TLR-9 Contributes to the Antiviral Innate Immune Sensing of Rodent Parvoviruses MVMp and H-1PV by Normal Human Immune Cells

The oncotropism of Minute Virus of Mice (MVMp) is partially related to the stimulation of an antiviral response mediated by type-I interferons (IFNs) in normal but not in transformed mouse cells. The present work was undertaken to assess whether the oncotropism displayed against human cells by MVMp and its rat homolog H-1PV also depends on antiviral mechanisms and to identify the pattern recognition receptor (PRR) involved. Despite their low proliferation rate which represents a drawback for parvovirus multiplication, we used human peripheral blood mononuclear cells (hPBMCs) as normal model specifically because all known PRRs are functional in this mixed cell population and moreover because some of its subsets are among the main IFN producers upon infections in mammals. Human transformed models consisted in lines and tumor cells more or less permissive to both parvoviruses. Our results show that irrespective of their permissiveness, transformed cells do not produce IFNs nor develop an antiviral response upon parvovirus infection. However, MVMp- or H-1PV-infected hPBMCs trigger such defense mechanisms despite an absence of parvovirus replication and protein expression, pointing to the viral genome as the activating element. Substantial reduction of an inhibitory oligodeoxynucleotide (iODN) of the latter IFN production identified TLR-9 as a potential PRR for parvoviruses in hPBMCs. However, neither the iODN treatment nor an antibody-induced neutralization of the IFN-triggered effects restored parvovirus multiplication in these cells as expected by their weak proliferation in culture. Finally, given that a TLR-9 activation could also not be observed in parvovirus-infected human lines reported to be endowed with a functional TLR-9 pathway (Namalwa, Raji, and HEK293-TLR9^+/+), our data suggest that transformed human cells do not sense MVMp or H-1PV either because of an absence of PRR expression or an intrinsic, or virus-driven defect in the endosomal sensing of the parvovirus genomes by TLR-9.     

Enhancement of interferon-induced 2–5 oligoadenylate synthetase activity by retinoic acid in human histiocytic lymphoma U937 cells and WISH cells

The effect of retinoic acid (RA) on the level of interferon (IFN)-induced 2-5 oligoadenylate (2-5A) synthetase activity was examined in human histiocytic lymphoma U937 cells and WISH cells** in order to ascertain the role of this polymerase in interaction between IFNs and RA. Cultures containing both IFNs (1-100 U/ml) and RA (0.1-10 microM) consistently had higher levels of enzyme activity than corresponding cells treated with IFN alone and this was true for all three types of IFNs in both cell lines. The potentiating effect of RA was dose- and time-dependent and under optimal conditions, the induction of the synthetase was synergistic between IFN-beta (10-100 U/ml) and RA (0.1-10 microM). Furthermore, pretreatment (but not posttreatment) with RA followed by subsequent treatment with IFNs preferentially induced higher levels of enzyme activity in U937 cells but not in WISH cells. In addition, our results indicated that the modulating effect of RA on IFNs did not involve interaction at the receptor level and the level of enhancement of 2-5A synthetase activity was not in parallel with either cell-growth arrest or promotion of differentiation. Lastly, the present study raises the possibility that interactions between IFNs and RA, in either a synergistic or antagonistic manner, may be mediated through amplification of the 2-5A system.     

Negative Regulation of Human Astrocytes by Interferon (IFN) α in Relation to Growth Inhibition and Impaired Glucose Utilization

