Dina G. Fischer

Increase of vulnerability to lymphotoxin in cells infected by vesicular stomatitis virus and its further augmentation by interferon.

The cytotoxic effect of lymphotoxin (LT) and its modulation by interferon (IFN) was quantitatively assessed in uninfected and vesicular stomatitis virus (VSV)-infected cultured cells. Preparations of human LT, which were depleted of IFN, had a significant cytotoxic effect on VSV-infected HeLa, SV-80, WISH, and Vero cells. IFN, most notably IFN-gamma, further potentiated destruction of the infected cells by these LT preparations, when applied on the cells at sub-antiviral IFN concentrations. In contrast, no cytotoxic effect could be observed in any of the examined cells, when applying LT, IFN, or their combination, in the absence of viral infection. Infected cells in which VSV replication was suppressed by treatment with antiviral concentrations of IFN also resisted destruction by LT. These findings indicate that LT cytotoxicity can be selectively directed against virus-infected cells and that IFN can augment this cell-killing mechanism when failing to exert an antiviral effect.

The protective effect of interferon against natural killing activity is not mediated via the expression of class I MHC antigens

Natural-killer cell mediated cytotoxicity (NK-CMC) is modulated by interferons both at the effector cell and at the target cell levels. Pretreatment of effector cells with interferon increases their cytotoxicity while pretreatment of target cells with interferons decreases their sensitivity to NK-CMC. Interferons are inducers of several genes including those of the major histocompatibility complex (MHC) and several earlier studies have established a circumstantial correlation between induction of class I MHC gene products and induction of resistance to NK-CMC. In the present study we demonstrate that interferon-alpha renders Daudi cells resistant to NK-CMC without inducing the surface expression of class I MHC antigens. Therefore we suggest that these two phenomena are independent responses to interferon treatment.

Spontaneous production of interferon-γ and acid-labile interferon-α by subpopulations of human mononuclear cells

A low-density subpopulation of normal human peripheral blood mononuclear leukocytes obtained by percoll gradients was found to produce significant levels (up to 10,000 units/ml) of interferon in the absence of an external inducer. The interferon (IFN) was characterized as a mixture of IFN-gamma (immune) and a pH-sensitive IFN-alpha by neutralization with specific antibodies, by lability to low pH, and by cross-reactivity on bovine cells. Significant levels of IFN were obtained only at high (greater than or equal to 5 X 10(6) cells/ml) cell densities. These levels were not affected by the presence of small T lymphocytes and sharply decreased with cell dilution in a nonlinear fashion. A release of IFN in the absence of an external inducer, indicates additional physiological roles besides its involvement in pathological conditions.

ISOLATION AND CHARACTERIZATION OF A SOLUBLE FORM OF THE LDL RECEPTOR, AN INTERFERON-INDUCED ANTIVIRAL PROTEIN

Abstract Interferons (IFNs) act by inducing several intracellular antiviral proteins. We report here that IFNs also induce an extracellular soluble protein that inhibits vesicular stomatitis virus (VSV) infection. This protein accounts for 25%-50% of the total antiviral activity elicited by IFN. The antiviral protein was purified to homogeneity from culture supernatants of IFN-treated cells by several chromatographic steps, to give a single 28-kDa active polypeptide. Upon sequencing, this novel protein corresponded to the N-terminal ligand-binding domain of the human 160-kDa low-density lipoprotein receptor (LDLR). In addition, we find that IFN induces the cell surface LDLR and this phenomenon may explain previous reports on reduction of serum cholesterol in IFN-treated patients. Viruses produce soluble cytokine receptors that inhibit their respective cytokines, thereby assisting virus infection. It appears now that host cells employ similar molecules for the opposite role of controlling virus infections.

Cyclosporin a regulates the expression of HLA-DR on human monocytes by two different mechanisms☆

Cyclosporin A (CsA), but not its nonimmunosuppressive analog cyclosporin H (CsH), inhibited the expression of HLA-DR in human monocytes. Induction of HLA-DR by interferon (IFN)-gamma in fresh monocytes was also inhibited by CsA and not by CsH. However, when monocytes were pretreated with either CsA or CsH for 16 hr prior to the addition of IFN-gamma, HLA-DR expression was increased, probably because of a cyclosporin-induced increase in the number of IFN-gamma receptors. Down-regulation of the HLA-DR mRNA by CsA was found to be dependent on continuous protein synthesis. IFN-alpha also inhibited the IFN-gamma-induced HLA-DR mRNA expression and showed synergy with CsA at low concentrations but not at high concentrations of the drugs. A common mechanistic element in the pathways of CsA and IFN-alpha is proposed.

