A novel gene constitutively expressed in human lymphoid cells is inducible with interferon-γ in myeloid cells

A cluster of at lest six interferon- (IFN)-inducible genes designated Ifi201-204 and located on mouse chromosome 1 has recently been described. Here , we report a human IFN--inducible gene, IFI 16, which has nucleotide sequence similarity with portions of two of the mouse genes, Ifi202 and Ifi204. A full-length cDNA clone derived from IFI 16 [2.709 kilobases (kb)] contained a single open reading frame of 2.187 kb which encoded a putative polypeptide of 729 amino acids and a predicted non-glycosylated M _r of 80020. IFI 16 mRNA was found to be constitutively expressed in lymphoid cells and in cell lines of both the T and B lineages. By contrast, the mRNA was not expresed by the cell lines HL-60, U937, and K562, which represent early stages of myeloid development, but was strongly inducible in HL-60 and U937 with IFN-. The IFI 16 protein demonstrated a putative domain structure with patchy similarity to the proteins expressed from gene Ifi202 and Ifi204. The mouse and human proteins each contain two analogous 200 amino acid domains which are imperfect copies, but IFI 16 demonstrated additional unique regions, including a Lys-rich N-terminal portion and a spacer region between the reiterated domains, analogous to spacer regions in the CD5 and CD8 molecules. Using a panel of inter-species somatic cell hybrid cell lines, IFI 16 was localized to the chromosomal region 1q121qter, a region systenic between mouse an man. DNA blotting indicated that, in contrast to the mouse, IFI 16 is present as a single copy gene in the human genome.The authors are pleased to make the cDNA clones described in this paper available to interested investigators.The nucleotide sequence data reported in this paper have been submitted to the Genbank database and have been assigned the accession number M63838. Correspondence to: J. A. Trapani.     

A novel anti-proliferative role of HMGA2?in induction of apoptosis through caspase 2?in primary human fibroblast cells

The HMGA2 (high-mobility group AT-hook) protein has previously been shown as an oncoprotein, whereas ectopic expression of HMGA2 is found to induce growth arrest in primary cells. The precise mechanisms underlying this phenomenon remain to be unravelled. In the present study, we determined that HMGA2 was able to induce apoptosis in WI38 primary human cells. We show that WI38 cells expressing high level of HMGA2 were arrested at G2/M phase and exhibited apoptotic nuclear phenotypes. Meanwhile, the cleaved caspase 3 (cysteine aspartic acid-specific protease 3) was detected 8 days after HMGA2 overexpression. Flow cytometric analysis confirmed that the ratio of cells undergoing apoptosis increased dramatically. Concurrently, other major apoptotic markers were also detected, including the up-regulation of p53, Bax and cleaved caspase 9, down-regulation of Bcl-2; as well as release of cytochrome c from the mitochondria. We further demonstrate that the shRNA (small-hairpin RNA)-mediated Apaf1 (apoptotic protease activating factor 1) silencing partially rescued the HMGA2-induced apoptosis, which was accompanied by the decrease of cleaved caspase-3 level and a decline of cell death ratio. Our results also reveal that γH2A was accumulated in nuclei during the HMGA2-induced apoptosis along with the up-regulation of cleaved caspase 2, suggesting that the HMGA2-induced apoptosis was dependent on the pathway of DNA damage. Overall, the present study unravelled a novel function of HMGA2 in induction of apoptosis in human primary cell lines, and provided clues for clarification of the mechanistic action of HMGA2 in addition to its function as an oncoprotein.     

A putative role for platelet-derived PPARγ in vascular homeostasis demonstrated by anti-PPARγ induction of bleeding, thrombocytopenia and compensatory megakaryocytopoiesis

