Structure of the carbohydrate moiety of human interferon-beta secreted by a recombinant Chinese hamster ovary cell line

The carbohydrate structure of the major oligosaccharide of human interferon-beta (IFN-beta) synthesized by a genetically engineered Chinese hamster ovary cell line has been determined. Analysis of the glycopeptidase F-released carbohydrates by sequential exoglycosidase treatment, methylation analysis, and fast atom bombardment-mass spectrometry revealed that 95% of the IFN-beta oligosaccharides had the following structure: (Formula: see text). The remaining 5% of the carbohydrates are probably tri- or higher antennary oligosaccharide chains. The major oligosaccharide of the recombinant IFN-beta is remarkably homogeneous with respect to terminal galactose sialylation. NeuAc, which is alpha 2-3-linked to galactose in the human IFN-beta secreted by Chinese hamster ovary cells, can be re-incorporated with an alpha 2-6 linkage in vitro, into enzymatically desialylated IFN-beta using rat liver Gal beta 1-4GlcNAc alpha 2-6 sialyltransferase. The sugar chain is important for maintaining protein solubility as shown by the fact that IFN-beta protein precipitates after deglycosylation with glycopeptidase F.     

Interferon-beta (INF-beta) antibodies in interferon-beta1a- and interferon-beta1b-treated multiple sclerosis patients. Prevalence, kinetics, cross-reactivity, and factors enhancing interferon-beta immunogenicity in vivo

We analysed the role of dosage, route and frequency of administration of clinical grade interferon-beta (IFN-beta) preparations in inducing anti-IFN-beta antibodies (IFN-beta-Abs) in 5 groups of relapsing-remitting multiple sclerosis (RRMS) patients who were respectively treated as follows: 1) weekly intramuscular (i.m.) injections of 30 mg of recombinant IFN-beta1a (Avonex), 2) subcutis (s.c.) injections of 250 mg IFN-beta1b (Betaferon) every other day, 3) weekly i.m. injections of 250 mg IFN-beta1b (Betaferon), 4) s.c. injections of 22 mg of IFN-beta1a (Rebif) three times a week, and 5) i.m. injections of 22 mg of IFN-beta1a (Rebif) twice a week. IFN-beta-Abs were determined by ELISA. IFN-beta1b was more immunogenic than IFN-beta1a not only when administered s.c. every other day, but also when administered i.m. at a lower weekly dose; i.m. injection, however, significantly delayed the appearance, and induced lower serum levels of IFN-beta-Abs. In patients treated with s.c. IFN-beta1b, Ab levels peaked 3 to 9 months after therapy initiation, and then slowly, but progressively, declined to pre-therapy levels that in some patients were reached after three years. Patients treated with i.m. or s.c. IFN-beta1a only rarely developed IFN-beta-Abs, and then at very low titers. Overall, the i.m. weekly administration of IFN-beta1a was the less immunogenic treatment. In IFN-beta1b-treated patients, a wash-out period of two/three months was sufficient to bring the IFN-beta-Ab levels below the cut-off. Our findings suggest that the immunogenicity of IFN-beta1a is low, regardless of the route of administration and the dosage, while that of IFN-beta1b is high, and is significantly, but not completely reduced by i.m. administration. As IFN-beta-Abs are cross-reactive, a wash-out period is suggested when the preparation is changed from IFN-beta1b to IFN-beta1a in order to maintain the clinical benefits of the therapy.     

Coupling mitochondrial respiratory chain to cell death: an essential role of mitochondrial complex I in the interferon-beta and retinoic acid-induced cancer cell death

Combination of retinoic acids (RAs) and interferons (IFNs) has synergistic apoptotic effects and is used in cancer treatment. However, the underlying mechanisms remain unknown. Here, we demonstrate that mitochondrial respiratory chain (MRC) plays an essential role in the IFN-beta/RA-induced cancer cell death. We found that IFN-beta/RA upregulates the expression of MRC complex subunits. Mitochondrial-nuclear translocation of these subunits was not observed, but overproduction of reactive oxygen species (ROS), which causes loss of mitochondrial function, was detected upon IFN-beta/RA treatment. Knockdown of GRIM-19 (gene associated with retinoid-interferon-induced mortality-19) and NDUFS3 (NADH dehydrogenase (ubiquinone) Fe-S protein 3), two subunits of MRC complex I, by siRNA in two cancer cell lines conferred resistance to IFN-beta/RA-induced apoptosis and reduced ROS production. In parallel, expression of late genes induced by IFN-beta/RA that are directly involved in growth inhibition and cell death was also repressed in the knockdown cells. Our data suggest that the MRC regulates IFN-beta/RA-induced cell death by modulating ROS production and late gene expression.     

