Priming of leukocytes selectively increases the level of some interferon-α subtypes and not others

The effect of pretreatment with interferon (IFN) (‘priming’) on the production of individual IFN subtypes was studied in subpopulations of human peripheral blood mononuclear cells and in the myeloid cell line KG-1. It was found that priming had a selective enhancing effect on the production of certain IFN-α subtypes (IFN-α20K and IFN-α21K) and not on others. KG-1 cells produce both IFN-α and -β; however, only the production of IFN-α was enhanced by priming with either IFN-α,β or γ.     

A vesicular stomatitis virus replicon-based bioassay for the rapid and sensitive determination of multi-species type I interferon

Type I interferons (IFN) comprise a family of cytokines that signal through a common cellular receptor to induce a plethora of genes with antiviral and other activities. Recombinant IFNs are used for the treatment of hepatitis C virus infection, multiple sclerosis, and certain malignancies. The capability of type I IFN to suppress virus replication and resultant cytopathic effects is frequently used to measure their bioactivity. However, these assays are time-consuming and require appropriate biosafety containment. In this study, an improved IFN assay is presented which is based on a recombinant vesicular stomatitis virus (VSV) replicon encoding two reporter proteins, firefly luciferase and green fluorescent protein. The vector lacks the essential envelope glycoprotein (G) gene of VSV and is propagated on a G protein-expressing transgenic cell line. Several mammalian and avian cells turned out to be susceptible to infection with the complemented replicon particles. Infected cells readily expressed the reporter proteins at high levels five hours post infection. When human fibroblasts were treated with serial dilutions of human IFN-β prior to infection, reporter expression was accordingly suppressed. This method was more sensitive and faster than a classical IFN bioassay based on VSV cytopathic effects. In addition, the antiviral activity of human IFN-λ (interleukin-29), a type III IFN, was determined on Calu-3 cells. Both IFN-β and IFN-λ were acid-stable, but only IFN-β was resistant to alkaline treatment. The antiviral activities of canine, porcine, and avian type I IFN were analysed with cell lines derived from the corresponding species. This safe bioassay will be useful for the rapid and sensitive quantification of multi-species type I IFN and potentially other antiviral cytokines.     

Anti-interferon-α neutralizing antibody induced telaprevir resistance under the interferon-α plus telaprevir treatment in vitro

Although the development of anti-interferon (IFN)-α neutralizing antibodies (NAbs) is likely to be a common clinical problem for patients with various diseases treated with IFN, anti-IFN-α NAb has been exceptionally considered to have no clinical significance in the treatment of chronic hepatitis C with pegylated IFN-α (Peg-IFN-α). However, we recently clarified that the presence of NAb was associated with a non-response to the Peg-IFN plus ribavirin (RBV) therapy. In this study, we used the HCV-replicon system with genotype 1b, and investigated the role of anti-IFN-α NAb in the response to telaprevir (TVR)-containing new antiviral therapy for hepatitis C virus (HCV). Anti-IFN-α NAb-positive sera specifically inhibited the anti-HCV effects of IFN-α, without any effect on the activity of IFN-β in vitro. The NAb-positive sera also inhibited the IFN-α-dependent induction of interferon-stimulated genes, MxA and OAS-1, in a dose-dependent manner. Although TVR monotherapy decreased the HCV-RNA in vitro, the HCV-RNA was increased again with the development of TVR-resistant mutations. When IFN-α was administrated with TVR, the replication of HCV was continuously suppressed for more than a month. However, in the presence of anti-IFN-α NAb-positive sera, even when IFN-α was combined with TVR, the levels of HCV-RNA exhibited a time-course similar to that with TVR monotherapy, and HCV with TVR-resistant mutations emerged. In conclusion, our findings suggest that the presence of IFN-α NAb decreases the antiviral effects of IFN-α and may be related to the development of TVR-resistant mutated viruses.     

