytolytic effects of macrophages, NK cells and T-lymphocytes on tumor cells reveals a decay in antitumor immunity from the 7th day to the 12th day and a marked resistance of B16F10 melanoma cells derived from hepatic metastases to T-lymphocytes and NK cells. The 1st-5th day CsA treatment of tumor-bearing mice produced a reduction in both T-lymphocyte and macrophage reactions against tumor cells and a significant increase in the 7th day micrometastasis incidence in the liver. Once micrometastases have been established the CsA-treatment suppression on the 5th day allows the tumor growth rate in these mice to become the same as in controls. However, the 7th-12th day CsA treatment produces a clear inhibitory effect on focal metastatic development which may correspond to the in vitro antiproliferative effect of CsA, detected on cultured B16F10 melanoma cells.
Maintaining B16F10 tumor cells in stirring culture for 48 h leads to an increase in lung and liver colonizing capacity in comparison with cells in adherent culture. Parallel to the increased metastatic capacity, we have observed a decrease in the proliferative rate of tumor cells (as the percentage of proliferating cell nuclear antigen-positive cells) and an increase in the population of tumor cells expressing la antigen. These results are not exclusive to B16F10 cells, since the same results were obtained when we analyzed 3LL cells maintained in identical culture conditions. In all the tumor lines tested, we found an association between the nonproliferating and the la-positive cell populations. We induced la expression by treating B16F10 cells in adherent culture with the lectin concanavalin A and again, coincident with an increase in metastatic capacity, we found the same association between the two parameters analyzed – nonproliferating state and la antigen expression. In addition, it was found that B16F10 cells induce lymphocytic proliferation, and a direct relationship was established between the number of Ia+ cells and lymphocytic proliferation.
In the present study, the effect of in vitro cyclosporin A (CsA) treatment on IL-2R expression and the metastatic behavior of B16F10 melanoma cells has been reported. CsA treatment was found to increase the percentage of B16F10 cells expressing the α-subunit of IL-2R on the cell surface and also at the mRNA level. Moreover, CsA treated B16F10 cells also express the β-subunit of IL-2. In vivo experiments showed that CsA increases the affinity of B16F10 metastazing cells for the liver and decreases that for the lung. CsA modulated the expression of MHC class I and class II antigens, but no significant differences in the resistance of CsA-treated B16F10 cells to NK lysis were observed. Finally, proliferation of B16F10 cells in the presence of several doses of CsA did not vary and CsA increased the amount of IL-1β mRNA expression. These results suggest that CsA, through the modulation of cytokines and MHC antigen expression on B16F10 cells, could have an effect upon the metastatic progression of the B16F10 melanoma.
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