Interferon-induced Expression Research Articles

An intronic LINE-1 regulates IFNAR1 expression in human immune cells

BackgroundDespite their origins as selfish parasitic sequences, some transposons in the human genome have been co-opted to serve as regulatory elements, contributing to the evolution of transcriptional networks. Most well-characterized examples of transposon-derived regulatory elements derive from endogenous retroviruses (ERVs), due to the intrinsic regulatory activity of proviral long terminal repeat regions. However, one subclass of transposable elements, the Long Interspersed Nuclear Elements (LINEs), have been largely overlooked in the search for functional regulatory transposons, and considered to be broadly epigenetically repressed.ResultsWe examined the chromatin state of LINEs by analyzing epigenomic data from human immune cells. Many LINEs are marked by the repressive H3K9me3 modification, but a subset exhibits evidence of enhancer activity in human immune cells despite also showing evidence of epigenetic repression. We hypothesized that these competing forces of repressive and activating epigenetic marks might lead to inducible enhancer activity. We investigated a specific L1M2a element located within the first intron of Interferon Alpha/Beta Receptor 1 (IFNAR1). This element shows epigenetic signatures of B cell-specific enhancer activity, despite being repressed by the Human Silencing Hub (HUSH) complex. CRISPR deletion of the element in B lymphoblastoid cells revealed that the element acts as an enhancer that regulates both steady state and interferon-inducible expression of IFNAR1.ConclusionsOur study experimentally demonstrates that an L1M2a element was co-opted to function as an interferon-inducible enhancer of IFNAR1, creating a feedback loop wherein IFNAR1 is transcriptionally upregulated by interferon signaling. This finding suggests that other LINEs may exhibit cryptic cell type-specific or context-dependent enhancer activity. LINEs have received less attention than ERVs in the effort to understand the contribution of transposons to the regulatory landscape of cellular genomes, but these are likely important, lineage-specific players in the rapid evolution of immune system regulatory networks and deserve further study.

Abstract 1370: Targeting LSD1 rescues MHC-I antigen presentation in small cell lung cancer

Abstract Purpose: Small cell lung cancer (SCLC) is a highly aggressive cancer with early primary resistance and modest clinical benefit to immune checkpoint blockade (ICB). Mutation or transcriptional repression of the major histocompatibility complex class I (MHC-I) is a key mechanism driving resistance to T cell-based therapies. Lysine Demethylase 1 (LSD1) regulates gene expression through modulating mono- and di- methylated lysine 4 and 9 of histone H3. LSD1 is a therapeutic target of interests in SCLC due to its oncogenic requirement for tumor growth, and there has been growing evidence pointing to LSD1 as a repressor of tumor-intrinsic immunogenicity. Here investigated the role of LSD1 as a transcriptional regulator of antigen presentation in SCLC. Method: To perturb LSD1 function, we employed the pharmacological inhibitor ORY-1001 and shRNA-mediated knockdown. We then assessed changes in MHC-I expression on SCLC cell lines by flow cytometry and western blot. To analyze transcriptional changes following LSD1 inhibition, we performed RNA-seq on SCLC cells pre- and post-treatment with ORY-1001. To observe for antigen-specific T cell killing, we engineered SCLC cells to express endogenous antigens and co-cultured these cells with primary cognate CD8+ T cells. Finally, we treated genetically engineered mouse model (GEMM)-derived tumors on immunocompetent syngeneic mice with ORY-1001 with or without anti-PD-L1 blockade to assess for anti-tumor effects. Results: We discovered a significant and strong negative correlation between expression of LSD1 and genes encoding the MHC-I antigen presentation pathway. Inhibition of LSD1 induces expression of MHC-I genes and restores expression of genes encoding the antigen presentation machinery. Perturbation of LSD1 further activates interferon signaling and interferon-inducible expression of NLRC5, a well-characterized transcriptional activator of MHC-I genes. We further showed that targeting LSD1 sensitizes SCLC cells to MHC-I-restricted T cell-dependent cytolysis and enhances ICB efficacy in refractory SCLC tumors. Conclusion: Our data define a role for LSD1 as a potent negative regulator of MHC-I-mediated antigen presentation and provide rationale for the use of LSD1 inhibitors to augment response to immunotherapy in SCLC. Citation Format: Minh N. Nguyen, Hirokazu Taniguchi, Andrew Chow, Yingqian A. Zhan, Triparna Sen, Charles M. Rudin. Targeting LSD1 rescues MHC-I antigen presentation in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1370.

