IFN-lambda (IFN-lambda) is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo

Interferons (IFN) exert antiviral, immunomodulatory and cytostatic activities. IFN-alpha/beta (type I IFN) and IFN-lambda (type III IFN) bind distinct receptors, but regulate similar sets of genes and exhibit strikingly similar biological activities. We analyzed to what extent the IFN-alpha/beta and IFN-lambda systems overlap in vivo in terms of expression and response. We observed a certain degree of tissue specificity in the production of IFN-lambda. In the brain, IFN-alpha/beta was readily produced after infection with various RNA viruses, whereas expression of IFN-lambda was low in this organ. In the liver, virus infection induced the expression of both IFN-alpha/beta and IFN-lambda genes. Plasmid electrotransfer-mediated in vivo expression of individual IFN genes allowed the tissue and cell specificities of the responses to systemic IFN-alpha/beta and IFN-lambda to be compared. The response to IFN-lambda correlated with expression of the alpha subunit of the IFN-lambda receptor (IL-28R alpha). The IFN-lambda response was prominent in the stomach, intestine and lungs, but very low in the central nervous system and spleen. At the cellular level, the response to IFN-lambda in kidney and brain was restricted to epithelial cells. In contrast, the response to IFN-alpha/beta was observed in various cell types in these organs, and was most prominent in endothelial cells. Thus, the IFN-lambda system probably evolved to specifically protect epithelia. IFN-lambda might contribute to the prevention of viral invasion through skin and mucosal surfaces.     

Induction of genes mediating interferon-dependent extracellular trap formation during neutrophil differentiation

Interferons (IFNs) are cytokines that possess potent anti-viral and immunoregulatory activities. In contrast, their potential role(s) in anti-bacterial defense and neutrophil activation mechanisms is less well explored. By comparing gene expression patterns between immature and mature human neutrophils, we obtained evidence that intracellular proteases and other anti-bacterial proteins are produced at earlier stages of maturation, whereas the genes for receptors and signaling molecules required for the release of these effector molecules are preferentially induced during terminal differentiation. For instance, mature neutrophils strongly expressed genes that increase their responses to type I and type II IFNs. Interestingly, granulocyte/macrophage colony-stimulating factor was identified as a repressor of IFN signaling components and consequently of IFN-responsive genes. Both IFN-alpha and IFN-gamma induced strong tyrosine phosphorylation of STAT1 in mature but not in immature neutrophils. Functional in vitro studies suggested that IFNs act as priming factors on mature neutrophils, allowing the formation of extracellular traps upon subsequent stimulation with complement factor 5a (C5a). In contrast, both IFN-alpha and IFN-gamma had only little capacity to prime immature cells in this system. Moreover, both IFNs did not have significant anti-proliferative effects on immature neutrophils. These data contribute to our understanding regarding changes of gene expression during neutrophil differentiation and IFN-mediated anti-bacterial defense mechanisms.     

Expression of IFN-alpha and IFN-gamma receptors on normal human small resting T lymphocytes and large granular lymphocytes

The expression of interferon (IFN) receptors was studied on freshly isolated human lymphocytes from normal donors. Highly enriched populations of small resting T lymphocytes and large granular lymphocytes (LGL) were found to constitutively express high-affinity receptors for IFN-alpha and IFN-gamma. Both types of lymphocytes also had lower-affinity IFN-alpha binding sites. T lymphocytes had a mean of 230 IFN-alpha and 520 IFN-gamma high-affinity receptors per cell, whereas LGL had 520 IFN-alpha and 760 IFN-gamma receptors. However, because LGL were larger than the T lymphocytes, the IFN receptor density was similar on the two types of lymphocytes. The affinity of binding was similar on the two types of normal lymphocytes and on the cultured lymphoblastoid cell line Daudi. The number of IFN receptors per cell and the affinities of the IFN-receptor interactions varied little among the normal donors. Both the freshly isolated normal lymphocytes and the cultured cell line Daudi had separate receptors for type I (alpha and beta) and type II (gamma) IFN. Taken together, our data indicate that two types of freshly isolated normal lymphocytes constitutively express IFN receptors that are similar to those present on the lymphoblastoid cell line Daudi derived from a patient with Burkitt's lymphoma.     