Cytokines and adhesion molecules in multiple sclerosis patients treated with interferon-beta1b

Multiple sclerosis (MS), an inflammatory, demyelinating disease of the central nervous system (CNS), is thought to be caused by a T cell-mediated attack on CNS myelin and axons. Recombinant interferon (IFN)-beta is an established treatment of multiple sclerosis, and is known to reduce the number of disease relapses and the development of irreversible symptoms and signs of disease. The mechanism of action of IFN-beta treatment is, however, not completely understood. Previous studies have suggested major effects on mononuclear cell cytokine production and T cell migration, but results have been inconsistent. We found decreases in CD4 and CD8 T cell expression of the CD49d/VLA-4 molecule, increases in plasma concentrations of soluble vascular cell adhesion molecule (sVCAM-1), and increases in plasma concentrations of tumor necrosis factor and interleukin (IL)-12 p40 chain in patients with MS who were initiated on de novo treatment with IFN-beta1b. We found only minor associations between the different changes induced by IFN-beta1b-treatment. Our findings are consistent with changes in T cell expression of CD49d/VLA-4 and induction of sVCAM-1 as important effects of treatment with IFN-beta1b in multiple sclerosis, whereas the role of changes in TNF and IL-12 p40 chain concentrations is more difficult to interpret.     

IFN-beta differentially regulates CD40-induced cytokine secretion by human dendritic cells

We have previously shown that IFN-beta, a key cytokine associated with the early phase of the innate host defense, can prevent the generation of human Th1 cells. Specifically, we demonstrated that IFN-beta prevents the in vitro monocyte-derived mature dendritic cell (DC)-dependent differentiation of naive Th cells into IFN-gamma-secreting Th cells, as a result of its ability to inhibit DC IL-12 secretion. The goal of the present study was to identify how IFN-beta negatively regulates IL-12 secretion by DC. We report that in our Th cell differentiation model, DC IL-12 secretion is dependent on the CD40L/CD40 accessory pathway, and, utilizing a Th cell-free system, we find that IFN-beta inhibits anti-CD40 mAb-induced DC secretion of the p40 chain of the IL-12 heterodimer. In addition, we show that IFN-beta-mediated inhibition of CD40 signaling does not interfere with all signaling pathways emanating from CD40, since anti-CD40 mAb-induced DC IL-6 secretion is augmented by IFN-beta. Thus, our results demonstrate that signaling from CD40 is differentially regulated by IFN-beta. A second critical element of innate immunity involves the response against components of bacterial membranes such as LPS. DC respond to LPS by secreting IL-6 and IL-12. In contrast to CD40-dependent IL-6 and IL-12 secretion, we find that LPS-induced DC secretion of p40 IL-12 and IL-6 is not affected by IFN-beta. Our findings show that IFN-beta influences the generation of acquired immune responses through its regulation of CD40-dependent DC functions.     

Receptor for activated C-kinase (RACK-1), a WD motif-containing protein, specifically associates with the human type I IFN receptor

The cytoplasmic domain of the human type I IFN receptor chain 2 (IFNAR2c or IFN-alphaRbetaL) was used as bait in a yeast two-hybrid system to identify novel proteins interacting with this region of the receptor. We report here a specific interaction between the cytoplasmic domain of IFN-alphaRbetaL and a previously identified protein, RACK-1 (receptor for activated C kinase). Using GST fusion proteins encoding different regions of the cytoplasmic domain of IFN-alphaRbetaL, the minimum site for RACK-1 binding was mapped to aa 300-346. RACK-1 binding to IFN-alphaRbetaL did not require the first 91 aa of RACK-1, which includes two WD domains, WD1 and WD2. The interaction between RACK-1 and IFN-alphaRbetaL, but not the human IFN receptor chain 1 (IFNAR1 or IFN-alphaRalpha), was also detected in human Daudi cells by coimmunoprecipitation. RACK-1 was shown to be constitutively associated with IFN-alphaRbetaL, and this association was not effected by stimulation of Daudi cells with type I IFNs (IFN-beta1b). RACK-1 itself did not become tyrosine phosphorylated upon stimulation of Daudi cells with IFN-beta1b. However, stimulation of cells with either IFN-beta1b or PMA did result in an increase in detectable immunofluorescence and intracellular redistribution of RACK-1.     