Targeting the Bromodomain of BRG-1/BRM Subunit of the SWI/SNF Complex Increases the Anticancer Activity of Temozolomide in Glioblastoma.

Glioblastoma (GBM) is a deadly and incurable brain cancer with limited therapeutic options. PFI-3 is a small-molecule bromodomain (BRD) inhibitor of the BRM/BRG1 subunits of the SWI/SNF chromatin remodeling complex. The objective of this study is to determine the efficacy of PFI-3 as a potential GBM therapy. We report that PFI-3 binds to these BRDs when expressed in GBM cells. PFI-3 markedly enhanced the antiproliferative and cell death-inducing effects of temozolomide (TMZ) in TMZ-sensitive GBM cells as well as overcame the chemoresistance of highly TMZ-resistant GBM cells. PFI-3 also altered gene expression in GBM and enhanced the basal and interferon-induced expression of a subset of interferon-responsive genes. Besides the effects of PFI-3 on GBM cells in vitro, we found that PFI-3 markedly potentiated the anticancer effect of TMZ in an intracranial GBM animal model, resulting in a marked increase in survival of animals bearing GBM tumors. Taken together, we identified the BRG1 and BRM subunits of SWI/SNF as novel targets in GBM and revealed the therapeutic potential of applying small molecule inhibitors of SWI/SNF to improve the clinical outcome in GBM using standard-of-care chemotherapy.

Tissue-specific and interferon-inducible expression of nonfunctional ACE2 through endogenous retroelement co-option.

Angiotensin-converting enzyme 2 (ACE2) is an entry receptor for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and a regulator of several physiological processes. ACE2 has recently been proposed to be interferon-inducible, suggesting that SARS-CoV-2 may exploit this phenomenon to enhance viral spread and questioning the efficacy of interferon treatment in Coronavirus disease 2019 (COVID-19). Using a recent de novo transcript assembly that captured previously unannotated transcripts, we describe a novel isoform of ACE2, generated by co-option of intronic retroelements as promoter and alternative exon. The novel transcript, termed MIRb-ACE2, exhibits specific expression patterns across the aerodigestive and gastrointestinal tracts and is highly responsive to interferon stimulation. In stark contrast, canonical ACE2 expression is unresponsive to interferon stimulation. Moreover, the MIRb-ACE2 translation product is a truncated, unstable ACE2 form, lacking domains required for SARS-CoV-2 binding and is therefore unlikely to contribute to or enhance viral infection.

Acute Kidney Injury in Children after Hematopoietic Cell Transplantation Is Associated with Elevated Urine CXCL10 and CXCL9.

Acute kidney injury (AKI) is nearly universally associated with worse outcomes, especially among children after hematopoietic stem cell transplant (HCT). Our objective was to examine urinary immune biomarkers of AKI after HCT to provide insights into novel mechanisms of kidney injury in this population. Studying patients undergoing allogeneic HCT provides a unique opportunity to examine immune markers of AKI because the risk of AKI is high and the immune system newly develops after transplant. Children (>2 years old) and young adults undergoing their first allogeneic HCT and enrolled in a prospective, observational cohort study at 2 large children's hospitals had urine collected pre-HCT and monthly for the first 4 months after HCT. Urine samples at each monthly time point were assayed for 8 immune-related biomarkers. AKI was defined as a 1.5-fold increase in the monthly serum creatinine value, which was recorded ±1 day from when the research urine sample was obtained, as compared with the pre-HCT baseline. Generalized estimating equation regression analysis evaluated the association between the monthly repeated measures (urinary biomarkers and AKI). A total of 176 patients were included from 2 pediatric centers. Thirty-six patients from 1 center were analyzed as a discovery cohort and the remaining 140 patients from the second center were analyzed as a validation cohort. AKI rates were 18% to 35% depending on the monthly time point after HCT. Urine CXCL10 and CXCL9 concentrations were significantly higher among children who developed AKI compared with children who did not (P < .01) in both cohorts. In order to gain a better understanding of the cellular source for these biomarkers in the urine, we also analyzed in vitro expression of CXCL10 and CXCL9 in kidney cell lines after stimulation with interferon-γ and interferon-α. HEK293-epithelial kidney cells demonstrated interferon-induced expression of CXCL10 and CXCL9, suggesting a potential mechanism driving the key finding. CXCL10 and CXCL9 are associated with AKI after HCT and are therefore promising biomarkers to guide improved diagnostic and treatment strategies for AKI in this high-risk population.