The present study assessed the direct effects of IFNs on human astrocytes. Human astrocytes were exposed to human recombinant IFNs, and the proliferation of cells was measured. Type I IFN receptor mRNA and protein expression, the phosphoprotein levels of signaling molecules including JNK, ERK1/2, IκB, p38MAPK, Stat3, and the expression of cytokines were determined respectively. In addition, cellular glucose consumption was measured as well as Glut-1 protein and activation of GSK-3β/mTOR signal were determined. The expression of Type I IFN receptor was detected in cultured human astrocytes. 2?IU/ml IFNα2a and IFNα2b significantly decreased the proliferation of human astrocytes respectively, compared to control. IFNβ had no significant effect on the proliferation of the cells. The phosphorylation of JNK stimulated by all IFNs detected was more pronounced and sustained than ERK1/2 and IκB. No effects were observed on the activation of p38MAPK and Stat3. Moreover, Treatment with IFNα, especially with IFNα2b, decreased glucose consumption and stimulated phosphorylation of GSK-3β and mTOR, but decreased the expression of Glut-1. In contrast, IFNβ had no significant effect on either glucose consumption or activation of GSK-3β/mTOR signals. INFα2b significantly decreased the levels of IL-8 whereas the levels of GM-CSF were increased. The present study demonstrates direct inhibitory effects of IFNα on cell proliferation, cell signaling and glucose utilization in human astrocytes.     

Interferons and interferon inhibitory activity in disease and therapy

Interferon (IFN) resistance is an important factor in the pathophysiology of neoplastic disorders, certain viral infections (e.g., AIDS), and autoimmune diseases (e.g., lupus erythematosus and Wegner's granulomatosis). In addition, in some of these disorders, there is also decreased ability to produce IFNs. The capacity of viruses and neoplastic processes to interfere with the IFN system are thought to represent a "virus-against-host" or "cancer-against-host" defense mechanism. Four resistance factors have been identified: 1) release of free IFN-alpha/beta type 1 receptors into the circulation that, at appropriate concentrations, capture and inactivate IFNs; 2) a new IFN inhibitory protein has been isolated and its chemical structure is under study; 3) prostaglandin E2, which is produced by certain tumor cells, inhibits IFN production; and 4) high levels of cAMP phosphodiesterases present, for example in certain tumor cells, reduces cAMP, an important second messenger in IFN synthesis. Studies are under way to reverse these inhibitory effects and to increase endogenous interferon production.     

Influence of retinoic acid on protein synthesis and transport of l-methionine in cultured L cells

The effects of retinoic acid (RA) on cell proliferation, activity of acid phosphatase, protein synthesis and methionine uptake were studied in transformed murine LPA cells. Early inhibition of protein synthesis was demonstrated under experimental conditions in which the rate of cell proliferation was diminished and non-specific effects of vitamin action could be excluded. Measurements of l-methionine uptake revealed a decrease to approximately one-half of that in control cultures after treatment with RA at the concentrations of 5 × 10^?5 M and 10^?5 M.     

Selective inhibitory effect of Hu-IFN-gamma on the agarose clonability of tumor-derived lymphoid cell lines

Recombinant human interferon alpha (IFN-alpha) and interferon gamma (IFN-gamma) were compared for their ability to influence the proliferative capacity of tumor-derived cell lines and of normal B lymphocytes infected in vitro by Epstein-Barr virus (EBV). EBV-induced B-cell proliferation was suppressed almost completely when 10(2) U/ml IFN-alpha were added to the culture medium while the same dose of IFN-gamma had significantly lower inhibitory activity. The pure IFNs differed in their ability to influence the growth of three Burkitt lymphoma-derived cell lines, Raji, Daudi, and Namalwa, depending on whether the cells were propagated in suspension or in semisolid cultures. IFN-alpha inhibited cell proliferation under both culture conditions with thresholds of sensitivity characteristics for each cell line. In contrast, IFN-gamma had no effect on the growth in suspension but it abolished the clonogenic potential of tumor cell lines in semisolid agarose. The results suggest that the two IFN types may exert their growth inhibitory activity through different mechanisms of action.     

