Ion Gresser

Genetic transfer of a functional human interferon α receptor into mouse cells: Cloning and expression of its c-DNA

Abstract A cDNA coding for the human interferon a receptor has been cloned using a gene transfer approach. This consists of transferring human DNA to mouse cells and selecting for cells sensitive to human interferon α. The transfected cells expressed the human interferon α receptor, and a 5 kb human DNA was isolated from a secondary transfectant. This DNA detects an mRNA present in human cells and was used to clone a 2.7 kb cDNA from a library constructed from human Daudi cells. The sequence of this cDNA is presented. It codes for a glycoprotein of 557 amino acids with an N-terminal hydrophobic region and a single transmembrane-spanning segment. Mouse cells expressing the cDNA become sensitive to the antiviral activity of and express binding sites for human interferon a, demonstrating that the cloned cDNA encodes a functional human interferon a receptor.

The neglected role of type I interferon in the T-cell response: implications for its clinical use

Originally described as an antiviral substance, type I interferon (IFN) was subsequently shown to exert multiple biological effects and is now the most frequently used cytokine in the treatment of some viral and neoplastic diseases. Although early studies described various effects on the immune system, the role of type I IFN as an immunoregulatory molecule has long been neglected. Here, Filippo Belardelli and Ion Gresser summarize recent experimental results on the interactions of type I IFN with T cells, which may prove important in its use in patients.

Type 1 Interferon (IFNα/β) and Type 2 Nitric Oxide Synthase Regulate the Innate Immune Response to a Protozoan Parasite

Abstract Type 2 nitric oxide synthase (NOS2) is required for the Th1-dependent healing of infections with intracellular microbes, including Leishmania major . Here, we demonstrate the expression and define the function of NOS2 during the innate response to L. major . At day 1 of infection, genetic deletion or functional inactivation of NOS2 abolished the IFNγ and natural killer cell response, increased the expression of TGFβ, and caused parasite spreading from the skin and lymph node to the spleen, liver, bone marrow, and lung. Induction of NOS2 was dependent on IFNα/β. Neutralization of IFNα/β mimicked the phenotype of NOS2 −/− mice. Thus, IFNα/β and NOS2 are critical regulators of the innate response to L. major.

Prevention of diabetes in NOD mice treated with antibody to murine IFNγ

The NOD mouse is studied as an animal model of human insulin-dependent diabetes mellitus (IDDM). To evaluate the role of IFN gamma in the pathogenesis of the disease, we have studied the effect of anti-IFN gamma mAb on the expression of insulitis and clinical diabetes. Treatment of mice with anti-IFN gamma mAb prevented the induction of early IDDM by cyclophosphamide as well as the adoptive transfer of diabetes by spleen cells from diabetic NOD mice. The protection against induction of diabetes by cyclophosphamide was observed in animals treated with the anti-IFN gamma mAb within 24 h following the first cyclophosphamide injection but not in animals in which mAb treatment was started 7 days later. Transfer of disease was prevented both in adult irradiated and in newborn recipients. The absence of clinical signs in these mice was corroborated by a significant reduction of both the extent and severity of insulitis. Over-expression of Ia antigen on endothelial cells lining the islets was also considerably reduced in mice treated with mAb. These data strongly suggest a role for IFN gamma during the autoimmune process leading to beta cell destruction in diabetes and prompt further investigation of the use of such antibodies in the immunoprevention of IDDM.

Genes for Interleukin-1, Interleukin-6, and Tumor Necrosis Factor are Expressed at Markedly Reduced Levels in the Livers of Patients with Severe Liver Disease

The genes for interferon (IFN) alpha, IFN gamma, IL-1 beta, IL-6, and TNF alpha were transcribed at readily detectable levels both in liver biopsies from individuals with normal liver function and in samples of normal viable liver taken for transplantation. These results provided evidence for the concept that such multifunctional cytokines play a role in homeostasis in normal human tissues. In normal human liver, in situ hybridization studies showed that, in the absence of a detectable inflammatory response, both hepatocytes and mononuclear cells exhibited a similar degree of expression of IL-6 mRNA in keeping with the finding that IL-6 is produced by cells of different lineages. The levels of IL-1, IL-6, and TNF mRNA were found to be markedly reduced in extracts of the livers of patients with primary biliary cirrhosis and other forms of autoimmune liver disease at a time when extensive liver lesions were apparent, compared to the levels of expression of these cytokines in the livers of normal individuals. The reduced expression of IL-1, IL-6, and TNF mRNAs appeared to be a specific effect and not due to a general reduction in RNA synthesis as the IFN alpha, IFN gamma and actin mRNAs were expressed at similar levels in both normal and diseased livers. The levels of IL-1 beta, IL-6, and TNF mRNAs were also reduced in samples of liver from a patient with a drug induced fulminant hepatitis suggesting that this specific pattern of altered cytokine gene expression was characteristic of the advanced stage of severe liver disease.