Genome-wide, CRISPR-Cas9-based genetic screening for regulators of interferon-induced PD-L1 expression in AML cells.

116 Background: Expression of PD-L1 in AML cells is induced by pro-inflammatory signals, but how the induction is regulated is incompletely known. Genome-wide screening using the CRISPR-Cas9 technology could provide a strategy to interrogate the regulators of PD-L1 expression. Methods: A lentiviral CRISPR-Cas9 library, containing 3 sgRNA targeting each of 19,050 human genes (Nat Methods 2014, 11:783), was used to transduce THP-1 AML cells. Transduced cells were treated with IFN-γ and stained with an anti-PD-L1 antibody. Two subsets of cells were isolated by cell sorting according to their levels of PD-L1 expression. One subset consisted of cells that showed the median 5% of PD-L1 expression and the other subset, the lowest 5%. sgRNA integrated in the sorted subsets were amplified and their frequencies quantified by deep sequencing. Results: As expected, sgRNA targeting the PD-L1 gene itself were highly enriched in the subset that expressed a low level of PD-L1. sgRNA that targeted IFNGR2, β-chain of the IFN-γ receptor, were also enriched in this subset, whereas those for IFNGR1 were not. sgRNA targeting both JAK1 and JAK2 were enriched. By contrast, those for JAK3 and TYK2 were not. Enrichment was seen for sgRNA that targeted STAT1, a transcriptional activator downstream of JAK1/2, whereas sgRNA targeting the other STAT activators (STAT-2, 3, 4, 5A, 5B and 6) showed no enrichment. sgRNA targeting several serine kinases that regulate STAT1 phosphorylation (PKCδ, PKCε, PKCα, CAMKII, ERK, JNK, and CDK8) were not enriched. Nor were sgRNA targeting several phosphatases that modulate STAT1 activity (TC-PTP, PTPN1, SHP1 and SHP2). sgRNA targeting IRF1 (IFN regulatory transcription factor 1) were highly enriched, whereas sgRNA for IRF-2 to IRF-9 were not. In each of the affected genes, at least 2 out of the 3 sgRNA targeting the gene were enriched, indicating a generality of the findings. Conclusions: A CRISPR-Cas9-based screening strategy provided an unbiased interrogation of genes essential for PD-L1 expression on AML cells. It confirmed the importance of the IFNGR2-JAK1/2-STAT1-IRF1 axis, which could serve as targets for the development of drugs to suppress the ability of AML cells to evade host immunity.

Interferon Inducible IFI16 Expression in p16 Positive Squamous Cell Carcinoma of the Oropharynx

Human-papillomavirus- (HPV-) positive oropharyngeal squamous cell carcinomas (OPSCC) are reported to be more responsive to treatment and to be related to a favorable prognosis compared with non-HPV carcinomas. However, the molecular basis of the responsiveness is unclear. Interferon inducible IFI16, which is implicated in the control of cell growth, apoptosis, angiogenesis, and immunomodulation in various types of cancers, is reported to be frequently expressed in the HPV-positive head and neck SCC and to correlate with a better prognosis. In this study, we hypothesized that HPV related OPSCC expresses IFI16 resulting in favorable prognosis. To clarify the relationship between the prognosis of HPV related OPSCC patients and IFI16 status, we examined immunohistologically the pretreatment specimens of OPSCC for the expression of p16 as a surrogate marker of HPV infection and IFI16. We could not show that the expression of IFI16 is associated with that of p16. There was no significant difference in the survival rate between IFI16 positive and negative groups. Patients with p16 negative tumor exhibited worse survival rate regardless of IFI16 status. In this limited case series, we could not conclude that IFI16 expression is altered in p16 positive OPSCC and that it would be a new predictive marker or a useful therapeutic tool.

