Expression, purification, and characterization of rat interferon-β, and preparation of an N-terminally PEGylated form with improved pharmacokinetic parameters

Arduini, Robert M.; Li, Zhifang; Rapoza, Alan; Gronke, Robert S.; Hess, Donna M.; Wen, Dingyi; Miatkowski, Konrad; Coots, Caroline; Kaffashan, Azita; Viseux, Nelly; Delaney, James C.; Domon, Bruno; Young, Carmen N.; Boynton, Raymond E.; Chen, Ling Ling; Chen, Liqing; Betzenhauser, Matthew J.; Miller, Shannon M.; Gill, Alan; Pepinsky, R. Blake; Hochman, Paula S.; Baker, Darren P.

doi:10.1016/j.pep.2003.11.004

Cited by 47 publications

(38 citation statements)

References 45 publications

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“…Biological Reagents and Cell Culture-Recombinant rat IFN␤ was obtained from Biogen-Idec, Inc. (7). Antibodies directed against the following proteins were used: p65, p50, IRF1, STAT1, STAT2, Tap1, and ␤-actin were from Santa Cruz Biotechnology (Santa Cruz, CA); Mx1 was from Dr. Otto Haller; phospho-Stat2 was from Upstate Biotechnology (Charlottesville, VA); and TFIIB was from ActiveMotif (Carlsbad, CA).…”

Section: Methodsmentioning

confidence: 99%

NFκB Negatively Regulates Interferon-induced Gene Expression and Anti-influenza Activity

Wei

Sandbulte

Thomas

et al. 2006

Journal of Biological Chemistry

103

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Type I IFNs 2 (IFN␣, IFN␤, IFN, and IFN) are multifunctional cytokines that are critical in the host defense to infectious agents by modulating innate and adaptive immune responses. IFNs induce their biological effects by regulating the expression of a family of early response genes, called IFN-stimulated genes (ISGs), through JAK-mediated tyrosine-phosphorylation of the STAT factors, STAT1 and STAT2. The phosphorylated STAT proteins dimerize, translocate into the nucleus, and bind to the conserved IFN-stimulus response element (ISRE) within the promoters of ISGs (1). In addition, transcription factors of the IFN regulatory factor (IRF) family have been shown to regulate ISG expression (2). Accumulating evidence indicates that IFNs also activate the nuclear factor B (NFB) transcription factor in a serine/threonine kinase-dependent signaling pathway (3, 4).The mammalian NFB proteins, p50, p52, RelA (p65), RelB, and c-Rel, form homodimers and heterodimers to regulate the expression of genes involved in the immune response, inflammation and cell survival (5). In most cell types, the predominant form of NFB, the p50:p65 heterodimer, is bound to IB inhibitory proteins in the cytoplasm of unstimulated cells. Similar to various other stimuli, IFN␣/␤ activates a phosphatidylinositol 3-kinase/Akt pathway, which results in the dissociation of the inactive cytosolic NFB⅐IB complexes followed by NFB nuclear translocation and DNA binding (3, 4). We previously demonstrated that mouse embryonic fibroblasts (MEFs) derived from mice with germ line deletions of the p50 and p65 genes were resistant to IFN-induced NFB activation and were sensitized to the antiviral action of IFN␤ against vesicular stomatitis virus (6). The present study was undertaken to further define the role of NFB in IFN-induced gene expression and the biological actions of IFN. Gene expression profiling identified 35 ISGs whose induction was highly regulated by NFB. A subset of genes that were induced higher by IFN in NFB knock-out (NFB-KO) cells encoded GTP-binding and antigen presentation proteins, which play critical roles in the antiviral and immunomodulatory activities of IFN, respectively. Quantitative RT-PCR demonstrated that these ISGs were induced more rapidly and at significantly lower IFN concentrations in NFB-KO cells relative to wild-type (WT) MEFs. Chromatin immunoprecipitation (ChIP) assays demonstrated that NFB dimers containing p50 and p65 were basally bound to the promoters of these ISGs. IFN induced the binding of STAT1 and STAT2 to these promoters. However, the kinetics of ISG induction in NFB-KO MEFs by IFN correlated with the promoter binding of IRF1. These results suggest that IFN induction of these genes is negatively regulated by NFB. We also found that NFB suppressed not only the direct antiviral action of IFN against influenza virus but also IFN-induced influenza-specific MHC class I antigen presentation. Together, our results suggest that NFB not only regulates the induction of a subset of IFN-induced genes but also the antiviral an...

