Functional Characterization of Murine Cell Lines Expressing High, Intermediate, or Negative Levels of Surface Receptors for Interferon-γ

Abstract

Using radioiodinated murine interferon-gamma (IFN-gamma), various cell lines were analyzed for their capacity to bind IFN-gamma and to respond to its biological activity as measured by activation of the 2',5'-oligoadenylate (2-5A) synthetase, induction of the antiviral activity, and inhibition of [3H]thymidine uptake. A T-cell lymphoma, EL-4, was found to express relatively high number of specific membrane receptors for IFN-gamma (60,000/cell) with a dissociation constant of around 9 X 10(-10) M. By contrast, a fibroblast cell line, K-BALB, transformed by the Kirstein murine sarcoma virus, was found negative for binding of radioiodinated IFN-gamma, even in the presence of high ligand concentrations. Other cell lines displayed numbers of receptors varying between 650 to 20,000 binding sites/cell with KD ranging between 5 X 10(-9) to 9 X 10(-10) M. When incubated with EL-4 expressing high levels of specific receptors, IFN-gamma caused stimulation of cell growth, but not resistance to viral (vesicular stomatitis virus) infection and expression of 2-5A synthetase. By contrast, when L1210 and TS/A cells, expressing intermediate levels of specific receptors, were incubated with IFN-gamma, inhibition of cell growth, induction of antiviral resistance and activation of 2-5A synthetase was observed. Finally, K-BALB cells, lacking specific receptors for IFN-gamma, were completely unsensitive to its biological activity. The present system could represent a useful model for the characterization of the interaction of IFN-gamma with target cells at molecular levels.

A possible novel pathway of regulation by murine T helper type‐2 (Th2) cells of a Th1 cell activity via the modulation of the induction of nitric oxide synthase on macrophages

“…The number of cells per well was estimated by counting the contents of identical wells put through the assay without labeled EN-y using trypsin to release the cells. Preliminary experiments established that under these conditions, equilibrium binding was achieved and that untreated M@ displayed 19000 receptor sites per cell with a Kd of 5.3 x consistent with that reported previously [26].…”

“…Murine rIFN-y was iodinated using 12sI Bolton and Hunter's reagent as described by Fassio et al [26]. This procedure was reported to preserve biological activity and routinely yielded labeled IFN-y with a specific activity of about 2 pCi/pg.…”

Interferon Suppresses Sympathetic Neuronal Cell Death Caused by Nerve Growth Factor Deprivation

“…The specific activity of the iodinated IFN-y was -47 X lo3 cpm/ ng or 9,300 cpm/unit. Preliminary experiments with L929 cells indicated this iodinated IFN-y binds to its receptors with a KD of 1.2 X lo-'' M, which is similar to the published value (Fassio et al, 1988).…”

“…Based on the experiments using 1251-IFN-7 autoradiography, we conclude that the binding sites are distributed both on the cell bodies and on neurites of these cells. Receptor cross-linking data indicate a major protein species in the region of 100,000 daltons, which is similar to the results from the crosslinking of IFN-?/ to other murine cell types (Basu et al, 1988;Fassio et al, 1988). These findings strongly suggest that the effects of 1FN are on the neuronal cells directly.…”

Cell and type specificity of interferon action. Unusual characteristics of the transcriptional control of gene expression by interferon‐γ in T cells*

“…A trivial explanation of the IFN resistance of these cell lines would be a possible absence of IFN receptors. However, Scatchard analysis revealed that the IFN-y-resistant T cell lines and the other IFN-y-responsive cell lines expressed similar numbers of receptors with similar Kd values [41]. Thus, to explain the cell specificity of the 2-5A synthetase gene activation, a few other hypothesis can be proposed, such as the absence in T lymphocytes of transcriptional factors recognizing consensus sequences in the 5' flanking region of the 2-5A synthetase gene.…”