W. Robert Fleischmann

The Interferons: Mechanisms of Action and Clinical Applications

The interferons (IFN) are one of the bodys natural defensive responses to such foreign components as microbes, tumors, and antigens. The IFN response begins with the production of the IFN proteins (alpha, beta, and gamma), which then induce the antiviral, antimicrobial, antitumor, and immunomodulatory actions of IFN. Recent advances have led to Food and Drug Administration approval of five clinical indications for IFN. Interferon alfa is approved for hairy-cell leukemia, condyloma acuminatum, Kaposis sarcoma in the acquired immunodeficiency syndrome, and non-A, non-B (type C) viral hepatitis. Interferon gamma has properties distinctive from those of IFNs alpha and beta and is approved as an immunomodulatory treatment for chronic granulomatous disease. Promising clinical results with IFNs have also been reported for basal cell carcinoma, chronic myelogenous leukemia, cutaneous squamous cell carcinoma, early human immunodeficiency virus infection, hepatitis B, and laryngeal papillomatosis. Future clinical uses of IFNs may emphasize combination therapy with other cytokines, chemotherapy, radiation, surgery, hyperthermia, or hormones.

Murine B16 Melanoma Vaccination-Induced Tumor Immunity: Identification of Specific Immune Cells and Functions Involved

Vaccination using inactivated B16 melanoma cells that have been treated in vitro for > 2 weeks with interferon-alpha (IFN-alpha) (B16alpha cells) has been shown to elicit a protective host antitumor immunity. In these studies, vaccination with B16alpha cells has been shown to provide protection against primary B16 tumor challenge, established B16 tumors, and metastatic B16 tumors. Specific immune cells and factors that might mediate this tumor immunity have now been evaluated. Macrophage depletion studies suggest that macrophage function is required for expression of tumor immunity either for processing of antigen or for cytokine production but that macrophage function is not involved in direct cytotoxicity against the B16 challenge tumor. CD8(+) T cell depletion studies show that cytotoxic T cell function is required for expression of tumor immunity. Syngeneic knockout mouse experiments offer further insights into the immune cells and factors that mediate the development and expression of tumor immunity. First, interleukin-12 (IL-12) knockout mouse experiments identify IL-12 as an important cytokine in mediating the development of tumor immunity. Second, specific knockout mouse experiments show that tumor immunity requires the function of CD4(+) T cells, CD8(+) T cells, and natural killer (NK) cells. Third, specific knockout mouse experiments show that tumor immunity does not require the function of B cells. The results suggest that vaccination with inactivated B16alpha cells induces an active, cell-mediated immunity to B16 melanoma cells. The tumor vaccination protocol with B16alpha cell vaccinations establishes a potent tumor immunity against B16 melanoma tumors in mice and may serve as a model for induction of tumor immunity against primary or secondary melanoma tumors in humans.

A simple and rapid method to determine hematopoietic growth factor activity

A rapid and simple colorimetric microassay method for the determination of hematopoietic growth factor activities was established. The assay was used to detect CSF-1, GM-CSF, and IL-3 activities. The assay was based on the metabolism of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan by metabolically active cells. Results obtained with the colorimetric microassay are comparable with those obtained with the soft agarose assay. Advantages of the colorimetric microassay include the conservation of reagents, the shorter incubation time for the experiment, the shorter assay time, and the ability to evaluate large numbers of samples.

The activity of interferon on ultraviolet light-induced squamous cell carcinomas in mice

The activity of interferons was tested in ultraviolet light-induced skin tumors in mice. After the tumors were well established, they were injected and measured daily for 19 days. Mouse virus type (IF-alpha + IF-beta) and immune (IF-gamma) interferons were injected intralesionally into three groups of test animals and compared with a fourth group which received mock interferon (control). When used separately, virus type and immune interferons did not affect tumor growth; however, we observed regression in tumor size when the two interferons were used in combination.

Potentiation of interferon action by mixtures of recombinant DNA-derived human interferons

Natural and essentially pure recombinant DNA-derived HuIFN-alpha and HuIFN-gamma were examined for their relative abilities to potentiate interferon action. Potentiation of human interferons antiviral and antiproliferative activities were studied. The essentially pure recombinant DNA-derived human interferons were found to be as effective in their potentiating interactions as their natural counterparts. The results demonstrate that it is the human interferons themselves which interact to potentiate human interferons varied activities.

Potentiating effect of murine interferon-γ-containing lymphokine preparations on the antiviral and antiproliferative effects of murine interferon-α/β: identification of the potentiation factor as murine interferon-γ itself

Abstract Mixed preparations of murine interferon-γ (MuIFN-γ) and murine interferon-α/β (MuIFN-α/β) have been shown to induce more than additive levels of antiviral protection, when compared to those induced by these interferons given separately. MuIFN-γ preparations contain many lymphokines and several of these as well as MuIFN-γ itself may participate in this potentiation. In the present study, natural as well as three recombinant DNA-derived MuIFN-γs, in combination with antibody to MuIFN-γ have been employed to examine the precise role of MuIFN-γ. The antiviral effect was examined with a single cycle virus yield reduction assay and the antiproliferative effect with a colony formation inhibition assay. Recombinant DNA-derived MuIFN-γ was as effective as natural MuIFN-γ at participating in the potentiation of both the antiviral and antiproliferative activities. Antibody to MuIFN-γ effectively blocked the potentiation of both the antiviral and the antiproliferative activities of natural and recombinant DNA-derived MuIFN-γs. Since the recombinant DNA-derived preparations from E. colie can be assumed not to contain mammalian proteins other than MuIFN-γ, the data conclusively demonstrate that the potentiation factor in MuIFN-γ preparations is MuIFN-γ itself.