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Section: Methodsmentioning

confidence: 99%

NFκB Negatively Regulates Interferon-induced Gene Expression and Anti-influenza Activity

Wei

Sandbulte

Thomas

et al. 2006

Journal of Biological Chemistry

103

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“…Biological Reagents and Cell Culture-Recombinant Chinese hamster ovary cell-expressed rat IFN-␤ was obtained from Biogen Idec, Inc. (6). The biological activity of IFN was assayed by protection against the cytopathic effect of vesicular stomatitis virus (VSV) on murine fibroblasts and was expressed in units/ml using the murine National Institutes of Health IFN-␤ standard for reference.…”

Section: Methodsmentioning

confidence: 99%

Role of Nuclear Factor-κB in the Antiviral Action of Interferon and Interferon-regulated Gene Expression

Pfeffer¹,

Kim²,

Pfeffer³

et al. 2004

Journal of Biological Chemistry

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Interferons (IFNs)1 are a family of multifunctional cytokines that block viral infection, inhibit cell proliferation, and modulate cell differentiation. Whereas type I IFNs (IFN-␣, IFN-␤, and IFN-) bind to a common cell-surface receptor, the receptor for type II IFN (IFN-␥) is a distinct entity (1). IFNs transduce signals from the cell surface, resulting in selective gene induction through the activation of JAK tyrosine kinases and STAT transcription factors (1-3). Upon JAK-mediated tyrosine phosphorylation, the STAT proteins (STAT1, STAT2, and STAT3) dimerize and translocate to the nucleus to activate transcription of IFN-stimulated genes (3). IFN also activates the nuclear factor-B (NF-B) transcription factor in a serine/threonine kinase-dependent signaling pathway that protects cells against apoptosis (4, 5).NF-B regulates the expression of genes involved in cell signaling, stress responses, growth, survival, and apoptosis by binding to cis-acting B sites in the promoters and enhancers of these genes. Viruses, cytokines, lipopolysaccharides, and other stimulating agents promote NF-B translocation to the nucleus and DNA binding. Active DNA-binding forms of NF-B are dimeric combinations of Rel proteins (p50, p52, c-Rel, v-Rel, RelA/p65, and RelB). In most cell types, the predominant form of NF-B is the p50⅐p65 heterodimer. NF-B dimers are retained in the cytoplasm of unstimulated cells in an inactive state by the binding of a family of inhibitory IB proteins.Whereas STAT proteins play crucial roles in the transcriptional response to IFN-␣/␤ and in the induction of antiviral activity, the role of NF-B in IFN signaling and action has not been studied extensively. To identify the functional role of NF-B in IFN action, sensitivity to the antiviral effect of IFN was examined in fibroblasts that either had normal NF-B function or had a functional deletion of the IFN-induced NF-B pathway by germ line disruption of the Rel p50 and p65 proteins. Antiviral assays demonstrated that NF-B knockout (KO) fibroblasts were sensitized to the antiviral action of IFN-␤. To determine the relationship between gene regulation by IFN-␤ and antiviral activity, microarray analysis was performed on RNA samples collected from IFN-treated murine fibroblasts. Microarray analysis identified several classical IFN-stimulated genes (ISGs) involved in the antiviral action of IFN. Quantitative real-time PCR demonstrated that, whereas the IFN-induced expression of some ISGs was enhanced in NF-B KO cells relative to mouse wild-type fibroblasts, the IFN-induced expression of other ISGs was lower in NF-B KO cells. Our results demonstrate the distinctive role of NF-B in the regulation of ISGs and in the induction of antiviral activity. Thus, the IFN-activated NF-B pathway not only counterbalances apoptosis, but also regulates the expression of ISGs and the induction of antiviral activity. EXPERIMENTAL PROCEDURESBiological Reagents and Cell Culture-Recombinant Chinese hamster ovary cell-expressed rat IFN-␤ was obtained from Biogen Idec, Inc. (6). The b...

