Interferon-α/β upregulate IL-15 expression in vitro and in vivo: analysis in human hepatocellular carcinoma cell lines and in chronic hepatitis C patients during interferon-α/β treatment

Type I interferon (IFN) possesses antiviral and antitumor activities and also having an immune regulatory effect, activating cellular immune response and upregulating several cytokines. Recent study has shown that type I IFN upregurates the dendritic cell production of IL-15 capable of activating natural killer cells and CD8⁺ memory T lymphocytes. However, it is still unknown if type I IFN induces IL-15 production in non-immune cells and if type I IFN affects IL-15 production in vivo. The present study investigated the effect of type I IFNs on IL-15 expression in hepatocellular carcinoma (HCC) cell lines in vitro and in patients with chronic hepatitis C in vivo. When three HCC cell lines, Huh7, HepG2, and JHH4 were cultured in vitro, IFN upregulation of IL-15 expression was observed at both the mRNA and protein levels. In experiments using Huh7 cells, upregulation of IL-15 expression occurred within 24 h of the start of IFN stimulation, and both IFN-α and -β dose-dependently increased IL-15 production in the range from 100 U/ml to 10,000 U/ml of concentration. IFN-β showed stronger activity in IL-15 production induction in vitro than IFN-α. For in vivo examination, sera were obtained from 21 chronic hepatitis C patients treated with IFN and 29 healthy individuals, and the serum IL-15 level was quantified by ELISA. The serum IL-15 level of chronic hepatitis C patients before IFN treatment was similar to that of the healthy controls and significantly increased only during the IFN administration period. These results confirm that IFN-α/β induce IL-15 production and also suggest that IL-15 may be associated with type I IFN-induced immune response.     

Purification and characterization of prokaryotically expressed human interferon-λ2

A system for the production of soluble interferon (IFN)-λ2 was developed by fusing the IFN-λ2, NusA protein, polyhistidine and S peptide genes and then expressing the fused product (Nus-His-S-tagged IFN-λ2) in Escherichia coli. The expressed fusion protein was purified by Ni-NTA affinity chromatography. The fusion tag was removed from recombinant IFN-λ2 by cleavage with enterokinase. N-Terminal sequencing confirmed the identity of the purified protein. When compared with commercial IFN-α2b, IFN-λ2 had similar antiviral activity. The production and characterization of biologically active IFN-λ2 will be beneficial for its potential role in clinical applications.     

NK1.1+ cells mediate the antitumor effects of a dual Toll-like receptor 7/8 agonist in the disseminated B16-F10 melanoma model

Innate immune stimulation with Toll-like receptor (TLR) agonists is a proposed modality for immunotherapy of melanoma. Here, a TLR7/8 agonist, 3M-011, was used effectively as a single systemic agent against disseminated mouse B16-F10 melanoma. The investigation of the mechanism of antitumor action revealed that the agonist had no direct cytotoxic effects on tumor cells tested in vitro. In addition, 3M-011 retained its effectiveness in scid/B6 mice and scid/NOD mice, eliminating the requirement for T and B cells, but lost its activity in beige (bg/bg) and NK1.1-immunodepleted mice, suggesting a critical role for natural killer (NK) cells in the antitumor response. NK cytotoxicity was enhanced in vivo by the TLR7/8 agonist; this activation was long lasting, as determined by sustained expression of the activation marker CD69. Also, in human in vitro studies, 3M-011 potentiated NK cytotoxicity. TLR7/8-mediated NK-dependent antitumor activity was retained in IFN-α/β receptor-deficient as well as perforin-deficient mice, while depletion of IFN-γ significantly decreased the ability of 3M-011 to delay tumor growth. Thus, IFN-γ-dependent functions of NK cell populations appear essential for cancer immunotherapy with TLR7/8 agonists. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. All authors are or were employed by 3M while this work was being conducted.     

Recombinant bacillus Calmette-Guérin (BCG) expressing interferon-alpha 2B enhances human mononuclear cell cytotoxicity against bladder cancer cell lines in vitro