Essential Role for Stat5a/b in Myeloproliferative Neoplasms Induced by BCR-ABL1 and Jak2 V617F.

Abstract 312The Signal Transducer and Activator of Transcription 5 (STAT5a/b) proteins are latent transcription factors that are constitutively activated in malignant cells from patients with a variety of hematologic malignancies, including the myeloproliferative neoplasms (MPNs) chronic myeloid leukemia (CML) and polycythemia vera (PV), but the role of STAT5a/b in the pathogenesis of these diseases has not been fully elucidated. While therapy with tyrosine kinase inhibitors like imatinib mesylate has revolutionized the treatment of CML, the emergence of clinical resistance to imatinib has rekindled interest in STAT5a/b as a promising therapeutic target downstream of dysregulated tyrosine kinases such as BCR-ABL1 and Jak2V617F.In this study, we used mice with a conditional (floxed, fl) null mutation in the Stat5a/b gene locus (Cui et al., Mol. Cell. Biol. 2004;24:8037) to determine the requirement for Stat5a/b in MPNs induced by BCR-ABL1 and Jak2V617F in retroviral bone marrow (BM) transduction/transplantation models of CML and PV, respectively. Two distinct strategies were used to express Cre recombinase in the mutant hematopoietic cells: retroviral expression of a GFP-Cre fusion protein, and interferon-inducible expression from an Mx-Cre transgene.In recipients of BCR-ABL1-transduced BM, loss of one Stat5a/b allele resulted in attenuation of CML-like MPN and the appearance of acute B-lymphoblastic leukemia (B-ALL). Complete deletion of the Stat5a/b gene resulted in the absence of any myeloid or lymphoid leukemia in recipients, with normal spleen weight and organ histopathology. However, these recipients had significant populations of GFP+ myeloid cells in peripheral blood and marrow with expression of BCR-ABL1 and phosphorylation of CrkL, indicative of engraftment with BCR-ABL1+ stem cells. Serial transplantation revealed that the BCR-ABL1-expressing Stat5a/b-deficient HSC can self-renew and progress to acute lymphoid leukemia in secondary recipients. These results demonstrate that Stat5a/b signaling is required for BCR-ABL1 to induce the massive expansion of myeloid progenitors that is characteristic of CML. However, our results also argue that inhibition of Stat5a/b signaling may be insufficient for eradication of leukemic stem cells in CML patients, or for preventing progression to blast crisis.To address the role of Stat5a/b in the pathophysiology of PV, we utilized a similar model of PV induced by retroviral transduction of Jak2V617F into mouse BM, followed by transplantation into recipient mice. Such recipients develop nonfatal polycythemia, reticulocytosis, and leukocytosis, and eventually progress to myelofibrosis (Zaleskas et al., PLoS One 2006;1:e18). Recipients of Stat5a/bfl/− donor transduced with Jak2V617F retrovirus that exhibited efficient Cre-mediated Stat5a/b deletion failed to develop polycythemia despite engraftment with transduced donor-derived stem cells and evidence of circulating GFP+ cells. Importantly, recipients of Jak2V617F-transduced Stat5a/bfl/− BM who engrafted with Jak2V617F+ cells had evidence of subclinical MPN, with infiltration of spleen and liver with myeloerythroid cells and substantial myelofibrosis in the BM. These results suggest that inhibition of Stat5a/b signaling in PV patients may normalize their red cell mass, but might be less effective at preventing progression to myelofibrosis.Together, these results demonstrate that Stat5a/b is necessary for the pathogenesis of CML-like disease induced by BCR-ABL1 and of polycythemia by Jak2V617F, and validate the Stat5a/b pathway as a target for therapy in MPNs associated with dysregulated TKs. However, targeting Stat5 alone may not eradicate CML stem cells or prevent Jak2V617F-induced myelofibrosis. Disclosures:No relevant conflicts of interest to declare.