Systemic Effects of Orally Administered Interferons and Interleukin-2

Orally administered interferons (IFN-alpha, IFN-beta, and IFN-gamma) have been shown to exert a number of systemic effects. Orally administered IFNs exert dose-dependent suppressive effects on the peripheral white blood cell (WBC) count. The suppression of the peripheral WBC count is mediated by a suppression of the function of the bone marrow, as measured in an in vitro bone marrow colony-forming assay. The peripheral WBC and bone marrow suppressive effects of orally administered IFNs are at least as potent as those occurring with parenterally administered IFNs. However, the mechanism by which orally administered IFNs exert these peripheral WBC suppressive and bone marrow suppressive effects differs significantly from that of parenterally administered IFNs: orally administered IFN is not detectable in the serum, the effect of orally administered IFN is not blocked by circulating antibody, the effect of orally administered IFN can be adoptively transferred by injection with peripheral white blood cells from donor mice, and the effect of orally administered IFN develops more slowly than that of parenterally administered interferon. Orally administered IFN-alpha employed alone and in synergistic combination with intraperitoneally administered IFN-gamma can exert an antitumor effect. Finally, orally administered interleukin-2 can exert a suppressive effect on both the peripheral white blood cell count and on the bone marrow. These observations suggest that the oral route may be an effective and novel mechanism for the efficacious administration of IFNs and other lymphokines/cytokines.

[75] A convenient microassay for cytolysis and cytostasis

Publisher Summary This chapter discusses convenient microassay for cytolysis and cytostasis. Apart from their many other properties, interferons are known to have both cytolytic and cytostatic activities. This is, particularly true of gamma interferon (IFN-γ). These activities have been measured by the inhibition of colony formation, radioisotope uptake or release, trypsinization and cell counting, or other methods generally requiring a relatively large amount of time, material, and preparation. The method described attempts to meet these needs by employing microhistoplates seeded with 100–200 target cells per well and then incubated with interferons (IFNs) alone or with effector cells for 1–3 days. Viable cells are counted microscopically to determine cytostasis or cytolysis. The most time-consuming portion of the test is the microscopic cell counting. The major advantages of this microassay for quantitating cytostasis and cytolysis lies in its simplicity and requirement for only minute quantities of test materials and its convenience while maintaining good sensitivity and reproducibility. Because a great number of assays can be run simultaneously by one person, one use for this method would be screening biopsy materials (also often available in only minute quantities) to determine which patients might benefit from IFN or other therapy. The method would also allow a rapid evaluation of the potential of various immunomodulators to stimulate leukocytes for killing tumor cells from biopsies.

Lack of mda-6/WAF1/CIP1-mediated inhibition of cyclin-dependent kinases in interferon-α resistant murine B16 melanoma cells

Previously we demonstrated that IFN-alpha augments mda-6/WAF1 and inhibits cyclin-dependent kinases in a p53-independent fashion in B 16 murine melanoma cells. On the other hand, IFN-gamma activates p53 expression without affecting the mda-6/WAF1 system. Combination of the two IFNs is additive. B16 cells acquire IFN-alpha resistant but IFN-gamma sensitive phenotype after long term IFN-alpha treatment (B16alpha cells). Here we demonstrate the absence of mda-6/WAF1-associated repression of cyclin-dependent kinases, but the existence of p53-dependent c-myc inhibition in IFN-gamma-treated B16alpha cells. Clearly, selective desensitization of IFN-alpha related growth regulation does not influence the IFN-gamma associated pathway. Our results further support the coexistence of distinct growth regulatory mechanisms in B16 cells that can be activated by different IFN-types independently of each other.

Regulation of interferon-β activity by fibroblast cells

Exogenously administered interferons are rapidly cleared from the body. Several pharmacological mechanisms have been implicated in this clearance; however, they do not entirely explain the different clearance rates of the interferons. Cultured cells were studied for their ability to regulate interferon levels in vitro. Preparations of MuIFN-alpha, MuIFN-beta, and MuIFN-gamma were exposed to cells in culture and monitored for any loss in titer. MuIFN-beta titers were found to be significantly reduced following exposure to mouse L-929 fibroblast cells. The reduction of MuIFN-beta activity appeared to be specific for fibroblasts, since the reduction occurred following exposure to L-cells and to mouse embryo fibroblasts, but not to mouse reticuloendothelial cells. Moreover, the ability of the mouse fibroblast cells to reduce MuIFN-beta titers was blocked if the cells were pre-treated with actinomycin D, suggesting that de novo RNA synthesis was required. The titers of IFN-alpha and IFN-gamma were not reduced following exposure to either fibroblast or reticuloendothelial cells. Thus, the reduction of interferon titer by fibroblasts was IFN-beta specific. Similarly, HuIFN-beta titers were reduced following exposure to human fibroblasts. The ability of fibroblast cells to reduce IFN-beta titers was also found to be species-specific, since human fibroblast cells reduced the titer of HuIFN-beta but not MuIFN-beta while murine fibroblasts reduced the titer of MuIFN-beta but not HuIFN-beta. These results suggest that IFN-beta-treated fibroblasts specifically regulate their response to IFN-beta by reducing the titer of the IFN-beta activity.