Purpose  The proper induction of cellular immunity is required for effective bacillus Calmette-Guérin (BCG) immunotherapy of bladder cancer. It has been known that BCG stimulation of human peripheral blood mononuclear cells (PBMC) leads to the generation of effector cells cytotoxic to bladder cancer cells in vitro. To improve BCG therapy, we previously developed human interferon (IFN)-α 2B secreting recombinant (r) BCG (rBCG-IFN-α). We demonstrated that rBCG-IFN-α augmented T helper type 1 (Th1) cytokine IFN-γ production by PBMC. In this study, we further investigated whether rBCG-IFN-α could also enhance PBMC cytotoxicity toward bladder cancer cells. Materials and methods  PBMC were prepared from healthy individuals, left alone or stimulated with rBCG-IFN-α or control MV261 BCG, and used as effector cells in ⁵¹Cr-release assays. Human bladder cancer cell lines T24, J82, 5637, TCCSUP, and UMUC-3 were used as target cells. To determine the role of secreted rIFN-α as well as endogenously expressed IFN-γ and IL-2 in inducing the cytotoxicity, PBMC were stimulated with rBCG-IFN-α in the presence of neutralizing antibodies to IFN-α, IFN-γ or IL-2. To determine the role of natural killer (NK) and CD8⁺ T cells in inducing the cytotoxicity, both cell types were isolated after BCG stimulation of PBMC and used as effector cells in ⁵¹Cr-release assays. Results  Non-stimulated PBMC showed basal levels of cytotoxicity against all target cell lines tested. MV261 BCG increased the PBMC cytotoxicity by 1.8- to 4.2-fold. rBCG-IFN-α further increased the PBMC cytotoxicity by up to 2-fold. Elevated production of IFN-γ and IL-2 by PBMC was observed after rBCG-IFN-α stimulation. Blockage of IFN-α, IFN-γ or IL-2 by neutralizing antibodies during rBCG-IFN-α stimulation reduced or abolished the induction of PBMC cytotoxicity. Both NK and CD8⁺ T cells were found to be responsible for the enhanced PBMC cytotoxicity induced by rBCG-IFN-α with the former cell type being more predominant. Conclusions  rBCG-IFN-α is an improved BCG agent that induces enhanced PBMC cytotoxicity against bladder cancer cells in vitro. This rBCG strain may serve as an alternative to BCG for the treatment of superficial bladder cancer.     

Characteristics of cell-mediated,anti-listerial immunity induced by a naturally avirulent <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> serotype 4a strain HCC23

The characteristics of cell-mediated, anti-listerial immune response initiated by an avirulent Listeria monocytogenes serotype 4a strain HCC23 was assessed. Similar to virulent strain EGD, avirulent strain HCC23 grew readily within macrophage-like J774 cells, but nonhemolytic strain ATCC 15313 did not. Compared with EGD, HCC23 induced a relatively low level of gamma interferon (IFN-γ) in mice, and ATCC 15313 stimulated no detectable IFN-γ. The percentages of gated CD4 T cells from mice immunized with EGD and HCC23 showed a notable drop (to 30%) at 21 days post exposure in comparison with that (about 50%) from ATCC 15313-injected or untreated mice; and the percentage of gated NK cells from EGD-immunized group was markedly higher than those from other treatment groups. Mice immunized with HCC23 and EGD developed an equally strong protective immunity against listeriosis that was effective in both short and long terms, but those injected with ATCC 15313 or saline succumbed to listeriosis within 6 days of challenge.     

Targeted Induction of Interferon-λ in Humanized Chimeric Mouse Liver Abrogates Hepatotropic Virus Infection

Background & Aims

The interferon (IFN) system plays a critical role in innate antiviral response. We presume that targeted induction of IFN in human liver shows robust antiviral effects on hepatitis C virus (HCV) and hepatitis B virus (HBV).

Methods

This study used chimeric mice harboring humanized livers and infected with HCV or HBV. This mouse model permitted simultaneous analysis of immune responses by human and mouse hepatocytes in the same liver and exploration of the mechanism of antiviral effect against these viruses. Targeted expression of IFN was induced by treating the animals with a complex comprising a hepatotropic cationic liposome and a synthetic double-stranded RNA analog, pIC (LIC-pIC). Viral replication, IFN gene expression, IFN protein production, and IFN antiviral activity were analyzed (for type I, II and III IFNs) in the livers and sera of these humanized chimeric mice.

Results

Following treatment with LIC-pIC, the humanized livers of chimeric mice exhibited increased expression (at the mRNA and protein level) of human IFN-λs, resulting in strong antiviral effect on HBV and HCV. Similar increases were not seen for human IFN-α or IFN-β in these animals. Strong induction of IFN-λs by LIC-pIC occurred only in human hepatocytes, and not in mouse hepatocytes nor in human cell lines derived from other (non-hepatic) tissues. LIC-pIC-induced IFN-λ production was mediated by the immune sensor adaptor molecules mitochondrial antiviral signaling protein (MAVS) and Toll/IL-1R domain-containing adaptor molecule-1 (TICAM-1), suggesting dual recognition of LIC-pIC by both sensor adaptor pathways.