Upregulation of interferon-induced indoleamine 2,3-dioxygenase in human macrophage cultures by lipopolysaccharide, muramyl tripeptide, and interleukin-1

The tryptophan decyclizing enzyme indoleamine 2,3-dioxygenase (IDO) was induced in human monocyte-derived macrophages (MDM) treated with human recombinant interferon-β (IFN-β) or interferon-γ (IFN-γ). Treated cells exhibited dose-dependent increases in IDO when assayed 48 hr after treatment. Cells exposed to IFN-γ were observed to exhibit consistently higher peak levels of IDO when compared with cells incubated in the presence of IFN-β. When IFN-β-treated cells were incubated in the presence of specified amounts of bacterial lipopolysaccharide (LPS) or liposome-encapsulated muramyl tripeptide (MTP), peak IDO activity increased such that enzyme activity was comparable to maximal activity observed with IFN-γ-treated cells. LPS and MTP also upregulated IFN-γ-mediated IDO activity when suboptimal amounts of IFN-γ were used. When macrophages were costimulated with various concentrations of human recombinant interleukin 1α (IL-1α), along with either maximum-stimulating amounts of IFN-β or suboptimal amounts of IFN-γ, IDO activity was upregulated in a manner similar to results obtained using the microbial products as stimuli. While neither IL-1α or IL-1β was detected in culture supernatants from macrophages treated with either LPS or MTP (alone or in combination with IFN), IL-1α was detected in cell lysates of macrophages treated with these upregulators. Although neutralizing antibody to IL-1α abolished the upregulatory effect of exogenous IL-1α, it had no effect on upregulation by LPS or MTP. This suggests that although LPS and MTP may induce production of cell-associated IL-1α, upregulation of IDO activity by these agents is independent of IL-1α production and may be mediated through distinct pathways.     

Production of IFN-β during Listeria monocytogenes infection is restricted to monocyte/macrophage lineage

The family of type I interferons (IFN), which consists of several IFN-α and one IFN-β, are produced not only after stimulation by viruses, but also after infection with non-viral pathogens. In the course of bacterial infections, these cytokines could be beneficial or detrimental. IFN-β is the primary member of type I IFN that initiates a cascade of IFN-α production. Here we addressed the question which cells are responsible for IFN-β expression after infection with the intracellular pathogen Listeria monocytogenes by using a genetic approach. By means of newly established reporter mice, maximum of IFN-β expression was observed at 24 hours post infection in spleen and, surprisingly, 48 hours post infection in colonized cervical and inguinal lymph nodes. Colonization of lymph nodes was independent of the type I IFN signaling, as well as bacterial dose and strain. Using cell specific reporter function and conditional deletions we could define cells expressing LysM as the major IFN-β producers, with cells formerly defined as Tip-DCs being the highest. Neutrophilic granulocytes, dendritic cells and plasmacytoid dendritic cells did not significantly contribute to type I IFN production.     

Modulation by Polymyxin B of the Effects of Interferon on Human Myelogenous Leukemia Cells

Natural and recombinant human interferon-α (IFN-α) and -γ (IFN-γ) exert differentiation-inducing and cytocidal effects in vitro on cells of the human myeloblastic leukemia cell line ML-2. These activities of IFNs are modulated by polymyxin B (PMB), a cyclic polycationic peptide antibiotic effective on Gram-negative bacilli. The modulating effect of PMB varies according to the species of IFN, namely, PMB enhances the activities of either natural IFN-γ or recombinant IFN-γ, while it inhibits the effects of either natural IFN-α or recombinant IFN-α. The cause of this variety in PMB effect on IFNs remains to be clarified.     

Induction of hepatocyte growth factor/scatter factor by interferon-γ in human leukemia cells

Induction of hepatocyte growth factor/scatter factor (HGF/SF) may be one of the critical steps in organ regeneration, wound healing, and embryogenesis. We previously reported the production of HGF/SF from various human leukemia cell lines and a high level of the growth factor in blood and bone marrow plasma from patients with various types of leukemia. We determined here the effects of hematopoietic cytokines on HGF/SF production in human leukemia cell lines, KG-1, a myeloid cell line, and RPMI-8226, a B cell line. Interferon (IFN)-γ remarkably stimulated HGF/SF production in both cell lines at concentrations of more than 0.1 or 1 IU/ml. IFN-α and IFN-β were as effective as IFN-γ in RPMI-8226 cells, but less than IFN-γ in KG-1 cells. HGF/SF gene expression in KG-1 cells was also up-regulated by IFN-γ. Granulocyte colony-stimulating factor (G-CSF), granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-5 and IL-6 had no effect on HGF/SF production in the 2 leukemia cell lines. We also determined the effects of HGF/SF inducers known for human fibroblasts on the growth factor production in leukemia cells. Out of phorbol 12-myristate 13-acetate (PMA), cholera toxin, IL-1β, and tumor necrosis factor (TNF)-α, the former three were as effective as IFN-γ in KG-1 cells, but only TNF-α stimulated HGF/SF production in RPMI-8226 cells, whose effect was less than those of IFN-α, IFN-β, and IFN-γ. The effect of IFN-γ in KG-1 cells was synergistic with that of PMA. In contrast with the effect in leukemia cells, HGF/SF induction by IFN-γ in human skin fibroblasts was much less than that by PMA or cholera toxin. These results indicated that IFN-γ is a potent inducer of HGF/SF in human leukemia cells. This finding suggests the presence of a homeostatic control mechanism in liver regeneration and repair: hepatic injury, DNA synthesis inhibition, or apoptosis caused by IFN-γ is subsequently overcome by cytokine-induced HGF/SF, a potent promoter of liver DNA synthesis. J. Cell. Physiol. 174:107–114, 1998. © 1998 Wiley-Liss, Inc.     

