Interferon (IFN) exhibits a potent antiviral activity in vitro and plays a major role in the early defense against viruses. Like IFN, the proinflammatory chemokine, interleukin (IL)-8, is induced by viruses and appears in circulation during viral infections. In an in vitro cytopathic effect assay for IFN, we found that IL-8 can inhibit IFN-α activity in a dose-dependent manner. This action was reversed by specific monoclonal antibodies to IL-8. The chemokine was able to attenuate the IFN-mediated inhibition of viral replication as determined by measuring infectious virus yield. IL-8 also diminished the ability of IFN to inhibit an early stage of viral replication since IL-8 attenuated the inhibition of the formation of viral proteins. It appeared that IL-8 interfered with a late rather than an early step of IFN-mediated pathway such as early gene expression. The IL-8 inhibitory action on IFN-α antiviral activity was associated with reduced 2′,5′-A oligoadenylate synthetase activity, a pathway well correlative with the anti– encephalomyocarditis virus action of IFN-α. Understanding pathways that antagonize IFN action may lead to novel approaches to potentiate endogenous and therapeutic IFN.

While production of engineered carbon nanotubes (CNTs) has escalated in recent years, knowledge of risk associated with exposure to these materials remains unclear. We report on the cytotoxicity of four CNT variants in human lung epithelial cells (A549) and murine macrophages (J774). Morphology, metal content, aggregation/agglomeration state, pore volume, surface area and modifications were determined for the pristine and oxidized single-walled (SW) and multi-walled (MW) CNTs. Cytotoxicity was evaluated by cellular ATP content, BrdU incorporation, lactate dehydrogenase (LDH) release, and CellTiter-Blue (CTB) reduction assays. All CNTs were more cytotoxic than respirable TiO2 and SiO2 reference particles. Oxidation of CNTs removed most metallic impurities but introduced surface polar functionalities. Although slopes of fold changes for cytotoxicity endpoints were steeper with J774 compared to A549 cells, CNT cytotoxicity ranking in both cell types was assay-dependent. Based on CTB reduction and BrdU incorporation, the cytotoxicity of the polar oxidized CNTs was higher compared to the pristine CNTs. In contrast, pristine CNTs were more cytotoxic than oxidized CNTs when assessed for cellular ATP and LDH. Correlation analyses between CNTs' physico-chemical properties and average relative potency revealed the impact of metal content and surface area on the potency values estimated using ATP and LDH assays, while surface polarity affected the potency values estimated from CTB and BrdU assays. We show that in order to reliably estimate the risk posed by these materials, in vitro toxicity assessment of CNTs should be conducted with well characterized materials, in multiple cellular models using several cytotoxicity assays that report on distinct cellular processes.

(1) and mRNA turnover regulation by the AU-rich elements in the 3Ј-untranslated regions (2). The IFN antiviral response is a highly regulated process involving the transient inhibition of viral and cellular protein synthesis. The translational inhibition is mediated predominately by the activity of PKR, the double-stranded RNAdependent protein kinase. PKR is a serine/threonine protein kinase of 65 and 68 kDa in murine and human cells, respectively, that is induced by IFN treatment of cells and phosphorylates itself and other proteins, notably eIF2␣, in response to viral double-stranded RNA (3). DsRNA is produced as the replicative intermediates of many RNA viruses and also by annealing of complementary RNA strands transcribed from some DNA viruses (3). Phosphorylated eIF2␣ sequesters the guanine nucleotide exchange factor, eIF2B, which becomes trapped as inactive complex with GDP resulting in translational arrest (4, 5).Among the principal effectors of the IFN-induced antiviral state are the 2Ј,5Ј-oligoadenylate (2-5A) synthetases that convert ATP to 2-5A, activators of RNase L, in response to viral double-stranded RNA (6, 7). Thus, 2-5A is an alarmone that alerts the cells to the presence of virus by signaling to RNase L. Both RNase L and PKR have been implicated in the action of IFN-␣ against a variety of viruses (reviewed in Refs. 8 and 9). RNase L is widely distributed in different tissues, and it has been suggested that low levels of 2-5A lead to RNase L-mediated selective degradation of viral mRNA (10), whereas higher levels may lead to broader effects such as cleavage of 18 S and 28 S ribosomal RNAs (11). During the course of experiments on the role of RNase L in the inhibition of viral protein synthesis during acute infections, we observed that an absence of RNase L led to selective stabilization of PKR mRNA, extended kinetics of eIF2␣ phosphorylation, and potent inhibition of viral protein synthesis. Our findings suggest that RNase L truncates and limits the induction of PKR, possibly contributing to the transient nature of the IFN response against viral infections. Cell Culture, Viral Infections, and IFN Treatments-RNase L ϩ/ϩ and RNase L Ϫ/Ϫ mouse embryonic fibroblast (MEF) cell lines were of mixed or C57BL/6 genetic backgrounds. The cell lines are post-crisis derivatives of primary MEFs as described previously (12). MEFs were cultured in DMEM with high glucose supplemented with 10% FBS and antibiotics (Invitrogen, Gaithersburg, MD). Bone marrow macrophages collected from the femurs of RNase L ϩ/ϩ and RNase L Ϫ/Ϫ mice, both on a background of C57BL/6, were cultured in L-cell-conditioned medium for 8 days and plated at a density of 10 6 cells per 10-cm plate. WISH cell line (HeLa markers) was obtained from the American Type Culture Collection (ATCC, Rockville, MD) and cultured in RPMI 1640 supplemented with 10% FBS and antibiotics. EXPERIMENTAL PROCEDURES