Interferon αβ‐induced abnormalities in adipocytes of suckling mice

Summary— The modification induced by interferon (IFN) in brown adipose tissue (BAT) was studied by high spatial resolution magnetic resonance imaging (MRI), histology, and transmission electron microscopy (TEM). In IFN‐treated mice, at MRI, the interscapular BAT was slightly enlarged and showed non‐homogeneous areas of lipid accumulation. The thickness of the subcutaneous white adipose tissue was reduced with respect to control mice. In the liver, MRI showed a lipid accumulation. In IFN‐treated mice, by light microscopy, brown adipocytes showed a larger lipid deposit with respect to control mice. At TEM, in BAT, the mitochondria were reduced in number, smaller and the number of cristae was also significantly reduced with respect to the controls (9.1 ± 1.5 vs 20.1 ± 1.9, P < 0.01). The inclusions in the mitochondrial matrix were significantly less numerous in IFN‐treated than in control animals (1.9 ± 0.7 vs 0.9 ± 0.7 for mitochondrial section, P < 0.01). Abnormalities of endoplasmic reticulum described in hepatocytes were not found in brown adipocytes of IFN‐treated mice. The present work demonstrates that, in the BAT of sucking mice, IFN‐treatment induces morphologic alterations and that brown adipocytes have MRI and TEM features resembling those found in the lipid laden BAT of aged animals.

Interferon α/β induces changes in the metabolism of polyenoic phospholipids and diacylglycerols in the livers of suckling mice

Suckling mice were injected daily from birth for 10 days with potent preparations of mouse interferon α/β. Interferon treatment resulted in a markedly lower concentration of polyunsaturated fatty acids (20∶4ω6 and 22∶6ω3) in the two principal liver phospholipids, phosphatidylcholine and phosphatidylethanolamine, than in livers of control-treated mice. This effect appeared to correlate with a low level of synthesis of polyunsaturated phospholipids in the livers of interferon-treated mice. Thus, in control mice, synthesis of species of polyunsaturated phospholipids increased markedly in the first 10 days of life, whereas in 10-day-old interferontreated mice, the level of synthesis of species of polyunsaturated phospholipids was comparable to that in newborn mice. In parallel, a marked increase in the diacylglycerol content without change of its renewal was observed in the livers of interferon-treated mice. We suggest that interferon treatment results in an inhibition of one of the processes that leads to activation of the enzymatic systems responsible for the synthesis of species of polyunsaturated phosphatidylcholine and phosphatidylethanolamine in the liver of suckling mice. It seems likely that these results are related to the inhibition of liver cell maturation and the marked cell necrosis that are observed in interferon-treated suckling mice.

Reversibility of the transformed and neoplastic phenotype. Iii. Long-term treatment with electrophoretically pure mouse interferon leads to the progressive reversion of the phenotype of x-ray transformed c3h/10t1/2 cells.

Electrophoretically pure mouse interferon induced the progressive reversion of the transformed phenotype of a clone of X-ray transformed C3H/10T1/2 cells. Cells of this clone did not harbor C-type particles and reverse transcriptase activity was not detected. Interferon-treated transformed cells were aligned without cellular overlapping and attained low cell densities. Morphologic changes were associated with the appearance of a thick layer of submembranous microfilaments. The tumorigenicity of interferon-treated cells was markedly reduced. Back reversion to the transformed phenotype occurred progressively when these cells were passaged in the absence of interferon. These results suggest that interferon may induce the reversion of the transformed phenotype by a mechanism other than by its antiviral activity.

Interferon and Tumor Necrosis Factor Alter the Phospholipid Metabolism in Friend Leukemia Cell Tumors in Mice

Interferon (IFN) has been shown to inhibit the growth of different tumors in experimental animals (reviewed in 1 and 2) and it is currently used to treat some patients with cancer in clinical trials. However the mechanisms of antitumor effects of IFN are still unknown (2). We have previously shown that administration of highly purified α/β IFN was equally effective in inhibiting tumor growth in mice injected with either IFN-sensitive or IFN-resistant Friend leukemia cells (3,4). These data suggested that the antitumor effects of IFN in this system were not due to a direct effect of IFN on tumor cells but that these effects were in some way host-mediated (4). Daily injection of IFN into FLC tumors implanted subcutaneously (s.c.) caused an arrest of tumor growth and tumor necrosis and resulted in a complete tumor regression in some mice transplanted with either IFN-sensitive or IFN-resistant FLC (5).