The nonstructural protein 1 papain-like cysteine protease was necessary for porcine reproductive and respiratory syndrome virus nonstructural protein 1 to inhibit interferon-β induction

Porcine reproductive and respiratory syndrome virus nonstructural protein 1 (nsp1) could be auto-cleaved into nsp1α and nsp1β, both of which had the papain-like cysteine protease activities. Previous studies have shown that porcine reproductive and respiratory syndrome virus nsp1 was an interferon (IFN) antagonist. However, the mechanism by which nsp1 inhibited IFN-β production was unclear. Here, we used site-directed mutagenesis that inactivated the papain-like cysteine protease activities of nsp1 to explore whether the papain-like cysteine protease activities were required for nsp1 to disrupt IFN-β production. The results showed that mutations that inactivated papain-like cysteine protease activity of nsp1α made nsp1 lose its IFN antagonism activity, whereas mutations that inactivated papain-like cysteine protease activity of nsp1β did not influence the IFN antagonism activity of nsp1. In conclusion, our present work indicated that the papain-like cysteine protease activity of nsp1α was necessary for nsp1 to inhibit IFN-β induction.     

Characterization of L1210 S Cells with Low Sensitivity to Mouse Interferon-γ

A mouse leukemic cell line L1210 Sg with a low sensitivity to interferon-γ (IFN-γ) was described. On the nature of the antiviral action and binding of IFN, L1210 Sg cells were compared with L1210m cell line which is sensitive to IFN-γ. For a half reduction of the vesicular stomatitis virus-RNA synthesis, L1210 Sg cells required 500–fold more IFN-γ than L1210m cells did. However, both cell lines were induced to the antiviral state to the same extent with IFN-α or -β. L1210 Sg and L1210m cells were sensitive to the anti-proliferative action of IFN-α and -β, but insensitive to IFN-γ. (2′-5′)Oligoadenylate synthetase was induced in these cell lines by IFN-β, but not by IFN-γ, which suggests that the induction of this synthetase may not be responsible for the antiviral action of IFN-γ. No substantial difference between L1210 Sg and L1210m cells was found in IFN receptors for IFN-γ and IFN-β either in number per cell or in their affinity to corresponding IFN type. However, differences were noted in time course profiles of cell-associated IFN-γ at 37 C: in L1210m cells, a rise-and-decay profile of IFN-γ bound to cells was observed at 37 C, but in L1210 Sg cells, rise and slight decay was observed. On the other hand, a similar rise-and-decay curve of IFN-β bound to these cells was observed. These results indicated that the low sensitivity of L1210 Sg cells to IFN-γ may be due to this slight decay of receptor-bound IFN-γ.     

Beta interferon controls West Nile virus infection and pathogenesis in mice

Studies with mice lacking the common plasma membrane receptor for type I interferon (IFN-αβR(-)(/)(-)) have revealed that IFN signaling restricts tropism, dissemination, and lethality after infection with West Nile virus (WNV) or several other pathogenic viruses. However, the specific functions of individual IFN subtypes remain uncertain. Here, using IFN-β(-)(/)(-) mice, we defined the antiviral and immunomodulatory function of this IFN subtype in restricting viral infection. IFN-β(-)(/)(-) mice were more vulnerable to WNV infection than wild-type mice, succumbing more quickly and with greater overall mortality, although the phenotype was less severe than that of IFN-αβR(-)(/)(-) mice. The increased susceptibility of IFN-β(-)(/)(-) mice was accompanied by enhanced viral replication in different tissues. Consistent with a direct role for IFN-β in control of WNV replication, viral titers in ex vivo cultures of macrophages, dendritic cells, fibroblasts, and cerebellar granule cell neurons, but not cortical neurons, from IFN-β(-)(/)(-) mice were greater than in wild-type cells. Although detailed immunological analysis revealed no major deficits in the quality or quantity of WNV-specific antibodies or CD8(+) T cells, we observed an altered CD4(+) CD25(+) FoxP3(+) regulatory T cell response, with greater numbers after infection. Collectively, these results suggest that IFN-β controls WNV pathogenesis by restricting infection in key cell types and by modulating T cell regulatory networks.     