Identification of a linear epitope of interferon-alpha2b recognized by neutralizing monoclonal antibodies.

Four monoclonal antibodies (mAbs) directed against the recombinant human interferon-alpha2b (IFN-alpha2b) were used as probes to study the interaction of the IFN molecule to its receptors. The [125I]IFN-alpha2b binding to immobilized mAbs was completely inhibited by IFN-alpha2b and IFN-alpha2a but neither IFNbeta nor IFNgamma showed any effect. Gel-filtration HPLC of the immune complexes formed by incubating [125I]IFN-alpha2b with paired mAbs revealed the lack of simultaneous binding of two different antibodies to the tracer, suggesting that all mAbs recognize the same IFN antigenic domain. Furthermore, the mAbs were also able to neutralize the IFN-alpha2b anti-viral and anti-proliferative activities as well as [125I]IFN-alpha2b binding to WISH cell-membranes. As [125I]mAbs did not recognize IFN exposed epitopes in the IFN:receptor complexes, mAb induction of a conformational change in the IFN binding domain impairing its binding to receptors was considered unlikely. In order to identify the IFN region recognized by mAbs, IFN-alpha2b was digested with different proteolytic enzymes. Immunoreactivity of the resulting peptides was examined by Western blot and their sequences were established by Edman degradation after blotting to poly(vinylidene difluoride) membranes. Data obtained indicated that the smallest immunoreactive region recognized by mAbs consisted of residues 107-132 or 107-146. As this zone includes the sequence 123-140, which has been involved in the binding to receptors, and our mAbs did not show an allosteric behaviour, it is concluded that they are directed to overlapping epitopes located close to or even included in the IFN binding domain.     

Characterization and regulation of alpha-interferon receptor expression in interferon-sensitive and -resistant human lymphoblastoid cells

Interferon-sensitive (IFN-S) and IFN-resistant (IFN-R) Daudi lymphoblastoid cells were studied for IFN-alpha receptor expression and regulation by steady state and kinetic procedures, utilizing a homogeneous 125I-IFN-alpha 2 probe. Heterogeneity in the binding of this probe to IFN-S cells was determined to result from negatively cooperative interactions between an initially homogeneous class of IFN receptor. No such heterogeneity was noted in the IFN-R cells, indicating an apparent difference in the interaction of IFN-alpha 2 with these cells. The apparent dissociation constants (Kd) for IFN-S cell receptors were calculated to be 1 X 10(-10) M and 1 X 10(-8) M, for the high and low affinity sites, respectively. The Kd for sites on the IFN-R cells was estimated to be 4 X 10(-9) M. IFN-R and IFN-S cells expressed 2.4 X 10(4) and 3.5 X 10(4) binding sites per cell, respectively, representing an increase of at least 6-fold over previous reports of IFN-S Daudi IFN receptor density. Both IFN-S and IFN-R cells were capable of down-regulating expression of the IFN-alpha receptor in response to low concentrations of IFN-alpha 2. Furthermore, both cell lines were shown to be capable of internalizing specifically bound 125I-IFN-alpha 2 to an equivalent degree. Accordingly, we propose that the relative insensitivity of the Daudi IFN-R phenotype involves the loss of a high affinity interaction between cellular receptors and IFN-alpha 2, in addition to the reduced level of expressed low affinity binding sites.     