Widely known for its role in adipogenesis and energy metabolism, PPARγ also plays a role in platelet function. To further understand functions of platelet-derived PPARγ, we produced rabbit polyclonal (PoAbs) and mouse monoclonal (MoAbs) antibodies against PPARγ 14mer/19mer peptide-immunogens. Unexpectedly, our work produced two key findings. First, MoAbs but not PoAbs produced against PPARγ peptide-immunogens displayed antigenic crossreactivity with highly conserved PPARα and PPARβ/δ. Similarly, Santa Cruz PoAb sc-7196 was monospecific for PPARγ while MoAb sc-7273 crossreacted with PPARα and PPARβ/δ. Second, immunized rabbits and mice exhibited unusual pathology including cachexia, excessive bleeding, and low platelet counts leading to thrombocytopenia. Spleens from immunized mice were fatty, hemorrhagic and friable. Although passive administration of anti-PPARγ PoAbs failed to induce experimental thrombocytopenia, megakaryocytopoiesis was induced 4-8-fold in mouse spleens. Similarly, marrow megakaryocytopoiesis was enhanced 1.8-4-fold in immunized rabbits. These peptide-immunogens are 100% conserved in human, rabbit and mouse; thus, immune-mediated platelet destruction via crossreactivity with platelet-derived PPARγ likely caused bleeding, thrombocytopenia, and compensatory megakaryocytopoiesis. Such overt pathology would cause significant problems for large-scale production of anti-PPARγ PoAbs. Furthermore, a major pitfall associated with MoAb production against closely related molecules is that monoclonicity does not guarantee monospecificity, an issue worth further scientific scrutiny.     

Cutting edge: the role of IFN-α receptor and MyD88 signaling in induction of IL-15 expression in vivo

IL-15 plays a multifaceted role in immune homeostasis, but the unreliability of IL-15 detection has stymied exploration of IL-15 regulation in vivo. To visualize IL-15 expression, we created a transgenic mouse expressing emerald-GFP (EmGFP) under IL-15 promoter control. EmGFP/IL-15 was prevalent in innate cells including dendritic cells (DCs), macrophages, and monocytes. However, DC subsets expressed varying levels of EmGFP/IL-15 with CD8(+) DCs constitutively expressing EmGFP/IL-15 and CD8(-) DCs expressing low EmGFP/IL-15 levels. Virus infection resulted in IL-15 upregulation in both subsets. By crossing the transgenic mice to mice deficient in specific elements of innate signaling, we found a cell-intrinsic dependency of DCs and Ly6C(+) monocytes on IFN-α receptor expression for EmGFP/IL-15 upregulation after vesicular stomatitis virus infection. In contrast, myeloid cells did not require the expression of MyD88 to upregulate EmGFP/IL-15 expression. These findings provide evidence of previously unappreciated regulation of IL-15 expression in myeloid lineages during homeostasis and following infection.     

IDO induction in IFN-γ activated astroglia: A role in improving cell viability during oxidative stress

Abstract

In the central nervous system (CNS), astrocytes play an integral role in the maintenance of neuronal viability and function. Inflammation within the CNS increases the concentration of oxidative metabolites and, therefore, the potential for NAD depletion through increased poly-(ADP-ribose) polymerase (PARP) activity. However, the activity of indoleamine 2,3-dioxygenase (IDO), the rate limiting enzyme for de novo NAD synthesis, is also markedly increased in astrocytes during inflammation. This study investigated the role of IDO induction in the maintenance of intracellular NAD and its relationship to improved cell viability under conditions of increased oxidative stress in the human astroglioma cell line, HTB-138. Treatment with the pro-inflammatory cytokine IFN-γ increased IDO activity in these cells. Intracellular NAD levels also increased significantly after treatment with IFN-γ in the presence of a PARP inhibitor. Pretreatment of astroglial cells with IFN-γ significantly moderated both the drop in intracellular NAD concentration and cell death following exposure to hydrogen peroxide. These results suggest that induction of IDO and subsequent de novo NAD synthesis may contribute to the maintenance of intracellular NAD levels and cell viability under conditions of increased oxidative stress.     

Patterns of constitutive and IFN-γ inducible expression of HLA class II molecules in human melanoma cell lines

