Effect of IL-2 and IL-6 on parameters related to metastatic activity in a murine melanoma.

Previous results have shown that the metastatic colonization with B16F10 melanoma in vivo increased after in vitro treatment of the cells with IL-2 or IL-6. To further investigate the mechanisms underlying this effect, we have studied adhesion, invasion, and proliferation properties of B16 melanoma, using two sublines with different metastatic ability. Adhesion of tumor cells to Matrigel coats increased using IL-6, which also induced upregulation of VLA-4 expression in both sublines. Unexpectedly, invasion through Matrigel filters was almost completely inhibited by IL-6 and decreased in the presence of IL-2. Cell growth was not affected by these interleukins; however, IL-6 could partially overcome the proliferation blockade induced by stress conditions. Taken together, these results suggest that upregulation of adhesion properties and/or the protective effect induced by IL-6 could account for the enhancement of metastasis exerted by this interleukin.

Serum-soluble IL-2 receptor and IL-6 levels in patients with melanoma.

Elevated soluble IL-2 receptor (sIL-2R) and IL-6 serum concentrations have been reported as adverse prognostic factors in several types of cancer. In order to determine whether these factors are predictive of metastatic progression in melanoma, sIL-2R and IL-6 levels were measured in sera from 172 patients with melanoma and 60 in healthy controls. Mean sIL-2R values were significantly higher in the patients than in normal controls and the highest values were observed in those that developed metastasis during follow-up. However, no correlation was found with the stage of the disease. Serum IL-6 levels were found to be correlated with age and sex, but not correlated with sIL-2R levels. Statistical analysis was based on logistic and Cox regression models. The factors considered were age, sex, stage, disease-free interval and serum sIL-2R and IL-6 levels. The analysis showed that only the sIL-2R value is significantly linked to metastatic progression. This finding suggests that high serum levels of sIL-2R could be a predictive factor of metastatic progression in malignant melanoma.

IL-2 treatment of B16F10 melanoma cells stimulates metastatic colonization in the liver.

We had previously shown that murine B16F10 melanoma cells express the receptor for IL-2, transcribe the gene for IL-2 and respond to its factor by increasing their proliferation. In the present work we have investigated the effect of in vitro IL-2 treatment on the metastatic ability of B16F10 cells. In vivo experiments showed that the metastatic colonization of the liver was notably higher after intrasplenic inoculation of IL-2-treated cells. However, no change was observed when cells were intravenously inoculated. In vitro, cells became more resistant to NK lysis although the cytometric analysis of class 1 MHC molecules revealed a decrease in H-2Kb expression. In contrast IL-2 induced a two fold increment in the expression of la antigen. On the other hand slot-blot analysis showed that IL-2 gene expression could be upregulated, however no free IL-2 was released into the culture medium of B16F10 cells. We conclude that IL-2 increases the ability of B16F10 cells to metastase to the liver. The increase in the resistance to NK activity and in la antigen expression could be involved in the mechanisms underlying this effect.

B16F10 murine melanoma cells express interleukin-2 and a functional interleukin-2 receptor.

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 alpha subunit in B16F10 melanoma, with a mean positivity 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 alpha subunit, the IL-2R beta 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.

Enhancement of Ia antigen expression and nonproliferating cells correlates with metastatic capacity.

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 Ia 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 Ia-positive cell populations. We induced Ia 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 Ia 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.