Interferon-gamma-inducible regulation of the human invariant chain gene

Interferon-gamma (IFN-gamma) regulates a variety of immunoregulatory functions through the induction of a specific set of IFN-gamma response genes. This includes the invariant chain associated with the major histocompatibility complex class II molecules. To investigate the mechanism involved in the invariant chain (In) response to IFN-gamma we constructed chloramphenicol acetyltransferase (CAT) hybrid genes in which the CAT gene is under the control of the In promoter. The glioblastoma cell line, U-373 MG, transfected with a CAT construct having the In promoter sequence -790 to +1 bp showed over 3-fold increased CAT activity when treated with IFN-gamma indicating that this region confers IFN-gamma responsiveness to the CAT gene. The IFN-gamma response element in the promoter was further sublocalized to the region -120 to -61 base pairs (bp). This region contains homology to the interferon-stimulated response elements identified in other IFN responsive genes. By gel shift analyses, an IFN-gamma-induced sequence-specific DNA-binding factor was identified. This induced complex binds to an oligonucleotide corresponding to -107 to -79 bp of the In promoter. Mutations of this binding site at -94 and -92 bp drastically decreased binding of the constitutive and IFN-gamma-induced complexes. This IFN-gamma induced factor also binds to an oligonucleotide corresponding to -91 to -62 bp of the interferon-beta (IFN-beta) gene promoter, a region necessary for the induction of the IFN-beta gene by virus and double-stranded RNA. This binding specificity is characteristic of a family of DNA binding factors that bind both the interferon-stimulated response elements and the IFN-beta gene promoter.     

Quantitative multiplex real-time PCR for the sensitive detection of interferon beta gene induction and viral suppression of interferon beta expression

Interferon-beta (IFN-beta) protein and activity can be detected by enzyme immunoassays and biological assays. However, precise quantification of low IFN-beta mRNA concentrations, which is advantageous for investigating IFN-beta gene expression in small tissue samples or during the early stage of a virus infection, remains a challenge. Therefore, we established a quantitative real-time PCR (qPCR) for IFN-beta and the housekeeping gene porphobilinogen deanimase (PBGD) in separated assays as well as in a multiplex procedure. Sensitivity for both the templates was less than 20 copies with an intra- and interassay variability of less than 5%. IFN-beta qPCR was utilized to optimize IFN-beta induction with dsRNA polyinosic-polycytidylic acid (poly I:C), delivered by a liposomal transfection agent for reproducible but low, non-cell-toxic IFN-beta concentrations. For studying coxsackievirus B3 (CVB3) interference with IFN-beta expression, CVB3 infected fibroblasts were induced with poly I:C. A significant reduction of IFN-beta mRNA but not PBGD mRNA was demonstrated 5 h after CVB3 infection, indicating a specific inhibition of IFN-beta expression by CVB3 on the mRNA level, in addition to previously reported effects on the translation/post-translation level. In conclusion, sensitive IFN-beta/PBGD multiplex qPCR proved to be a useful tool to study viral interaction with IFN-beta expression.     

Interferon-beta induction through toll-like receptor 3 depends on double-stranded RNA structure

Type I interferons (IFN-alpha/beta) play an essential role in both innate and adaptive antiviral immune responses. IFN- beta is produced by fibroblasts and myeloid dendritic cells (DCs) upon viral infection or in response to doublestranded RNA (dsRNA). Several intracellular molecules having a dsRNA-binding motif such as dsRNA-dependent protein kinase recognize dsRNA in a sequence-independent manner and induce antiviral innate responses. Toll-like receptor (TLR) 3, a member of TLR family proteins, recognizes extracellular dsRNA and activates NF- kappaB and the IFN-beta promoter leading to the induction of IFN-beta production. Here we analyzed the dsRNA structure capable of inducing TLR3-mediated IFN-beta production using various synthetic RNA duplexes. In contrast to the recognition of dsRNA by intracellular molecules, TLR3 preferentially recognizes polyriboinocinic:polyribocytidylic acid (poly(I:C)) rather than synthetic virus-derived dsRNAs. 2'-O-methyl or 2'-fluoro modification of cytidylic acid abolished the IFN-beta-inducing ability of the poly(I:C) duplex, and these modified dsRNAs inhibited poly(I:C)-induced TLR3-mediated IFN-beta production by fibroblasts and DCs. In addition, poly(dI:dC), a non-IFN inducer, also blocked poly(I:C)-induced IFN-beta induction. Since TLR3 is localized in the intracellular compartment of DCs where signaling occurs, modified dsRNAs may compete with poly(I:C) for binding to the cell-surface receptor that transfers dsRNA into TLR3-enriched vesicles. Thus, TLR3 recognizes a unique dsRNA structure that largely differs from those recognized by other dsRNA-binding proteins.