Interferon-Induced Expression of MxA in the Respiratory Tract of Rhesus Macaques Is Suppressed by Influenza Virus Replication

To determine the relationship between influenza A virus replication and innate antiviral immune responses, rhesus monkeys were given oseltamivir before influenza A/Memphis/7/01 (H1N1) challenge. We found that oseltamivir treatment significantly reduced viral replication in the trachea (p < 0.029). Further, in the trachea of both treated and untreated monkeys the mRNA levels of most innate antiviral molecules in the IFN-alphabeta pathway were dramatically increased by 24 h postinfection. However, the mRNA level of a single IFN-stimulated gene, MxA (myxovirus resistance A), the IFN-stimulated gene known to be critical in blocking influenza virus replication, was significantly lower in the tracheal lavages of untreated monkeys than in the oseltamivir-treated monkeys (p = 0.05). These results demonstrate for the first time that uncontrolled influenza A virus replication actively suppresses MxA gene expression and emphasize the critical role of innate immunity in controlling influenza virus replication in vivo.

Interferon-inducible IFI16 protein in human cancers and autoimmune diseases

Interferon-inducible IFI16 protein (encoded by IFI16 gene located at 1q21 region) is a member of the p200-protein family. The family includes structurally and functionally-related mouse (for example, p202, p203, and p204 proteins) and human (for example, MNDA, AIM2, and IFIX) proteins. Increased expression of p200-family proteins in a variety of cells is known to inhibit cell cycle progression and modulate cell survival. Consistent with this role of p200-family proteins, increased expression of IFI16 protein in normal human diploid fibroblasts and prostate epithelial cells is associated with cellular senescence-associated permanent cell growth arrest. Furthermore, reduced or loss of IFI16 expression in cells is associated with the development of certain cancers, such as breast and prostate cancer. Interestingly, recent studies have provided evidence that the constitutive and interferon-induced expression of the IFI16 gene varies among individuals and may depend on the race. These studies raise the possibility that alterations (increases or decreases) in the expression of IFI16 protein may contribute to the development of human diseases. In this review, we discuss how our understanding of the regulation of IFI16 expression and its role in cell growth regulation will help elucidate the molecular mechanisms that contribute to the development of various human diseases.

The interferon-induced expression of APOBEC3G in human blood–brain barrier exerts a potent intrinsic immunity to block HIV-1 entry to central nervous system

In the human genome, the APOBEC3 gene has expanded into a tandem array of genes termed APOBEC3A-H. Several members of this family have potent anti-HIV-1 activity. Here we demonstrate that APOBEC-3B/3C/3F and -3G are expressed in all major cellular components of the CNS. Moreover, we show that both interferon-α (IFN-α) and IFN-γ significantly enhance the expression of APOBEC-3G/3F and drastically inhibit HIV-1 replication in primary human brain microvascular endothelial cells (BMVECs), the major component of blood–brain barrier (BBB). As the viral inhibition can be neutralized by APOBEC3G-specific siRNA, APOBEC3G plays a key role to mediate the anti-HIV-1 activity of IFN-α and/or IFN-γ. Our findings suggest that, in addition to the restriction at viral entry level, the restriction from APOBEC3 family could account for the low-level replication of HIV-1 in BMVECs. The manipulation of IFN-APOBEC3 signaling pathway could be a potent therapeutic strategy to prevent HIV invasion to central nervous system (CNS).

Bone morphogenetic protein-7 and interferon-alpha synergistically suppress hepatitis C virus replicon

Various cytokines contribute to control hepatitis C virus (HCV) viral replication. HCV subgenomic replicon systems have been developed, and cell-cycle-dependent replication has been reported. But the molecules involved in this processes is not totally elucidated. The aim of this study is to investigate the involvement of the bone morphogenetic protein (BMP)-7, a member of TGF-β superfamily, to the in vitro HCV replication. BMP-7 dose-dependently suppressed the replication and protein expression from the HCV replicon in Huh7/Rep-Feo cells and was associated with cell-cycle arrest at the G1 phase. These results were consistent with the effect of TGF-β in a previous study. Combination of BMP-7 and interferon-alpha showed a synergic decrease of HCV replication, and was more effective compared to the treatment with interferon-alpha alone. This synergistic effect was also present in HCV-JFH1 virus cell culture. While BMP-7 alone did not stimulate expression of the interferon-stimulated genes (ISGs), it augmented interferon-induced expression of the ISGs independently of the interferon-induced Jak/STAT pathway. Taken together, BMP-7 may constitute a novel molecule to suppress HCV replication.