The interferon system of teleost fish

Interferons (IFNs) are secreted proteins, which induce vertebrate cells into an antiviral state. In mammals, three families of IFNs (type I IFN, type II IFN and IFN-lambda) can be distinguished on the basis of gene structure, protein structure and functional properties. Type I IFNs, which include IFN-alpha and IFN-beta, are encoded by intron lacking genes and have a major role in the first line of defense against viruses. The human IFN-lambdas have similar biological properties as type I IFNs, but are encoded by intron containing genes. Type II IFN is identical to IFN-gamma, which is produced by T helper 1 cells in response to mitogens and antigens and has a key role in adaptive cell mediated immunity. IFNs, which show structural and functional properties similar to mammalian type I IFNs, have recently been cloned from Atlantic salmon, channel catfish, pufferfish, and zebrafish. Teleost fish appear to have at least two type I IFN genes. Phylogenetic sequence analysis shows that the fish type I IFNs form a group separated from the avian type I IFNs and the mammalian IFN-alpha, -beta and -lambda groups. Interestingly, the fish IFNs possess the same exon/intron structure as the IFN-lambdas, but show most sequence similarity to IFN-alpha. Recently, IFN-gamma genes have also been cloned from several fish species and shown to have the same exon/intron structure as mammalian IFN-gamma genes. The antiviral effect of mammalian type I IFN is exerted through binding to the IFN-alpha/beta-receptor, which triggers signal transduction through the JAK-STAT signal transduction pathway resulting in expression of Mx and other antiviral proteins. Putative IFN receptor genes have been identified in pufferfish. Several interferon regulatory factors and members of the JAK-STAT pathway have also been identified in various fish species. Moreover, Mx and several other interferon stimulated genes have been cloned and studied in fish. Furthermore, antiviral activity of Mx protein from Atlantic salmon and Japanese flounder has recently been demonstrated.     

Dissociation between IFN-alpha-induced anti-viral and growth signaling pathways

The ability of IFN-alpha to induce an anti-viral state in a wide variety of cell types as well as to inhibit cellular growth has long been appreciated. It is less clear, however, whether both these effects lie downstream of a common signaling pathway. In this study we have taken advantage of an atypical human myeloma cell line (KAS-6/1) displaying a dramatic proliferative response to IFN-alpha in an effort to resolve the signaling requirements for IFN-alpha-induced anti-viral and growth regulatory effects. Thus, we have analyzed the ability of IFN-alpha to induce a number of known receptor-initiated events in this cell line and have compared these responses with those exhibited by a cell lineage- and maturation stage-matched myeloma cell line (ANBL-6) that displays typical IFN-alpha responsiveness. Despite the widely contrasting effects of IFN-alpha on cellular proliferation, IFN-alpha was shown to be comparable in its ability to induce the expression of early response genes as well as induce resistance to viral infection in both cell lines. By contrast, the effects of IFN-alpha on the activation of mitogen-activated protein kinase (MAPK) were strikingly distinct. Finally, although inhibition of MEK and MAPK activation had no effect on the induction of the anti-viral response, it completely blocked IFN-alpha-stimulated proliferation of the KAS-6/1 cells. In summary, our analysis of the role of the MAPK and anti-viral signaling pathways using these two cell lines suggests that the anti-viral and growth regulatory effects of IFN-alpha display a differential requirement for activation of the MAPK pathway.     

Kaposi's sarcoma-associated herpesvirus viral interferon regulatory factor confers resistance to the antiproliferative effect of interferon-alpha

BACKGROUND: Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a 442 amino acid polypeptide-designated viral interferon regulatory factor (vIRF) that displays homology to members of the interferon regulatory factor (IRF) family that bind to consensus interferon sequences and transactivate cellular genes that can modulate growth inhibition. Studies were conducted to determine whether vIRF affects the growth suppression mediated by interferon-alpha (IFN-alpha) in a human B lymphocyte cell line. MATERIALS AND METHODS: The human B lymphocyte cell line Daudi, which is sensitive to the antiproliferative effects of IFN-alpha, was stably transfected to express vIRF, and the proliferative response of vIRF expressing cells to IFN-alpha was compared with controls. The effect of vIRF on IRF- 1 transactivation was analyzed by co-transfection of an IFN-alpha-responsive chloramphenicol acetyltransferase reporter and expression plasmids encoding IRF-1 and vIRF. Electrophoretic mobility shift assays were conducted to determine whether vIRF interferes with the DNA binding activity of IRF-1. RESULTS: Daudi human B lymphocyte cells expressing vIRF were resistant to the antiproliferative effects of IFN-alpha, whereas wild-type Daudi or Daudi cells transformed with vector DNA were growth inhibited by IFN-alpha. The activation of an interferon-responsive reporter by IFN-alpha or IRF-1 was repressed by expression of vIRF. IRF-1 DNA binding activity was unaffected by vIRF, and vIRF alone did not bind to the interferon consensus sequence. CONCLUSIONS: These studies revealed that vIRF functions to inhibit interferon-mediated growth control of a human B lymphocyte cell line by targeting IRF-1 transactivation of interferon-inducible genes. Since KSHV is a B lymphotropic herpesvirus associated with two forms of B lymphocyte neoplasms, these effects of vIRF likely contribute to B cell oncogenesis associated with KSHV infection.     