Conclusions

These findings demonstrate that the expression and function of various IFNs differ depending on the animal species and tissues under investigation. Chimeric mice harboring humanized livers demonstrate that IFN-λs play an important role in the defense against human hepatic virus infection.     

Intratumoral interferon-α gene transfer enhances tumor immunity after allogeneic hematopoietic stem cell transplantation

One of the major challenges in the treatment of solid cancers by allogenic hematopoietic stem cell transfer (alloHSCT) is the specific enhancement of antitumor immunity. Interferon (IFN) is a cytokine with pleiotropic biological functions including an immunomoduration, and our preclinical studies have shown that an intratumoral IFN-α gene transfer induced strong local tumor control and systemic tumor-specific immunity. In the present study, we examined whether the IFN-α gene transfer could enhance recognition of tumor-associated antigens by donor T cells and augment the antitumor activity of alloHSCT. First, when a mouse IFN-α adenovirus vector (Ad-mIFN) was injected into subcutaneous xenografts of syngeneic renal and colon cancer cells, tumor growth was significantly suppressed in a dose-dependent manner. A significant tumor cell death and infiltration of immune cells was recognized in the Ad-mIFN-injected tumors, and the dendrtic cells isolated from the tumors showed a strong Th1-oriented response. The antitumor effect of Ad-mIFN was then examined in a murine model of minor histocompatibility antigen-mismatched alloHSCT. The intratumoral IFN-α gene transfer caused significant tumor suppression in the alloHSCT recipients, and this suppression was evident not only in the gene-transduced tumors but also in simultaneously inoculated distant tumors which did not receive the vector injection. A cytotoxicity assay showed specific tumor cell lysis by donor T cells responding to IFN-α. Graft-versus-host disease was not exacerbated serologically or clinically in the mice treated with IFN-α. This combination strategy deserves evaluation in future clinical trials for human solid cancers.     

Different physiology of interferon-α/-γ in models of liver regeneration in the rat

Liver regeneration may take place after liver injury through replication of hepatocytes or hepatic progenitor cells called oval cells. Interferons (IFN) are natural cytokines with pleiotrophic effects including antiviral and antiproliferative actions. No data are yet available on the physiology and cellular source of natural IFNs during liver regeneration. To address this issue, we have analyzed the levels and biologic activities of IFN-α/IFN-γ in two models of partial hepatectomy. After 2/3rd partial hepatectomy (PH), hepatic levels of IFN-α and IFN-γ declined transiently in contrast to a transient increase of the IFN-γ serum level. After administration of 2-acetylaminofluorene and partial hepatectomy (AAF/PH model), however, both IFN-α and IFN-γ expression were up-regulated in regenerating livers. Again, the IFN-γ serum level was transiently increased. Whereas hepatic IFN-γ was up-regulated early (day 1–5), but not significantly, in the AAF/PH model, IFN-α was significantly up-regulated at later time points in parallel to the peak of oval cell proliferation (days 7–9). Biological activity of IFN-α was shown by activation of IFN-α-specific signal transduction and induction of IFN-α specific-gene expression. We found a significant infiltration of the liver with inflammatory monocyte-like mononuclear phagocytes (MNP) concomitant to the frequency of oval cells. We localized IFN-α production only in MNPs, but not in oval cells. These events were not observed in normal liver regeneration after standard PH. We conclude that IFN-γ functions as an acute-phase cytokine in both models of liver regeneration and may constitute a systemic component of liver regeneration. IFN-α was increased only in the AAF/PH model, and was associated with proliferation of oval cells. However, oval cells seem not to be the source of IFN-α. Instead, inflammatory MNP infiltrating AAF/PH-treated livers produce IFN-α. These inflammatory MNPs may be involved in the regulation of the oval cell compartment through local expression of cytokines, including IFN-α.     

A crude extract from immature green tea (<Emphasis Type="Italic">Camellia sinensis</Emphasis>) leaves promotes Toll-like receptor 7-mediated interferon-α production in human macrophage-like cells

Toll-like receptor 7 (TLR7) senses viral single-stranded RNA (ssRNA), induces the production of type I interferons (IFNs), IFN-α and -β, in macrophages such as dendritic cells (DCs), and its immune system protects the host from virus infection. Here, we found that a crude extract from immature green tea leaves (iTPS) containing a macromolecule with ssRNA fragments, induces IFN-α production in human macrophage-like cells. In addition IFN-α production was inhibited by treatment with TLR7 inhibitors or a phagocytosis inhibitor.     

