The use of RNA blot hybridization with DNA or RNA probes of high specific activity has shown that interferon (IFN)-a mRNA is present constitutively in the spleen, kidney, liver, and peripheral blood leukocytes of normal individuals. A single band (41.2 kilobases) was detected in poly(A)+ RNA isolated from human organs. This RNA hybridized specifically to human IFN-al DNA and comigrated with mature IFN-a mRNA from virus-induced human peripheral blood leukocytes. No IFN-P RNA transcripts were detected in any of the tissues tested. IFN-y mRNA was detected in only one sample of normal human spleen, which also contained an unusually high level of IFN-a mRNA. The use of a modified S1 mapping technique revealed the presence of transcripts were detected in the same sample. The detection, in all the samples tested, of a characteristic pattern of expression of IFN genes, different from that obtained following induction, together with the low number of transcripts present (-0.03 copy per cell) suggest that specific IFN genes are transcribed constitutively in vivo.Interferons (IFNs) constitute an important group of regulatory cytokines that modulate the expression of a number of cellular genes (1) and exhibit pleiotropic biological activities both in vitro and in vivo (2). In view ofthe biological importance of IFNs, it is ofinterest to determine how the expression ofIFN genes is regulated in vivo under normal physiological conditions. Such information may also provide insight into more general aspects of gene control in eukaryotes.The production of IFNs is usually considered to be subject to stringent control, and significant quantities of IFNs are produced only after induction (3-5). Thus, although human cells contain 18 or more gene loci encoding potentially functional IFNs (6, 7), the expression of these genes is usually not detected. Induction leads to the activation of one or more IFN genes, and transient transcription of the corresponding mRNA culminates in the synthesis of IFN protein (4,5,8). Recent evidence suggests, however, that IFNs may be produced in vivo under conditions other than virus infection (9, 10) and that these endogenous IFNs may play a role in regulating such important physiological processes as fetal development and normal hematopoiesis. As biologically active interferons are detected only irregularly and then in small amounts in the organs of experimental animals or humans in the absence of induction (10-13), we developed techniques based on a modified S1 mapping procedure to detect low levels of specific IFN RNA transcripts. These techniques permitted the systematic study of the expression of IFN genes in normal individuals. We show herein that transcription of certain IFN genes, but not others, could be detected in the organs of normal individuals in the apparent absence of induction. The characteristic pattern of expression of IFN genes in vivo together with the low number of IFN transcripts produced per cell suggest that specific IFNs are produced constitutively under physiologica...

The antiviral and antiproliferative activities of human type I interferons (IFNs) are mediated by two transmembrane receptor subunits, IFNAR1 and IFNAR2. To elucidate the role of IFNAR1 in IFN binding and the establishment of biological activity, specific residues of IFNAR1 were mutated. Residues (62)FSSLKLNVY(70) of the S5-S6 loop of the N-terminal subdomain of IFNAR1 and tryptophan-129 of the second subdomain of IFNAR1 were shown to be crucial for IFN-alpha binding and signaling and establishment of biological activity. Mutagenesis of peptide (278)LRV in the third subdomain shows that these residues are critical for IFN-alpha-induced biological activity but not for ligand binding. These data, together with the sequence homology of IFNAR1 with cytokine receptors of known structure and the recently resolved NMR structure of IFNAR2, led to the establishment of a three-dimensional model of the human IFN-alpha/IFNAR1/IFNAR2 complex. This model predicts that following binding of IFN to IFNAR1 and IFNAR2 the receptor complex assumes a "closed form", in which the N-terminal domain of IFNAR1 acts as a lid, resulting in the activation of intracellular kinases. Differences in the primary sequence of individual IFN-alpha subtypes and resulting differences in binding affinity, duration of ligand/receptor association, or both would explain differences in intracellular signal intensities and biological activity observed for individual IFN-alpha subtypes.

Mucosal administration of the Th1 stimulatory cytokines interleukin-2 (IL-2), IL-12, IL-15, IL-18, or granulocyte-macrophage colony-stimulating factor (GM-CSF) induced antiviral activity in mice challenged systemically with a lethal dose of encephalomyocarditis virus (EMCV) similar to that observed following parenteral administration. In contrast, mucosal administration of the Th2 stimulatory cytokines IL-4, IL-5, IL-10, or IL-13 did not affect significantly the survival of EMCV-infected animals. Mucosal administration of IL-2 or IL-12 also exerted a marked antitumor activity in mice inoculated intravenously with Friend erythroleukemia cells. Recombinant IL-2 and IL-18, but none of the other recombinant cytokines tested, induced low levels of IFN in vitro. Polyclonal antibodies to both mouse and human interferon-alpha/beta (IFN-alpha/beta) abrogated the antiviral activity of IL-2 in vivo, even though the anti-human IFN-alpha/beta antibody did not neutralize mouse IFN-alpha/beta, and neither antibody bound to IL-2. IL-15 did not exhibit antiviral activity in IFN-alpha/beta R-/- mice, which are deficient in natural killer (NK) cell activity. These results suggest that mucosal Th1 cytokine therapy induces a soluble factor or activates a specific cell population in the lymphoid or epithelial tissue of the oropharyngeal cavity, which potentiates elimination of virus-infected or neoplasic cells systemically.

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.