Cell-specific regulation of APOBEC3F by interferons

Human cytidine deaminase APOBEC3F(A3F)has broad anti-viral activity against hepatitis Bvirus and retroviruses including human immunodeficiency virus type 1.However,its regulation in viralnatural target cells such CD4~ T lymphocytes,macrophages,and primary liver cells has not been wellstudied.Here we showed that A3F was up-regulated by interferon(IFN)-α in primary hepatocytes andmultiple liver cell lines as well as macrophages.Although the IFN-α signaling pathway was active in Tlymphoid cells and induction of other IFN stimulated genes such as PKR was detected,A3F and APOBEC3G(A3G)were not induced by IFN-α in these cells.Thus,additional factors other than known IFN-stimulatedgenes also regulated IFN-α-induced A3F expression distinctly.A3F and A3G expression levels in primaryhepatocytes,especially after IFN-α stimulation,were comparable to those in CD4~ T lymphocytes in someindividuals.Significant variations of A3F and A3G expression in primary hepatocytes from various subjectswere observed.Individual variations in A3F and/or A3G regulation and expression might influence the clinicaloutcomes of hepatitis B infection.     

Vanadate facilitates interferon alpha-mediated apoptosis that is dependent on the Jak/Stat pathway

Type I interferon (IFN)-dependent inhibition of cell growth can occur either in the absence or presence of apoptosis. The mechanisms that determine whether or not cells undergo apoptosis after exposure to IFN-alpha are not clear. This study shows that a variety of cell lines that display growth inhibition but not apoptosis in response to IFN-alpha will undergo programmed cell death when low concentrations of the protein-tyrosine phosphatase inhibitor vanadate are added with IFN-alpha. In contrast, the combination of tumor necrosis factor-alpha with vanadate did not trigger apoptosis in these cells. Caspase-3 activity was detected only in cells exposed to IFN-alpha and vanadate but not to IFN-alpha or vanadate alone. The ability of IFN-alpha and vanadate to induce apoptosis did not require expression of p53 and was blocked by N-acetyl-l-cysteine. Activation of the Jak/Stat pathway and expression of IFN-inducible genes was not altered by incubation of cells with IFN-alpha and vanadate compared with IFN-alpha alone. However, mutant cells lacking Stat1, Stat2, Jak1, or Tyk2, or cells expressing kinase inactive Jak1 or Tyk2 did not undergo apoptosis in the presence of IFN-alpha and vanadate. These results suggest that IFN-alpha stimulation of Stat-dependent genes is necessary, but not sufficient, for this cytokine to induce apoptosis. Another signaling cascade that involves the activity of a protein-tyrosine phosphatase and/or the generation of reactive oxygen species may play an important role in promoting IFN-alpha-induced apoptosis.     