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“…IFN-␤ pegylation was done as described previously 9 (with modifications). The pegylated compound was diluted, filtered, and purified by cation exchange and size exclusion chromatography.…”

Section: Drug Protocolmentioning

confidence: 99%

Interferon-β Fails to Protect in a Model of Transient Focal Stroke

Maier

Nishi

et al. 2006

Stroke

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Background and Purpose-Compelling evidence supporting the role of inflammation in the development of cerebral infarction has focused attention on the potential of antiinflammatory treatment strategies for stroke. Interferon (IFN)-␤, an immunomodulatory agent approved for treatment of multiple sclerosis, is being evaluated in a phase I clinical trial in acute ischemic stroke. In the present study, we evaluated the effects of wild-type rat IFN-␤ and its pegylated counterpart (PEG-IFN-␤) in a model of focal ischemia and reperfusion. Methods-After 60 minutes of middle cerebral artery occlusion, rats (nϭ12/group) were treated with IV tail injections of 8 or 16 g of IFN-␤ in 300 L of PBS once daily for 3 or 7 days or with IV or SC injections of PEG-IFN-␤ for 1 day. The animals were assessed daily for weight and for neurological findings. Additional animals underwent complete hematology and chemistry profiles, as well as complete multiorgan necropsy studies. All of the brain tissue was evaluated for assessment of infarct areas, neutrophil infiltration, and presence of hemorrhagic transformations. Results-IFN-␤ and PEG-IFN-␤ failed to protect against experimental ischemic brain injury as assessed by histopathology and neurological outcome. Furthermore, IFN-␤ treatment was associated with significant weight loss and alterations in hematology and chemistry profiles. Key Words: inflammation Ⅲ interferon ␤ Ⅲ ischemia Ⅲ neuroprotection I nflammation plays a key role in the pathophysiology of ischemic stroke, and several antiinflammatory treatment strategies have been shown to mitigate secondary brain injury after experimental cerebral ischemia 1 . Interferon (IFN)-␤, a cytokine with immunomodulatory properties, was approved by the US Food and Drug Administration for treatment of relapsingremitting multiple sclerosis Ͼ10 years ago. 2 A National Institute of Neurological Disorders and Stroke-sponsored phase I clinical trial is underway to determine the safety, tolerability profile, and optimal administration route of IFN-␤ in patients with acute ischemic stroke. The rationale for use of IFN-␤ in stroke patients comes from a limited number of animal studies in which systemic administration of IFN-␤ before or after (4 to 6 hours after) stroke onset reduced infarct volumes at 24 hours 3 and after 3 weeks of reperfusion. 4 The neuroprotective effects of IFN-␤ are associated with a reduction in neutrophil infiltration and decreased blood-brain barrier (BBB) disruption, 4 -6 as well as with the promotion of cell survival factors. 7 However, the exact mechanisms through which IFN-␤ might moderate the development of ischemic stroke remain unclear. One question is whether the drug can directly access the central nervous system, because it does not penetrate the BBB well unless the barrier is substantially compromised during the ischemic insult. To date, only 2 animal stroke models have been reported that evaluate the efficacy of IFN-␤: (1) transient (1 hour) occlusion of the middle cerebral artery (MCA) via direct placement of a microv...

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Expression, purification, and characterization of rat interferon-β, and preparation of an N-terminally PEGylated form with improved pharmacokinetic parameters

Cited by 47 publications

References 45 publications

NFκB Negatively Regulates Interferon-induced Gene Expression and Anti-influenza Activity

NFκB Negatively Regulates Interferon-induced Gene Expression and Anti-influenza Activity

Role of Nuclear Factor-κB in the Antiviral Action of Interferon and Interferon-regulated Gene Expression

Interferon-β Fails to Protect in a Model of Transient Focal Stroke

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