Major histocompatibility complex (MHC) class II proteins (HLA-DR, HLA-DP and HLA-DQ) play a fundamental role in the regulation of the immune response. The level of expression of human leukocyte antigen (HLA) class II antigens is regulated by interferon-gamma (IFN-gamma) and depends on the status of class II trans-activator protein (CIITA), a co-activator of the MHC class II gene promoter. In this study, we measured levels of constitutive and IFN-gamma-induced expression of MHC class II molecules, analysed the expression of CIITA and investigated the association between MHC class II transactivator polymorphism and expression of different MHC class II molecules in a large panel of melanoma cell lines obtained from the European Searchable Tumour Cell Line Database. Many cell lines showed no constitutive expression of HLA-DP, HLA-DQ and HLA-DR and no IFN-gamma-induced increase in HLA class II surface expression. However, in some cases, IFN-gamma treatment led to enhanced surface expression of HLA-DP and HLA-DR. HLA-DQ was less frequently expressed under basal conditions and was less frequently induced by IFN-gamma. In these melanoma cell lines, constitutive surface expression of HLA-DR and HLA-DP was higher than that of HLA-DQ. In addition, high constitutive level of cell surface expression of HLA-DR was correlated with lower inducibility of this expression by IFN-gamma. Finally, substitution A-->G in the 5' flanking region of CIITA promoter type III was associated with higher expression of constitutive HLA-DR (p<0.005). This study yielded a panel of melanoma cell lines with different patterns of constitutive and IFN-gamma-induced expression of HLA class II that can be used in future studies of the mechanisms of regulation of HLA class II expression.     

Expression of recombinant human interferon-γ with antiviral activity in the bi-cistronic baculovirus-insect/larval system

A bi-cistronic baculovirus-insect/larval system containing a polyhedron promoter, an internal ribosome entry site (IRES), and an egfp gene was developed as a cost-effective platform for the production of recombinant human interferon gamma (rhIFN-γ). There was no significant difference between the amounts of rhIFN-γ produced in the baculovirus-infected Spodoptera frugiferda 21 cells grown in serum-free medium and the serum-supplemented medium, while the Trichoplusia ni (T. ni) and Spodoptera exigua (S. exigua) larvae afforded rhIFN-γ amounting to 1.08±0.04 and 9.74±0.35 μg/mg protein respectively. The presence of non-glycosylated and glycosylated rhIFN-γ was confirmed by immunoblot and lectin blot. The immunological activity of purified rhIFN-γ, with 96% purity by Nickel (II)-nitrilotriacetic acid (Ni-NTA) affinity chromatography, was similar to that commercially available. Moreover, the rhIFN-γ protein from T. ni had more potent antiviral activity. These findings suggest that this IRES-based expression system is a simple and inexpensive alternative for large-scale protein production in anti-viral research.     

Performance of serum-supplemented and serum-free media in IFNγ Elispot Assays for human T cells

The choice of serum for supplementation of media for T cell assays and in particular, Elispot has been a major challenge for assay performance, standardization, optimization, and reproducibility. The Assay Working Group of the Cancer Vaccine Consortium (CVC-CRI) has recently identified the choice of serum to be the leading cause for variability and suboptimal performance in large international Elispot proficiency panels. Therefore, a serum task force was initiated to compare the performance of commercially available serum-free media to laboratories’ own medium/serum combinations. The objective of this project was to investigate whether a serum-free medium exists that performs as well as lab-own serum/media combinations with regard to antigen-specific responses and background reactivity in Elispot. In this way, a straightforward solution could be provided to address the serum challenge. Eleven laboratories tested peripheral blood mononuclear cells (PBMC) from four donors for their reactivity against two peptide pools, following their own Standard Operating Procedure (SOP). Each laboratory performed five simultaneous experiments with the same SOP, the only difference between the experiments was the medium used. The five media were lab-own serum-supplemented medium, AIM-V, CTL, Optmizer, and X-Vivo. The serum task force results demonstrate compellingly that serum-free media perform as well as qualified medium/serum combinations, independent of the applied SOP. Recovery and viability of cells are largely unaffected by serum-free conditions even after overnight resting. Furthermore, one serum-free medium was identified that appears to enhance antigen-specific IFNγ-secretion.     

Regulatory role of KEAP1 and NRF2 in PPARγ expression and chemoresistance in human non-small-cell lung carcinoma cells