Enhanced osteoclast development in collagen-induced arthritis in interferon-γ receptor knock-out mice as related to increased splenic CD11b+ myelopoiesis

Collagen-induced arthritis (CIA) in mice is accompanied by splenomegaly due to the selective expansion of immature CD11b⁺ myeloblasts. Both disease manifestations are more pronounced in interferon-γ receptor knock-out (IFN-γR KO) mice. We have taken advantage of this difference to test the hypothesis that the expanding CD11b⁺ splenic cell population constitutes a source from which osteoclast precursors are recruited to the joint synovia. We found larger numbers of osteoclasts and more severe bone destruction in joints of IFN-γR KO mice than in joints of wild-type mice. Osteoclast-like multinucleated cells appeared in splenocyte cultures established in the presence of macrophage colony-stimulating factor (M-CSF) and stimulated with the osteoclast-differentiating factor receptor activator of NF-κB ligand (RANKL) or with tumour necrosis factor-α (TNF-α). Significantly larger numbers of such cells could be generated from splenocytes of IFN-γR KO mice than from those of wild-type mice. This was not accompanied, as might have been expected, by increased concentrations of the intracellular adaptor protein TRAF6, known to be involved in signalling of RANKL- and TNF-α-induced osteoclast formation. Splenocyte cultures of IFN-γR KO mice also produced more TNF-α and more RANKL than those of wild-type mice. Finally, splenocytes isolated from immunised IFN-γR KO mice contained comparatively low levels of pro-interleukin-1β (pro-IL-1β) and pro-caspase-1, indicating more extensive conversion of pro-IL-1β into secreted active IL-1β. These observations provide evidence that all conditions are fulfilled for the expanding CD11b⁺ splenocytes to act as a source of osteoclasts and to be indirectly responsible for bone destruction in CIA. They also provide a plausible explanation for the higher susceptibility of IFN-γR KO mice to CIA.     

Stimulation of the natural immune system in normal mice by polysaccharide from maitake mushroom

 Maitake D-Fraction is a polysaccharide extracted from the maitake mushroom (Grifola frondosa S.F. Gray). It is a β-glucan with a β-1,6 main chain with β-1,3 branches. Using normal C3H/Hej mice, its effects on the natural immune system, including macrophages, dendritic cells, and natural killer (NK) cells, were investigated. NK cells attack cells infected with pathogens such as bacteria and virus and produce cytokines, such as interferon-gamma (IFN-γ), that can modulate natural and specific immune responses. D-Fraction was administered to the mice intraperitoneally for 3 consecutive days; spleen cells containing macrophages and dendritic cells were then cultured and the culture supernatants were analyzed for IL-12. At the same time, IFN-γ expression in splenic NK cells was investigated. The levels of these cytokines were increased by D-Fraction. To elucidate NK cell activation by D-Fraction, CD69 expression on the surface of activated NK cells was examined, resulting in an increase in CD69-positive ratio for splenic NK cells. These results indicate that D-Fraction stimulates the natural immunity related to the activation of NK cells indirectly through IL-12 produced by macrophages and dendritic cells. Therefore, administration of D-Fraction to healthy individuals may serve to prevent infection. Received: August 1, 2002 / Accepted: February 10, 2003     

Effects of interferon-alpha subtypes on the Th1/Th2 balance in peripheral blood mononuclear cells from patients with hepatitis virus infection-associated liver disorders

Summary Interferon-alpha (IFN-α) has recently been shown to modulate in vitro T helper (Th) 1-driven responses in the peripheral blood mononuclear cells (PBMC) of patients with hepatitis B virus or C virus infection. In this study, we examined the in vitro effects of IFN-α subtypes (IFN-α1, −α2, −α5, −α8, and −α10) on the Th1/Th2 balance in PBMC obtained from patients with hepatitis virus infection-associated liver disorders and chronic hepatitis (CH), in comparison with the effect on healthy control volunteer PBMC. The Th1-type cell percentages and Th1/Th2 ratios were significantly higher in the PBMC of patients when compared with controls both before and after cultivation in vitro, with the IFN-α subtypes. The IFNα-5 induced an increase in the Th2-type cell percentages in both control and patient PBMC, resulting in that IFN-α5 lowered the Th1/Th2 ratio in patients with CH. Furthermore, statistical analysis revealed that IFN-α8 significantly promoted an increase in the Th1/Th2 ratios of PBMC from patients with CH and liver cirrhosis (LC) but not that of PBMC from patients with LC-hepatocellular carcinoma (HCC) and HCC. These findings imply that hepatitis virus infection and its disease status modify the effects of IFN-α subtypes on Th1 and Th2 immune balance in patients. Our findings should help to elucidate the mechanisms underlying successful IFN therapy for hepatitis virus infection and prevention of hepatocellular carcinogenesis.     

