Mixed preparations of fibroblast and immune interferons interacted with cells synergistically to cause the development of a much greater level of protection than expected on the basis of their separate activities. This increased level of protection was 5to 20-fold greater than expected on the basis of a simple additive effect of the interferons. The potentiating factor copurified with both fibroblast interferon and immune interferon as they were partially purified. The potentiation was not an artifact of a more rapid development of immune interferon-induced antiviral resistance in the presence of fibroblast interferon. The results were consistent with the hypothesis that fibroblast and immune interferons mutually potentiate each other, thus supporting the supposition that they have different modes of action.

No abstract

Human lymphocytes were treated with human alpha (IFN-oa), beta (IFN-,), or recombinant gamma (IFN-y) interferons separately or in combination to determine their ability to enhance natural killing against mouse L cell targets. Our results showed that recombinant IFN-y was approximately 50 times more active per unit of antiviral activity than either IFN-a or IFN-P. Moreover, the levels of

Mouse IFN gamma preparations purified 30-fold were found to have direct cytolytic activity against a number of tumor and normal cells. Cell killing was determined using a sensitive, rapid and accurate assay which employed very low numbers of cells and very small quantities of interferon. The cytolytic activity of IFN gamma on 11 murine tumor cell lines was investigated. A 20-fold difference was found between the most-sensitive cell type, P-388 lymphoma, versus the most resistant cell type, C127v leukemia. A number of normal mouse cells was also found to have low to intermediate sensitivity to the cytolytic action of IFN gamma. Human IFN gamma was also shown to have cytolytic activity which, like mouse IFN gamma, was relatively species-specific. Direct cytolysis was not found to be a characteristic of IFN-alpha/beta. Different mechanisms of action for the antiviral and cytolytic activities of IFN gamma are indicated because the cytolytic titer of IFN gamma did not parallel its antiviral titer on most cell types and increasing the cell number produced a decrease in the cytolytic titer and an increase in the anti-viral titer. High concentrations of IFN gamma (i.e., 2,900 units/ml) resulted in complete lysis of cells within 24 h, while lower concentrations (i.e., 700 units/ml) resulted in a reversible inhibition of cell growth during this time period. Evidence that the cytolytic substance in the IFN gamma preparation was IFN gamma include the following: (1) both antiviral and anticellular activities copurified through a 30-fold purification; and both activities were (2) relatively species-specific; (3) sensitive to heat; (4) inactivated by low pH and (5) neutralized by antibodies to IFN gamma. Therefore, we propose the possibility that direct cytolysis is yet another of IFN gamma's distinctive antivities.

Mouse B16 melanoma cells maintained in vitro in the presence of interferon (IFN)-alpha become resistant to the in vitro antiproliferative effects of IFN-alpha. However, IFN-alpha-treated mice inoculated with these in vitro IFN-treated cells (B16 alpha res cells) have significantly increased life spans (ILS) and significantly higher cure rates than IFN-alpha-treated mice inoculated with B16 cells. This unexpectedly greater sensitivity of B16 alpha res cells to the in vivo antitumor effects of IFN-alpha was evaluated by in vivo cell depletion experiments. Depletion of either activated peritoneal macrophages or cytotoxic T lymphocytes (CTL) reduced the ILS of IFN-treated B16 alpha res-inoculated mice to a level comparable to that of IFN-treated B16-inoculated mice. Depletion of natural killer (NK) cells did not affect the ILS for IFN-treated B16 alpha res-inoculated mice. These studies indicate that activated macrophage and CD8 cell function, but not NK cell function, is important for the enhanced antitumor effects induced by IFN-alpha against B16 alpha res cells. Macrophage killing was unlikely to be mediated by TNF-alpha or IL-1 as B16 and B16 alpha res cells were equally sensitive to TNF-alpha and insensitive to IL-1 in vitro. Further, H-2K antigen expression is significantly more readily inducible on B16 alpha res cells than on B16 cells, consistent with enhanced CD8-mediated killing due to increased MHC class I antigen expression.