The nuclear factor-E2-related factor 2 (NRF2) serves as a master regulator in cellular defense against oxidative stress and chemical detoxification. However, persistent activation of NRF2 resulting from mutations in NRF2 and/or downregulation of or mutations in its suppressor, Kelch-like ECH-associated protein 1 (KEAP1), is associated with tumorigenicity and chemoresistance of non-small-cell lung carcinomas (NSCLCs). Thus, inhibiting the NRF2-mediated adaptive antioxidant response is widely considered a promising strategy to prevent tumor growth and reverse chemoresistance in NSCLCs. Unexpectedly, stable knockdown of KEAP1 by lentiviral shRNA sensitized three independent NSCLC cell lines (A549, HTB-178, and HTB-182) to multiple chemotherapeutic agents, including arsenic trioxide (As(2)O(3)), etoposide, and doxorubicin, despite moderately increased NRF2 levels. In lung adenocarcinoma epithelial A549 cells, silencing of KEAP1 augmented the expression of peroxisome proliferator-activated receptor γ (PPARγ) and genes associated with cell differentiation, including E-cadherin and gelsolin. In addition, KEAP1-knockdown A549 cells displayed attenuated expression of the proto-oncogene cyclin D1 and markers for cancer stem cells (CSCs) and reduced nonadherent sphere formation. Moreover, deficiency of KEAP1 led to elevated induction of PPARγ in response to As(2)O(3). Pretreatment of A549 cells with PPARγ agonists activated PPARγ and augmented the cytotoxicity of As(2)O(3). A mathematical model was formulated to advance a hypothesis that differential regulation of PPARγ and detoxification enzymes by KEAP1 and NRF2 may underpin the observed landscape changes in chemosensitivity. Collectively, suppression of KEAP1 expression in human NSCLC cells resulted in sensitization to chemotherapeutic agents, which may be attributed to activation of PPARγ and subsequent alterations in cell differentiation and CSC abundance.     

Roles of fascin in human carcinoma motility and signaling: prospects for a novel biomarker?

Fascin is a globular actin cross-linking protein that has a major function in forming parallel actin bundles in cell protrusions that are key specialisations of the plasma membrane for environmental guidance and cell migration. Fascin is widely expressed in mesenchymal tissues and the nervous system and is low or absent in adult epithelia. Recent data from a number of laboratories have highlighted that fascin is up-regulated in many human carcinomas and, in individual tissues, correlates with the clinical aggressiveness of tumours and poor patient survival. In cell culture, over-expression or depletion of fascin modulates cell migration and alters cytoskeletal organisation. The identification of biomarkers to provide more effective early diagnosis of potentially aggressive tumours, or identify tumours susceptible to targeted therapies, is an important goal in clinical research. Here, we discuss the evidence that fascin is upregulated in carcinomas, its contributions to carcinoma cell behaviour and its potential as a candidate novel biomarker or therapeutic target.     

Functional genomics of PPAR-γ in human immunomodulatory cells

Keeping in view the fact that peroxisome-proliferators activated receptors-PPARs (α,γ) play a crucial role in atherogenic inflammation, the present study was addressed to explore as to how selective and specific PPAR-γ gene silencing within human mononuclear cells affects genes involved in lipid metabolism and innate immune process. Such a study revealed that with respect to control cells, the PPAR-γ knock-out cells exhibited significant reduction in the expression of genes coding for PPAR- α, CD-36, LDL-R as well as significant increase in the expression of genes coding for IL-4, IL-8, IFN-γ, CX3CR1, hTERT. However, the expression of genes coding for LXR-α and Receptor-C _k could not be detected in PPAR-γ knock-out cells. Based on these results, we propose that PPAR-γ gene has the inherent capacity to influence the lipid mediated inflammation process in atherosclerotic lesions.     

Synergy between TLR3 and IL-18 promotes IFN-γ dependent TRAIL expression in human liver NK cells

Natural killer (NK) cells are a component of innate immunity against viral infections through their rapid cytotoxic activity and cytokine production. However, intra-hepatic NK cells’ ability to respond to virus is still mostly unknown. Our results show that the synthetic dsRNA polyinosinic–polycytidylic acid (poly I:C), a mimic of a common product of viral infections, activates NK cells directly in the context of cytokines found in the liver, i.e.: poly I:C plus inflammatory cytokines (IL-18, IL-12, and IL-2) induced NK cell IFN-γ production and TRAIL expression, and anti-inflammatory cytokines (TGF-β and IL-10) inhibit NK cell IFN-γ production. Neutralization of IFN-γ blocks poly I:C plus inflammatory cytokines-induced NK cell TRAIL expression, suggesting that IFN-γ is an autocrine differentiation factor for these cells. A better understanding of the intra-hepatic NK cell activation against viral infection may help in the design of therapies and vaccines for the control of viral hepatitis.