Sulindac, a nonsteroidal anti-inflammatory drug, selectively inhibits interferon-γ-induced expression of the chemokine CXCL9 gene in mouse macrophages

Sulindac, a non-steroidal anti-inflammatory drug, has been shown to exert an anti-tumor effect on several types of cancer. To determine the effect of sulindac on intracellular signaling pathways in host immune cells such as macrophages, we investigated the effect of the drug on interferon gamma (IFNγ)-induced expression of signal transducer and activator of transcription 1 (STAT1) and other genes in mouse macrophage-like cell line RAW264.7 cells. Sulindac, but not aspirin or sodium salicylate, inhibited IFNγ-induced expression of the CXC ligand 9 (CXCL9) mRNA, a chemokine for activated T cells, whereas the interferon-induced expression of CXCL10 or IFN regulatory factor-1 was not affected by sulindac. Luciferase reporter assay demonstrated that sulindac inhibited IFNγ-induced promoter activity of the CXCL9 gene. Surprisingly, sulindac had no inhibitory effect on IFNγ-induced STAT1 activation; however, constitutive nuclear factor κB activity was suppressed by the drug. These results indicate that sulindac selectively inhibited IFNγ-inducible gene expression without inhibiting STAT1 activation.

Critical role of the transcription factor AP-1 for the constitutive and interferon-induced expression of IFI 16.

IFI 16 is a member of the HIN-200 family of transcriptional regulators that suppress cell growth, modulate the cell cycle and have been linked to cellular differentiation. We hypothesized that the activity of IFI 16 depends on its level of expression and therefore studied the transcriptional activity of the IFI 16 promoter. A discrete sequence within the 5' untranslated region was required for constitutive activity of the promoter and the functional motif within this region was shown to be a consensus AP-1 site. Interestingly, this AP-1 site was also critical for IFN-induced activation of the promoter and consistent with these observations, treatment of cells with IFNgamma resulted in a rapid and robust induction of AP-1 activity that preceded expression of IFI 16. These experiments define the transcriptional mechanisms of IFI 16 gene regulation and provide evidence suggesting that AP-1 activation may be an important event in IFN signaling.

A highly efficient ligand-regulated Cre recombinase mouse line shows that LoxP recombination is position dependent.

Conditional gene inactivation using the Cre/loxP system is widely used, but the difficulty in properly regulating Cre expression remains one of the bottlenecks. One approach to regulate Cre activity utilizes a mutant estrogen hormone-binding domain (ERT) to keep Cre inactive unless the non-steroidal estrogen analog 4-hydroxytamoxifen (OHT) is present. Here we describe a mouse strain expressing Cre-ERT from the ubiquitously expressed ROSA26 (R26) locus. We demonstrate efficient temporal and spatial regulation of Cre recombination in vivo and in primary cells derived from these mice. We show the existence of marked differences in recombination frequencies between different substrates within the same cell. This has important consequences when concurrent switching of multiple alleles within the same cell is needed, and highlights one of the difficulties that may be encountered when using reporter mice as indicator strains.

Ethanol inhibits prolactin- and tumor necrosis factor-?-, but not gamma interferon-induced expression of intercellular adhesion molecule-1 in human astrocytoma cells