Stimulation of primary human endothelial cell proliferation by IFN

The IFN family of cytokines has pleiotropic roles in immunity and development. In this study, we provide evidence that IFN can stimulate the proliferation of primary human endothelial cells. This is in contrast to the growth-suppressive effects of IFN observed on transformed human cells, thereby underscoring the distinctive responses of primary human cells. The growth-stimulatory effect of IFN was determined by an increase in DNA synthesis assessed with [(3)H]thymidine incorporation, an increase in G(2) and M cell cycle phases assessed with flow cytometric analysis, and an increase in cell number. Distinct cell types, including primary human fibroblast and smooth muscle cells, were also growth stimulated by IFN. Neutralizing Abs to IFN were used to demonstrate the growth response was mediated specifically by the IFN cytokine. The signaling pathway of type I IFNs activates STAT1 and STAT2. In primary endothelial cells, we demonstrate that STAT3 and STAT5 are also activated, and these STATs may contribute to cellular proliferation. To evaluate possible effectors of positive growth, DNA microarray analyses were performed to assess gene induction in response to IFN. These results reveal changes in the RNA levels of genes in endothelial cells that encode proteins involved in cellular proliferation.     

Activation of the p38 mitogen-activated protein kinase mediates the suppressive effects of type I interferons and transforming growth factor-beta on normal hematopoiesis.

Type I interferons (IFNs) are potent regulators of normal hematopoiesis in vitro and in vivo, but the mechanisms by which they suppress hematopoietic progenitor cell growth and differentiation are not known. In the present study we provide evidence that IFN alpha and IFN beta induce phosphorylation of the p38 mitogen-activated protein (Map) kinase in CD34+-derived primitive human hematopoietic progenitors. Such type I IFN-inducible phosphorylation of p38 results in activation of the catalytic domain of the kinase and sequential activation of the MAPK-activated protein kinase-2 (MapKapK-2 kinase), indicating the existence of a signaling cascade, activated downstream of p38 in hematopoietic progenitors. Our data indicate that activation of this signaling cascade by the type I IFN receptor is essential for the generation of the suppressive effects of type I IFNs on normal hematopoiesis. This is shown by studies demonstrating that pharmacological inhibitors of p38 reverse the growth inhibitory effects of IFN alpha and IFN beta on myeloid (colony-forming granulocytic-macrophage) and erythroid (burst-forming unit-erythroid) progenitor colony formation. In a similar manner, transforming growth factor beta, which also exhibits inhibitory effects on normal hematopoiesis, activates p38 and MapKapK-2 in human hematopoietic progenitors, whereas pharmacological inhibitors of p38 reverse its suppressive activities on both myeloid and erythroid colony formation. In further studies, we demonstrate that the primary mechanism by which the p38 Map kinase pathway mediates hematopoietic suppression is regulation of cell cycle progression and is unrelated to induction of apoptosis. Altogether, these findings establish that the p38 Map kinase pathway is a common effector for type I IFN and transforming growth factor beta signaling in human hematopoietic progenitors and plays a critical role in the induction of the suppressive effects of these cytokines on normal hematopoiesis.     

Multi-step neoplastic transformation of normal human fibroblasts by Co-60 gamma rays and Ha-ras oncogenes

As reported previously (Namba et al., 1985; Namba, 1985), normal human fibroblasts were transformed into immortal cells with abnormal karyotypes by Co-60 gamma-ray irradiation. These immortally transformed cells (KMST-6) showed no clonability in soft agar and were not tumorigenic. However, by treatment with Ha-ras oncogenes derived from a human lung carcinoma or Harvey murine sarcoma virus, the KMST-6 cells acquired elevated clonability in soft agar and transplantability in nude mice. All the tumors produced grew progressively without showing regression and killed the mice. The tumors were also serially transplantable into other mice. The Ha-ras oncogene alone did not convert normal human fibroblasts into either immortal or tumorigenic cells. Our current data suggest that gamma rays worked as an initiator of carcinogenesis in normal human cells, giving rise to chromosome aberrations and immortality, and the Ha-ras oncogene played a role in the progression of the immortally transformed cell population to a neoplastic one showing enhanced colony formation in soft agar and tumorigenicity in nude mice.     