Human monocytes tumoricidal activity: the role of interferon-γ and bacterial lipopolysaccharide in its stimulation, preservation and decay

The effect of interferon-gamma (IFN-gamma) and bacterial lipopolysaccharide (LPS) on the cytotoxic activity of cultured monocytes was studied in a 6-h51Cr release assay with Actinomycin-D-treated tumor cells as targets. In this system, the lysis of target cells is mediated by a soluble factor (CF) which is similar or identical to human tumor necrosis factor (TNF). The spontaneous cytotoxic activity of freshly isolated monocytes declined after their maturation to macrophages during in vitro culturing. The decrease in the ability of cultured monocytes to lyse the targets is explained by a decrease in their ability to produce the soluble cytolytic factor. Both LPS and IFN-gamma modulated the effect of culturing. LPS exhibited a dual effect. Within 2-3 h after its addition, LPS enhanced the cytotoxic activity of monocytes by increasing the synthesis of CF. However, upon a longer incubation, the decay of the activity was more pronounced in the presence of LPS. IFN-gamma did not augment the cytotoxic activity of monocytes above the basal level, yet it prevented the loss of activity which accompanies the process of monocyte maturation to macrophages.

Isolation of two discrete human interferon-γ (immune) subtypes by high-performance liquid chromatography

A rapid procedure for isolation of two biologically active human interferon-gamma subtypes was developed. Crude interferon-gamma produced in a serum-free culture of peripheral blood mononuclear cells by mitogen stimulation was concentrated and partially purified by chromatography on controlled-pore glass. Following desalting and concentration by ultrafiltration, a step of cation-exchange high-performance liquid chromatography was performed. A linear NaCl gradient (0.01-0.4 M) at pH 7 was employed and four peaks of biological activity eluting at 0.17, 0.20, 0.26 (major peak), and 0.3 M were obtained. The major peak of biological activity coincided with two protein peaks. Analysis of one fraction from the major activity peak by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a protein band having an apparent molecular weight of 26,000, while an adjacent fraction of the same activity peak contained a protein band corresponding to a molecular weight of 21,000. The specific activity of both subtypes was 7-10 X 10(6) units/mg.

Pharmacokinetics of recombinant interferon alpha-C

SummaryRecombinant interferon alpha-C (rIFNα-C, Interpharm), is a new type of alpha-interferon that has a specific activity of 1–2×109 units/mg protein. The pharmacokinetics of rIFNα-C were studied in 11 patients with metastatic renal-cell carcinoma. A total of 10 million units IFNα-C were injected intramuscularly and the serum level of IFN was evaluated up to 72 h post-administration. Measurable IFN concentrations appeared in the serum as early as 0.5 h, and levels peaked at 4–6 h (Cmax=53.2±4.6 units/ml). Relatively high levels persisted for 24 h and declined thereafter with an apparent half-life of 3–4 h. The mean area under the serum-concentration curve (AUC) was 1,259±145 units h ml−1, indicating good bioavailability of the preparation from the intramuscular injection.

Purification of the Human Interferon-γ Receptor by Ligand Affinity

Interferon-γ(IFN-γ) is a product of activated T lymphocytes (Kasahara et al., 1983) and low density lymphocytes (Fischer and Rubinstein, 1983). In addition to its antiviral and growth inhibitory activities, IFN-γis one of the major immunoregulatory lymphokines. Its immunoregulatory functions include macrophage activation, growth, differentiation, and maturation of various immunocytes and induction of class I and II MHC gene products both in macrophages and in cells of nonhematopoietic origin (for a review see Trinchieri and Perrusia, 1985). A survey of its various activities reveals that IFN-γelicits 50% of its maximal effect at concentrations of 0.5-1 pM. Indeed, the level of IFN-γ in blood or in other tissue fluids rarely exceeds the lower limit of detection, which is about 10 antiviral U/mL (10 pM). Therefore, a highly sensitive and selective system must be present in the cells in order to trace IFN-γ and respond to it. Such a system is the cell surface receptor. In this respect, IFN-γ is similar to other polypeptide hormones, lymphokines, and other cytokines, all acting through specific cell-membrane receptors. This array of polypeptides and their receptors serve as an efficient chemical signaling network that is essential for the existence of multicellular organisms.

The Use of Ion Exchange and Reversed-Phase HPLC for the Isolation and Characterization of Interferon Gamma

Publisher Summary Human interferon-γ (IFN-γ) is a lymphokine that is produced by peripheral blood mononuclear cells upon stimulation with mitogens, such as phytohemagglutinin (PHA). IFN-γ consists of two molecular subtypes having apparent molecular weights of 26,000 and 21,000 as determined by sodium codicil sulfate -polyacrylamide gel electrophoresis (SDS-PAGE). On gel filtration, a single peak having an apparent molecular weight of 45,000 is obtained. Analysis of IFN-γ cDNA revealed the presence of only a single gene, coding for a polypeptide of 166 amino acid residues, which includes a leader peptide of about 20 amino acid residues. IFN-γ is a glycoprotein having two glycosylation sites and lacking disulfide bridges. Unlike IFN-α and IFN-β, it is labile at low pH. The present study characterizes the structure of IFN-γ.