Amino acid differences in interferon-tau (IFN-τ) of Bos taurus Coreanae and Holstein

Interferons (IFNs) are commonly grouped into type I and type II IFN. Type I IFNs are known as antiviral IFNs including IFN-α, IFN-β, and IFN-ω whereas type II IFN is referred to immune IFN and IFN-γ is only member of the type II IFN. Type I IFNs are induced by virus invading however type II IFN is produced by mitogenic or antigenic stimuli. IFN-τ was first identified in ruminant ungulates as a pregnancy recognition hormone, trophoblastin. IFN-τ constitutes a new class of type I IFN, which possesses the common features of type I IFN, such as the ability to prevent viral infection and to limit cell proliferation. In addition, IFN-τ is unique in that it is induced by pregnancy unlike other type I IFNs. We cloned Bos taurus (B. T.) Coreanae IFN-τ from peripheral blood mononuclear cells. The amino acid sequence of B. T. Coreanae IFN-τ shares only 90.3% identity with that of Holstein dairy cow. Recombinant B. T. Coreanae and Holstein IFN-τ proteins were expressed in Escherichia coli and the antiviral activity of IFN-τ proteins were examined. Both recombinant proteins were active and protected human WISH and bovine MDBK cells from the cytopathic effect of vesicular stomatitis virus. The recombinant IFN-τ protein of B. T. Coreanae and Holstein properly induced the expression of antiviral genes including 2',5'-oligoadenylate synthetase (OAS) and Mx GTPase 1 (Mx-1).     

Inhibition of transferrin receptor expression by interferon-alpha in human lymphoblastoid cells and mitogen-induced lymphocytes

125I-Transferrin binding to lymphoblastoid K562 and Daudi cells markedly increased after exposure of the cells to culture conditions that stimulated proliferation. Treatment of these cells with interferon-alpha (IFN-alpha) resulted in concurrent inhibition of cell growth and of the rise in transferrin binding. Scatchard analyses revealed that IFN reduced the number of transferrin receptors without altering the binding constant. When 125I-transferrin binding was measured using permeabilized cells, the IFN-induced reduction of binding was comparable to that observed with intact cells, indicating that IFN diminished the total number of cellular transferrin receptors. We also found that addition of IFN-alpha to phytohemagglutinin-stimulated human lymphocytes inhibited the mitogen-induced enhancement of [3H]thymidine incorporation as well as surface binding of 125I-transferrin. Our findings suggest that the decrease in transferrin receptor expression on IFN-alpha-treated cells may be one of the mechanisms responsible for the antiproliferative action of IFN.     

Interferon production in human T lymphoblastoid cells

Human T lymphoblastoid cell (RPMI 8402 cell) produced interferon (IFN) through the induction by Sendai virus. The priming effect on the interferon production in the RPMI 8402 cell could be found by the pretreatment of human leukocyte IFN (Hu IFN-alpha), but not by that of the IFN produced in the RPMI 8402 cell (T-IFN). The superinduction by the irradiation of ultraviolet rays or the treatment of antimetabolites (actinomycin D and cycloheximide) or 5-bromodeoxyuridine was not found. The T-IFN was completely neutralized by the anti-Hu IFN-beta serum, but not by the anti-Hu IFN-alpha serum at all. In conclusion, it was confirmed that the IFN produced in the RPMI 8402 cell through the induction by Sendai virus was antigenically identical to Hu IFN-beta.     

Reduction of transferrin receptor expression by interferon gamma in a human cell line sensitive to its antiproliferative effect

Interferon gamma (IFN gamma) reduced 125I-transferrin binding to WISH cells which are sensitive to its antiproliferative effect. IFN gamma did not affect transferrin binding to Daudi cells or phytohemagglutinin-stimulated human lymphocytes, neither of which respond to its antigrowth action. Scatchard analyses of the equilibrium binding of 125I-transferrin to WISH cells exposed to IFN gamma revealed a decrease in the number of cell surface receptors but no change in the apparent association constant compared with control cells. When 125I-transferrin binding was measured using detergent-extracted cells, the IFN-induced reduction of binding was smaller than with intact cells. This suggests that in WISH cells, IFN gamma not only reduced the total number of transferrin receptors, but also modified the process of receptor internalization and recycling. Labeling of newly synthesized receptors with [35S]-methionine indicated that a reduction in the biosynthesis might account for the decrease in the total number of transferrin receptors in IFN gamma-treated cells. Our results suggest that the antigrowth effect of IFN gamma is at least partly due to its inhibitory action on transferrin receptor expression leading to iron starvation.