(-)-Epigallocatechin-3-gallate (EGCG) sensitizes melanoma cells to interferon induced growth inhibition in a mouse model of human melanoma

Melanoma incidences have increased over the last few decades and metastatic melanoma is one of the hardest malignancies to treat. Thus, novel approaches are needed for an effective management of melanoma. Interferon-α2b (IFN), an immunomodulatory cytokine commonly used in melanoma treatment, has shown marginal efficacy and often results in discontinuation of therapy due to toxicity. We earlier demonstrated that epigallocatechin-3-gallate (EGCG), the major polyphenolic constituent of green tea, caused cell cycle arrest and apoptosis of human melanoma cells via modulation in cki-cyclin-cdk machinery and Bcl-2 family proteins. This study was undertaken to determine if EGCG could enhance the anti-proliferative effects of IFN. In this study, we demonstrated that EGCG and/or IFN treatments to melanoma cells resulted in a marked i) decrease in cell proliferation and colony formation ability, and ii) induction of apoptosis. Interestingly, the combination was found to be more effective than either of the agents alone. Further, the anti-proliferative effects of EGCG and/or IFN were accompanied with an increase in FAS protein levels and a decrease in nuclear factor NF-κB/p65 in the nucleus as well as NF-κB promoter activity. EGCG and/or IFN also resulted in an increase in FAS-L mediated apoptosis. Further, EGCG and/or IFN treatments resulted in a decrease in melanoma tumor growth and protein levels of proliferation marker PCNA, in athymic nude mice implanted with melanoma tumors. The combination of the two modalities demonstrated a better response than either of them alone. Our data suggest that EGCG could impart therapeutic advantage if used in conjunction with IFN.     

PSMA7 inhibits the tumorigenicity of A549 human lung adenocarcinoma cells

The gene for proteasome subunit alpha type-7 (PSMA7) is located in chromosomal 20q13.33, a region frequently amplified in tumor. In this study, we employed A549 human lung adenocarcinoma cells and showed that PSMA7 inhibits the proliferation, tumorigenicity and invasion of A549 cells in vitro. Moreover, both gain and loss of function studies demonstrated that PSMA7 modulates the tumorigenicity of A549 cells in a xenograft nude mice model. In conclusion, these results identify inhibitory effects associated with PSMA7 that affect the tumorigenicity of A549 cells, suggesting PSMA7 as a potential tumor biomarker.     

Different requirements for the cytostatic and apoptotic effects of type I interferons. Induction of apoptosis requires ARF but not p53 in osteosarcoma cell lines

The regulation of cell growth is one of the most important effects of type I interferons (IFNs). This response may involve a cytostatic effect or the induction of apoptosis depending on the cell context. Often the growth-inhibitory response of type I IFNs is studied in tumor cell lines carrying mutations of tumor suppressor genes, and therefore, the growth-inhibitory effect can be influenced by inactivation of these important regulators of cell proliferation. In this report, we explored the role of the ARF-p53 pathway in the growth-inhibitory effect of type I IFNs. We found that p53 is only induced in cells that express p14(ARF) (p19(ARF) in mouse cells). Surprisingly, mouse embryonal fibroblasts that are null for p19(ARF) or P53, even after transformation with oncogenic RAS, respond as well as wild type to the growth-inhibitory effect of type I IFNs. Similarly, human ARF(-/-) U2OS and P53(-/-) SAOS-2 cells show a significant decrease in cell proliferation. However, only SAOS-2 or U2OS reconstituted with inducible p14(ARF) undergo apoptosis in response to IFN beta treatment, and this effect was not inhibited by expression of dominant negative p53. These data suggest that (i) at least in specific cell types, the induction of apoptosis by type I IFNs requires an ARF pathway that is p53-independent and (ii) the cytostatic and pro-apoptotic effects of type I IFNs employ different pathways.     