In humans, alcohol consumption has multiple effects on the immune system. Despite an increase in our understanding of the effects of alcohol on the immune system, little is known about the effect of alcohol on the neuroimmune response. In the central nervous system (CNS), astrocytes and microglial function as immune effector cells. In response to infection of injury, astrocytes increase in number and size, express several proinflammatory cytokines, MHC class I and II antigens, and several adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1). Interactions between ICAM-1 and its counter-receptors play an important role in the regulation of neuroimmune response. In this study, cultured human astrocytoma cells were used to examine the effect of ethanol on ICAM-1 expression. Western blot analyses show that quiescent astrocytes express, at least, four immunoreactive ICAM-1 proteins with apparent molecular weights 55, 67, 82, and 90 kDa. Incubation of human astrocytoma cells with tumor necrosis factor-alpha (TNF-alpha) or prolactin (PRL) resulted in marked increases in all four immunoreactive ICAM-1 proteins. In the presence of ethanol, however, PRL- and TNF-alpha-induced increases in all four immunoreactive ICAM-1 proteins were markedly inhibited. ICAM-1 is a cell surface transmembrane glycoprotein. Using a cell surface specific ICAM-1 adhesion assay we found that in human astrocytoma cells TNF-alpha, interferongamma (IFN-gamma) and PRL increased cell surface ICAM-1 expression. Consistent with our Western blot analyses, ethanol significantly inhibited TNF-alpha- and PRL-induced cell surface ICAM-1 expression. By contrast, IFN-gamma-induced ICAM-1 expression was not inhibited by exposure of the cells to ethanol. Expression of ICAM-1 is regulated predominantly at the transcriptional level. In the present report, we show that TNF-alpha increased ICAM-1 mRNA levels in human astrocytoma cells and that ethanol markedly blocked TNF-alpha-induced increases in ICAM-1 mRNA levels. Further, we found that PRL-induced ICAM-1 expression was, at least in part, due to a PRL-induced increase in TNF-alpha syntheses and secretion. Our results clearly indicate that ethanol has a pronounced effect on ICAM-1 expression in human astrocytoma cells, thus suggesting that ETOH exposure may impair the immune response in the CNS by blocking leukocytes adhesion and migration into the CNS in response to injury or infection.

Ethanol inhibits prolactin- and tumor necrosis factor-α-, but not gamma interferon-induced expression of intercellular adhesion molecule-1 in human astrocytoma cellsPresented in part at the 79th Annual Meeting of the Endocrine Society, Minneapolis, Minnesota.

In humans, alcohol consumption has multiple effects on the immune system. Despite an increase in our understanding of the effects of alcohol on the immune system, little is known about the effect of alcohol on the neuroimmune response. In the central nervous system (CNS), astrocytes and microglial function as immune effector cells. In response to infection of injury, astrocytes increase in number and size, express several proinflammatory cytokines, MHC class I and II antigens, and several adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1). Interactions between ICAM-1 and its counter-receptors play an important role in the regulation of neuroimmune response. In this study, cultured human astrocytoma cells were used to examine the effect of ethanol on ICAM-1 expression. Western blot analyses show that quiescent astrocytes express, at least, four immunoreactive ICAM-1 proteins with apparent molecular weights 55, 67, 82, and 90 kDa. Incubation of human astrocytoma cells with tumor necrosis factor-α (TNF-α) or prolactin (PRL) resulted in marked increases in all four immunoreactive ICAM-1 proteins. In the presence of ethanol, however, PRL- and TNF-α-induced increases in all four immunoreactive ICAM-1 proteins were markedly inhibited. ICAM-1 is a cell surface transmembrane glycoprotein. Using a cell surface specific ICAM-1 adhesion assay we found that in human astrocytoma cells TNF-α, interferonγ (IFN-γ) and PRL increased cell surface ICAM-1 expression. Consistent with our Western blot analyses, ethanol significantly inhibited TNF-α- and PRL-induced cell surface ICAM-1 expression. By contrast, IFN-γ-induced ICAM-1 expression was not inhibited by exposure of the cells to ethanol. Expression of ICAM-1 is regulated predominantly at the transcriptional level. In the present report, we show that TNF-α increased ICAM-1 mRNA levels in human astrocytoma cells and that ethanol markedly blocked TNF-α-induced increases in ICAM-1 mRNA levels. Further, we found that PRL-induced ICAM-1 expression was, at least in part, due to a PRL-induced increase in TNF-α syntheses and secretion. Our results clearly indicate that ethanol has a pronounced effect on ICAM-1 expression in human astrocytoma cells, thus suggesting that ETOH exposure may impair the immune response in the CNS by blocking leukocytes adhesion and migration into the CNS in response to injury or infection. J. Cell. Biochem. 77:455–464, 2000. © 2000 Wiley-Liss, Inc.