Systemic lupus erythematosus and the type I interferon system

Patients with systemic lupus erythematosus (SLE) have ongoing interferon-α (IFN-α) production and serum IFN-α levels are correlated with both disease activity and severity. Recent studies of patients with SLE have demonstrated the presence of endogenous IFN-α inducers in such individuals, consisting of small immune complexes (ICs) containing IgG and DNA. These ICs act specifically on natural IFN-α-producing cells (NIPCs), often termed plasmacytoid dendritic cells (PDCs). Given the fact that the NIPC/PDC has a key role in both the innate and adaptive immune response, as well as the many immunoregulatory effects of IFN-α, these observations might be important for the understanding of the etiopathogenesis of SLE. In this review we briefly describe the biology of the type I IFN system, with emphasis on inducers, producing cells (especially NIPCs/PDCs), IFN-α actions and target immune cells that might be relevant in SLE. On the basis of this information and results from studies in SLE patients, we propose a hypothesis that explains how NIPCs/PDCs become activated and have a pivotal etiopathogenic role in SLE. This hypothesis also indicates new therapeutic targets in this autoimmune disease.     

Human Plasmacytoid Dendritic Cells Sense Lymphocytic Choriomeningitis Virus-Infected Cells In Vitro

We previously reported that exosomal transfer of hepatitis C virus (HCV) positive-strand RNA from human Huh-7 hepatoma cells to human plasmacytoid dendritic cells (pDCs) triggers pDC alpha/beta interferon (IFN-α/β) production in a Toll-like receptor 7 (TLR7)-dependent, virus-independent manner. Here we show that human pDCs are also activated by a TLR7-dependent, virus-independent, exosomal RNA transfer mechanism by human and mouse hepatoma and nonhepatoma cells that replicate the negative-strand lymphocytic choriomeningitis virus (LCMV).     

Opposite effects of serotonin and interferon-α on the membrane potential and function of human natural killer cells

Summary In an earlier article, we reported that serotonin (5-hydroxytryptamine, 5-HT) inhibits the natural killer cell (NK) cytotoxicity of human whole blood in a dose-dependent manner and that natural human interferon-α (IFN-α) partially eliminates this effect. Because natural IFN-α might contain factors other than IFN, we repeated these experiments with recombinant human interferon-α (rhIFN-α) and separated blood lymphocytes enriched with NK cells and then demonstrated that IFN really is responsible for this effect. Furthermore, this investigation was carried out to clarify the mechanisms of the action of 5-HT and of rhIFN-α on NK cells. The inhibition of the cytotoxicity was pronounced when 5-HT was added at the onset of the cytotoxic assay, whereas the pretreatment of lymphocytes for 18 h only led to a slight inhibition. Moreover, rhIFN-α applied 1 h before or 1 h after the addition of 5-HT decreased the inhibitory effect of 5-HT. Flow cytometric analysis involving the use of a voltage-sensitive dye, oxonol, revealed that 5-HT depolarized, whereas rhIFN-α hyperpolarized the plasma membrane of the lymphocytes. Thus, it seems likely that the inhibitory effect of 5-HT on the cytotoxicity of peripheral human lymphocytes is due to the depolarization on the plasma membrane of the effector cells and that rhIFN-α antagonizes this ability via its hyperpolarizing activity.     

NK cells rapidly remove B16F10 tumor cells in a perforin and interferon-gamma independent manner in vivo

Natural killer (NK) cells have been shown critical in reducing tumor lung metastasis in various murine cancer models. Effector molecules such as perforin and IFN-γ may play important roles in inhibition of metastasis. However, most of these conclusions were based on experiments that involved quantitation of metastatic colonies several weeks after tumor challenge. The roles of NK cells and their effector molecules (perforin and IFN-γ) in the initial immune responses against tumor metastasis in lungs are still unknown. By using the B16F10 melanoma tumor model combined with confocal microscopy, we observed an increase in numbers of B16F10 cells in NK-depleted mice at 60 min post tumor inoculation, but this effect was independent of perforin or IFN-γ. In addition, NK cell numbers in lungs after tumor injection rapidly increased suggesting a redistribution of NK cells in the lungs. However, NK cells were not found in contact with tumor cells until day 6 or later. Our data indicate that during early responses against B16F10 cells, NK cells use another mechanism(s) besides perforin and IFN-γ to prevent tumor metastasis.