Sensitivity of human germ-cell-tumor cell lines to human interferons

We examined the sensitivity of four human germ-cell-tumor cell lines exhibiting different stages of differentiation to human interferons (IFNs) in vitro. The cell lines were derived from two embryonal carcinomas (NEC 8 and NEC 14), a choriocarcinoma (IMa), and a yolk-sac tumor (HUOT). Treatment with poly I:C induced IFN production in IMa and HUOT cells, but not in NEC-8 and NEC-14 cells. In the two embryonal-carcinoma cell lines, the addition of IFN-alpha, IFN-beta, and IFN-gamma did not prevent infection by vesicular stomatitis virus and encephalomyocarditis virus. Also, in these two lines, 2-5A synthetase was not induced by the addition of IFN-alpha. In contrast, both IMa and HUOT showed sensitivity to the antiviral action of IFN-alpha and IFN-beta against the two viruses, and 2-5A synthetase was induced by IFN-alpha. IFNs added at doses of up to 1000 IU/ml had no antiproliferative effect on NEC 8, NEC 14, and HUOT, whereas colony formation by IMa cells was greatly suppressed by all three forms of IFN. These results indicate that the production of and sensitivity to IFN are developmentally regulated and are related to the level of differentiation of human germ-cell stem cells.     

Regulation of apoptosis by type III interferons

Abstract. Objective: Two types of interferons (IFNs), type I (IFN‐α/β) and type III (IFN‐λs), utilize distinct receptor complexes to induce similar signalling and biological activities, including recently demonstrated for IFN‐λs antitumour activity. However, ability of type III IFNs to regulate cell population growth remains largely uncharacterized.Materials and methods: Intact and modified human colorectal adenocarcinoma HT29 cells were used to study regulation of apoptosis by IFN‐λs. Results and Conclusions: We report that the IFN‐λR1 chain of the type III IFN receptor complex possesses an intrinsic ability to trigger apoptosis in cells. Signalling induced through the intracellular domain of IFN‐λR1 resulted in G₁/G₀ phase cell cycle arrest, phosphatidylserine surfacing and chromosomal DNA fragmentation. Caspase‐3, caspase‐8 and caspase‐9 were activated; however, pancaspase inhibitor Z‐VAD‐FMK did not prevent apoptosis. In addition, the extent of apoptosis correlated with the level of receptor expression and was associated with prolonged IFN‐λ signalling. We also demonstrated that the ability to trigger apoptosis is a unique intrinsic function of all IFN receptors. However, more robust apoptosis was induced by signalling through type III IFN receptor than through type I or type II (IFN‐γ) receptors, suggesting higher cytotoxic potential of type III IFNs. In addition, we observed that IFN‐γ treatment sensitized HT29 cells to IFN‐λ‐mediated apoptosis. These results provide evidence that type III IFNs, alone or in combination with other stimuli, have the potential to induce apoptosis.     

The interferons and their receptors--distribution and regulation

The interferons (IFNs) were originally described over 50 years ago, identified by their ability to confer viral resistance to cells. We now know that they are much more than just anti-viral cytokines collectively having roles in both innate and adaptive immune responses, in tumor surveillance and defense, and modulation of immune cell function. Three types of IFN have now been described, simply referred to as type I, II and III. Distinguishable by the unique receptors that they rely on for signal transduction, the three types of IFN have specific and varied roles in the maintenance of human health and defense against pathogens. In mounting an IFN-mediated immune response, the human body has developed the ability to regulate IFN-mediated signal transduction. Like all cytokines, the ability of a cell to respond to IFN is completely dependent on the presence of its cognate receptor on the surface of the target cell. Thus, one of the major mechanisms used by the human body to regulate the strength and duration of the IFN response is through regulation of receptor levels, thereby altering the cytokine-specific responsiveness of the target cell. This review will discuss the receptor system utilized by the type I IFNs and compare it with that of the type II and III IFNs, which also regulate immune responses through controlling receptor level on the cell surface.     

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).     