Identification of the murine Mx2 gene: interferon-induced expression of the Mx2 protein from the feral mouse gene confers resistance to vesicular stomatitis virus.

The mouse genome contains two related interferon-regulated genes, Mx1 and Mx2. Whereas Mx1 codes for the nuclear 72-kDa protein that interferes with influenza virus replication after interferon treatment, the Mx2 gene is nonfunctional in all laboratory mouse strains examined, since its open reading frame (ORF) is interrupted by an insertional mutation and a subsequent frameshift mutation. In the present study, we demonstrate that Mx2 mRNA of cells from feral mouse strains NJL (Mus musculus musculus) and SPR (Mus spretus) differs from that of the laboratory mouse strains tested. The Mx2 mRNA of the feral strains contains a single long ORF consisting of 656 amino acids. We further show that Mx2 protein in the feral strains is expressed upon interferon treatment and localizes to the cytoplasm much like the rat Mx2 protein, which inhibits vesicular stomatitis virus replication. Furthermore, transfected 3T3 cell lines of laboratory mouse origin expressing Mx2 from feral strains acquire slight resistance to vesicular stomatitis virus.

Globotriaosyl ceramide modulates interferon-alpha-induced growth inhibition and CD19 expression in Burkitt's lymphoma cells.

Previous studies have indicated that globotriaosyl ceramide (Gb3 or CD77) plays a role in alpha-interferon signal transduction and CD19-mediated homotypic adhesion in B cell lines derived from Burkitt's lymphoma. These roles for Gb3 may involve the proteins IFNAR-1 (subunit 1 of the interferon-alpha receptor) and CD19, respectively, both of which have potential Gb3-binding sites in their extracellular domains which resemble those of the verotoxin (Shiga toxin and Shiga-like toxin) B subunit. The majority of this work was performed using wild-type Daudi cells and a single, Gb3-deficient mutant cell line, VT500. In the present investigations, these and additional Daudi-derived cells with varying degrees of sensitivity to interferon-alpha were examined for Gb3 expression, interferon-induced growth inhibition and CD19 expression. The degree of interferon-induced growth inhibition and CD19 expression correlated with Gb3 expression in the various cell lines tested. In addition, reconstitution of the VT500 cell line with Gb3 but not other glycolipids partially restored the sensitivity of cells to IFN-induced growth inhibition. The degree to which reconstitution restored sensitivity to growth inhibition was similar to the results of previous studies in which Gb3 reconstitution restored sensitivity to verotoxin-induced cytotoxicity. These results demonstrate that Gb3 is specifically required for IFN-induced growth inhibition in Daudi cells and provide further evidence of a role for Gb3 in CD19 expression and function in these cells.

The p69/71 2-5A synthetase promoter contains multiple regulatory elements required for interferon-alpha-induced expression.

The p69/71 2-5A synthetase is an interferon-inducible enzyme that polymerizes ATP to form 2'-5'-linked oligoadenylates when activated by double-stranded RNA. A genomic clone was isolated that contained 12.5 kb of the 5'-flanking region and the first exon of the p69/71 2-5A synthetase gene. The major transcriptional start site was mapped to an A residue located 84 bp upstream of the translational start site within a sequence that matches both a consensus ISRE and an Inr element. Sequencing of the region 972 bp upstream of the translation start site revealed 4 imperfect direct repeats of 70 to 80 bp that contain several putative regulatory elements. This region does not have a TATA or CAAT box but does contain two IRF-1-like elements, an Ets-1 motif, an AP-1 site, an Sp1 binding site, an NF-kappaB-binding site, and a palindrome containing two overlapping NF-IL-6 consensus motifs in opposite orientation. The region -480 to -850 bp contains PuF, UBP-1, and PEA 3 motifs and another NF-IL-6 motif adjacent to an E2A-binding site. The 5'-flanking sequence binds proteins in DNase I foot printing assays and is a functional interferon-inducible promoter that requires multiple elements for maximal constitutive and interferon-inducible expression.