Different effects of the immunostimulatory drug Stimforte on infections of hepatitis C virus and herpes simplex virus type 1

Stimforte, an immune response-stimulating preparation, is active with respect to hepatitis C virus (HCV) and herpes simplex virus type I (HSV-1). The effects of Stimforte in animals infected with either HCV or HSV-1 are fundamentally different. In mice with acute herpes virus infection, Stimforte administration leads to a higher activity of natural killer cells and cytotoxic lymphocytes, and the amount of interferon (IFN) λ grows. In mice infected with HCV, Stimforte administration results in a significant increase in IFN-β but not IFN-λ in blood and affected organs. Stimforte has been found to affect directly HCV reproduction that causes the infected cell death, but it does not affect HSV-1 reproduction in the Vero cells (V).     

Interferon gamma encapsulated into liposomes enhances the activity of monocytes and natural killer cells and has antiproliferative effects on tumor cells in vitro

The effects of human interferon gamma (IFN gamma) encapsulated into liposomes were investigated in vitro. Monocytes were induced to release a cytotoxic factor with either IFN gamma encapsulated into liposomes, free IFN gamma or lipopolysaccharide (LPS). If IFN gamma was applied in the liposomal form, less IFN activity was required to stimulate monocytes. Most of the cytotoxic factor was secreted during the first 4 h of stimulation. The cytotoxic factor in supernatants from PMNLs was completely neutralized by a monospecific polyclonal antiserum to tumor necrosis factor (TNF). Combining subthreshold doses of IFN gamma liposomes or IFN gamma with lipopolysaccharide synergistically enhanced the release of TNF. In fluorescence analysis, altered expression of the class II HLA-DR antigen on LeuM3 positive monocytes was induced with IFN gamma liposomes as well as with IFN gamma. Not only monocytes but also natural killer (NK) cells were stimulated to higher cytotoxicity by IFN gamma liposomes in a dose-dependent manner. In comparison with IFN gamma, the same amount of activity was necessary for adequate stimulation of NK-cells against the K562 target cells. Furthermore, the antiproliferative effects of IFN gamma liposomes and free IFN gamma on several human tumor cell lines was compared. Among several cell lines tested, U937 and A549 turned out to be sensitive to IFN gamma, and both cell lines reacted with 50% growth inhibition at a lower amount of gamma presented by liposomes than in the free form. These data show production of IFN gamma liposomes which possess immunomodulatory and antiproliferative activity in vitro. In several of the test systems studied, liposome-encapsulated IFN gamma was more effective than free IFN gamma.     

Interleukin 2 induces gamma-interferon production: participation of macrophages and NK-like cells

Interleukin 2 (IL 2) has been shown to be a potent stimulator of natural killer (NK) cells. In the present studies, partially purified mouse and human IL 2 preparations were also found to induce interferon (IFN) from mouse spleen cells. By the criteria of sensitivity to treatment at pH 2 and failure to be neutralized by a potent anti-alpha, beta IFN serum, the species of IFN produced was of type gamma. Cooperation between two types of cell, a macrophage and an NK-like cell, was required for IFN production by murine spleen cells treated with IL 2. The requirement for macrophages could be replaced with supernatant obtained by incubating macrophages for 24 hr with lymphokine preparations containing IL 2. Interestingly, mature T cells apparently played no role in the process. Furthermore, the beige (bg/bg) mutation, which severely impairs NK cell lytic activity, had no effect on the ability of NK-like cells to participate in IFN production. Cell fractionation experiments revealed no dissociation between the requirements for augmentation of NK cytotoxic activity and for IFN production, and it is concluded that at least a portion of the NK boosting induced by IL 2-containing preparations is mediated through gamma-IFN.     