Apoptosis and interferons: role of interferon-stimulated genes as mediators of apoptosis

IFNs are a family of cytokines with pleiotropic biological effects mediated by scores of responsive genes. IFNs were the first human proteins to be effective in cancer therapy and were among the first recombinant DNA products to be used clinically. Both quality and quantity of life has been improved in response to IFNs in various malignancies. Despite its beneficial effects, unraveling the mechanisms of the anti-tumor effects of IFN has proven to be a complex task. IFNs may mediate anti-tumor effects either indirectly by modulating immunomodulatory and anti-angiogenic responses or by directly affecting proliferation or cellular differentiation of tumor cells. Both direct or indirect effects of IFNs result from induction of a subset of genes, called IFN stimulated genes (ISGs). In addition to the ISGs implicated in anti-viral, anti-angiogenic, immunomodulatory and cell cycle inhibitory effects, oligonucleotide microarray studies have identified ISGs with apoptotic functions. These include TNF- related apoptosis inducing ligand (TRAIL/Apo2L), Fas/FasL, XIAP associated factor-1 (XAF-1), caspase-4, caspase-8, dsRNA activated protein kinase (PKR), 2'5'A oligoadenylate synthetase (OAS), death activating protein kinases (DAP kinase), phospholipid scramblase, galectin 9, IFN regulatory factors (IRFs), promyelocytic leukemia gene (PML) and regulators of IFN induced death (RIDs). In vitro IFN-, IFN- and IFN- induced apoptosis in multiple cell lines of varied histologies. This review will emphasize possible mechanisms and the role of ISGs involved in mediating apoptotic function of IFNs.     

Signaling pathways regulating TC21-induced tumorigenesis

TC21(R-Ras2), a Ras-related GTPase with transforming potential similar to H-, K- and N-Ras, is implicated in the pathogenesis of human cancers. Transforming growth factor beta (TGF-beta), a cytokine that plays a significant role in modulating tumorigenesis, normally prevents uncontrolled cell proliferation but paradoxically induces proliferation in H-Ras-transformed cancer cells. Although TC21 activates some pathways that mediate cellular transformation by the classical Ras proteins, the mechanisms through which TC21 induces tumor formation and how TGF-beta regulates TC21 transformed cells is not known. To better understand the role of TC21 in cancer progression, we overexpressed an activated G23V mutant of TC21 in a nontumorigenic murine mammary epithelial (EpH4) cell line. Mutant TC21-expressing cells were significantly more oncogenic than cells expressing activated G12V H-Ras both in vivo and in vitro. TC21-induced transformation and proliferation required activation of p38 MAPK, mTOR (the mammalian target of rapamycin), and phosphoinositide 3-kinase but not Akt/PKB. Transformation by TC21 rendered EpH4 cells insensitive to the growth inhibitory effects of TGF-beta, and the soft agar growth of these cells was increased upon TGF-beta stimulation. Despite losing responsiveness to TGF-beta-mediated growth inhibition, both Smad-dependent and independent pathways remained intact in TC21-transformed cells. Thus, overexpression of active TC21 in EpH4 cells induces tumorigenicity through the phosphoinositide 3-kinase, p38 MAPK, and mTOR pathways, and these cells lose their sensitivity to the normal growth inhibitory role of TGF-beta.     

Differential induction of the 200-family proteins in Daudi Burkitt's lymphoma cells by interferon-alpha

Interferon (IFN)-inducible "effector" proteins mediate the biological activities of the IFNs. Therefore, the identification of the functional role(s) of IFN inducible proteins in IFN action is important to elucidate the molecular mechanisms by which IFNs inhibit cell growth. One family (the "200-family") of IFN-inducible proteins includes structurally related murine (p202a, p202b, p203, p204 and D3) and human (MNDA, IFI-16 and AIM2) proteins. However, their role in IFN action remains to be established. Here we report that IFN-alpha treatment of Daudi Burkitt's lymphoma cells resulted in differential induction of MNDA, IFI 16, and a p202-related protein (p202RP). Interestingly, IFN induction of p202RP preceded the induction of MNDA and IFI 16 proteins and the growth inhibition by IFN. Additionally, the induction of these proteins by IFN was accompanied by: (i) a transient increase in p21(WAF1/CIP1) levels; (ii) an increase in the functional form of pRb and p130; (iii) an inhibition of the sequence-specific DNA binding activity of E2F complexes; and (iv) a marked decrease in c-Myc levels. Our observations reported herein provide support to the hypothesis that IFN-inducible p202RP and MNDA proteins from the 200-family contribute to the growth inhibitory activities of the IFNs.