Inhibition of Varicella-Zoster Virus Glycoprotein Expression by Peripheral Blood Mononuclear Cells

The effect of peripheral blood mononuclear cells (PBMC) on expression of varicella-zoster virus (VZV) glycoproteins (Gps) was analyzed by flow cytometry. PBMC from VZV seropositive and seronegative donors and supernatant of PBMC co-cultured with VZV-infected human embryonic fibroblasts reduced VZV Gp expression. Neutralization of supernatant fluid with mixture of anti-interferons (IFN)-α, -β, -γ, and tumor necrosis factor (TNF)-α partially reduced inhibitory activity of supernatant on VZV Gp expression. Deletion of natural killer (NK) cells and adherent cells from PBMC reduced inhibitory activity of PBMC on VZV Gp expression. These results suggest that IFN-α, -β, -γ, TNF-α and other soluble factors released from NK cells and monocytes by co-cultivation with VZV-infected fibroblasts inhibit VZV Gp expression.     

Changes in DNA-polymerase and thymidine kinase activity during interferon treatment

Treatment of the human glioma cell line, U-251 MG, with human IFN-β resulted in a dose-dependent growth depression and a decreased activity of DNA-polymerase in exponentially growing cells, although paradoxally the number of cells in the S phase increased. In synchronized cells, a S block was confirmed. Both thymidine kinase and DNA-polymerase increased but with a lower rate during IFN treatment. No inhibitory effects on any of the enzymes could be seen when IFN-treated lysate was mixed with control lysate. The possible significance of depressed DNA synthesis during virus infection is discussed.     

In vitro protective and enhancing effects of interferons from several species on human lymphotoxin-induced target cell killing

In a previous study it was reported that human alpha-interferons (IFN) caused a significant enhancement of human lymphotoxin (LT)-induced in vitro killing of human target cells. This synergistic effect was dose dependent and was demonstrable on normal and tumor cell targets. The effects of IFNs from human and several animal species on human LT-induced cell killing of human, mouse, and rabbit target cells are examined. In addition to enhancement of IFN, a new finding was made showing protective effects of IFN on human LT activity. IFN-induced enhancement or protection depended on the particular IFN:target cell combination, with the highest degree of enhancement being observed in the homologous human combination. In this latter case, IFN-induced enhancement was blocked by antiserum to IFN. While a role for other soluble factors cannot be ruled out, the results suggest that, in the homologous human system, enhancement of LT activity was mediated by IFN. These results are discussed in relationship to previously observed enhancing and protective effects of IFN in natural killer cell systems.     

Ⅰ型IFN的结构、信号传导和功能

IFN是一种具有广谱抗病毒、抗肿瘤、抑制细胞增殖及免疫调节作用的活性蛋白,至少可分为α、β、δ、ε、κ、ω、τ和limitin八种,分子质量约为18.5 kDa,包括4个保守的半胱氨酸,形成2对分子内二硫键。IFN-α/βR是IFN-α和IFN-β共同结合受体,包括IFNAR1和IFNAR2,IFNAR1仅有1个亚单位,IFNAR2由3个亚单位组成。IFN与位于细胞膜上的特异性受体结合,主要经JAK-STAT途径介导信号传导,不仅能通过PKR、2-′5′OAS、Mx和ADAR1等途径发挥抗病毒作用,还能通过对淋巴细胞和巨噬细胞的调节以及诱导MHC分子表达等途径发挥直接或间接的免疫调节作用。     

Endogenous and recombinant type I interferons and disease activity in multiple sclerosis

Although treatment of multiple sclerosis (MS) with the type I interferon (IFN) IFN-β lowers disease activity, the role of endogenous type I IFN in MS remains controversial. We studied CD4+ T cells and CD4+ T cell subsets, monocytes and dendritic cells by flow cytometry and analysed the relationship with endogenous type I IFN-like activity, the effect of IFN-β therapy, and clinical and magnetic resonance imaging (MRI) disease activity in MS patients. Endogenous type I IFN activity was associated with decreased expression of the integrin subunit CD49d (VLA-4) on CD4+CD26(high) T cells (Th1 helper cells), and this effect was associated with less MRI disease activity. IFN-β therapy reduced CD49d expression on CD4+CD26(high) T cells, and the percentage of CD4+CD26(high) T cells that were CD49d(high) correlated with clinical and MRI disease activity in patients treated with IFN-β. Treatment with IFN-β also increased the percentage of CD4+ T cells expressing CD71 and HLA-DR (activated T cells), and this was associated with an increased risk of clinical disease activity. In contrast, induction of CD71 and HLA-DR was not observed in untreated MS patients with evidence of endogenous type IFN I activity. In conclusion, the effects of IFN-β treatment and endogenous type I IFN activity on VLA-4 expression are similar and associated with control of disease activity. However, immune-activating effects of treatment with IFN-β may counteract the beneficial effects of treatment and cause an insufficient response to therapy.