RESUMO
Antigen-driven immune responses are modulated by immune complexes (ICs), in part through their ability to inhibit IFN-gamma-dependent MHC Class II expression. We have demonstrated previously that ICs dramatically inhibit IFN-gamma-induced activation of human monocytes through the suppression of the JAK/STAT signaling pathway. In the current study, we further explore the mechanisms by which ICs regulate IFN-gamma activation of human monocytes. Consistent with previous studies in monocytes pretreated with ICs, there was a reduction in steady-state levels of RNA by real-time RT-PCR of the IFN-inducible protein 10 gene as well as the FcgammaRI gene. Pull-down assays confirm that IC pretreatment inhibits IFN-gamma-induced STAT1 phosphorylation without affecting the ability of STAT1 to bind to the STAT1-binding domain of the IFN-gamma receptor. In addition, the inhibitory function of ICs was reduced when cells from the FcR common gamma-chain knockout mice were used, supporting the role of the FcgammaRI in this inhibitory pathway. It is unexpected that ICs also require the phosphatase Src homology-2-containing tyrosine phosphatase 1 (SHP-1) to inhibit IFN-gamma induction, as demonstrated by studies with cells from the SHP-1 knockout (motheaten) mice. These data suggest a mechanism of IC-mediated inhibition of IFN-gamma signaling, which requires the ITAM-containing FcgammaRI, as well as the ITIM-dependent phosphatase SHP-1, ultimately resulting in the suppression of STAT1 phosphorylation.
Assuntos
Complexo Antígeno-Anticorpo/farmacologia , Interferon gama/metabolismo , Monócitos/metabolismo , Receptores de IgG/metabolismo , Transdução de Sinais , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Cadeias gama de Imunoglobulina , Fosforilação , Proteína Fosfatase 1 , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Fator de Transcrição STAT1/metabolismoRESUMO
Specific prion peptides have been shown to mimic the pathologic isoform of the prion protein (PrP) and to induce a neurotoxic effect in vitro and in vivo. As monocytic cells are thought to play a role in the transmission and pathogenesis of prion disease, the use of these peptides in regulating monocytic cell function is under intense investigation. In the current study, we characterize the ability of prion peptide PrP(106-126) to activate specific signaling pathways in human monocyte-derived dendritic cells (DCs). Electrophoretic mobility shift assays establish the activation of transcription factor nuclear factor-kappaB within 15 min of exposure, with as little as 25 micro M peptide. This signaling cascade results in the up-regulation of inflammatory cytokines interleukin (IL)-1beta, IL-6, and tumor necrosis factor alpha (TNF-alpha) at the mRNA and protein levels. Phenotypic activation of DCs exposed to PrP(106-126) is partly a result of an autocrine TNF-alpha response and results in an increased ability of these cells to induce lymphocyte proliferation. The effects of PrP(106-126) on DCs were elicited through a receptor complex distinct from that used by human monocytes, demonstrating the ability of this peptide to interact with a multiplicity of receptors on various cell types. Together, these data suggest an involvement of DCs in prion disease pathogenesis.
Assuntos
Citocinas/biossíntese , Células Dendríticas/efeitos dos fármacos , NF-kappa B/metabolismo , Fragmentos de Peptídeos/farmacologia , Príons/farmacologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica , Humanos , Inflamação/imunologia , Monócitos/citologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , NF-kappa B/fisiologia , Doenças Priônicas/etiologiaRESUMO
Dendritic cells (DCs) are ideal accessory cells in the developing field of gene therapy. Although viral transfection of DCs has become widespread, non-viral transfection of DCs has shown disappointing results. Recently, a new technique for transfecting primary cells has become available -- the Amaxa Nucleofector. Here, we describe the use of this device in the successful non-viral transfection of human monocyte-derived DCs. Using enhanced green fluorescent protein as a reporter gene DCs were transfectable with efficiencies approaching 60%, remaining responsive to lipopolysaccharide-stimulated cytokine production in short-term experiments (though long-term functional assays were hampered by loss of viability). Although these data demonstrate the ease and efficiency with which human monocyte-derived DCs can now be non-virally transfected, they also suggest the limitations of this technology due to the gradual loss of cell viability. The potential use of this system in the development of DC-based cell and gene therapies will be hampered until cell viability can be maintained.
Assuntos
Células Dendríticas/metabolismo , Eletroporação , Monócitos/metabolismo , Células Dendríticas/imunologia , Proteínas de Fluorescência Verde , Humanos , Imunofenotipagem , Cinética , Proteínas Luminescentes/metabolismo , Monócitos/imunologia , TransfecçãoRESUMO
Macrophages respond to bacterial lipopolysaccharide (LPS) by activating latent cis-acting factors that initiate transcription of immediate early genes. One such immediate early gene, junB, is induced by LPS in macrophages within 30 min. To identify elements that mediate the induction of junB by LPS, upstream and downstream sequences flanking the junB gene were examined by transient expression in the RAW264.7 murine macrophage cell line using a luciferase reporter gene vector containing the junB minimal promoter. A >10-fold enhancement was associated with a 222 bp region downstream of the junB promoter in response to LPS. Transient reporter assays demonstrated that multiple nuclear factor (NF) kappaB sites are required for inducibility of junB by LPS in RAW264.7 cells. Electrophoretic mobility shift assays confirmed binding of LPS-induced nuclear proteins included p50/p65 heterodimers at these NF-kappaB sites.
Assuntos
Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-jun/biossíntese , Animais , Linhagem Celular , Macrófagos/metabolismo , Camundongos , Proteínas Proto-Oncogênicas c-jun/efeitos dos fármacosRESUMO
An increasing number of monoclonal antibodies for cancer diagnosis and treatment are in clinical use and in the development pipeline, with more expected as new molecular targets are identified. As with all drugs, product quality, an appropriate pre-clinical pharmacology-toxicology testing program, and well-designed clinical trials are essential for a successful drug development program. However, protein products such as monoclonal antibodies present unique regulatory concerns. The derivation from biological sources as well as the constantly evolving technologies utilized to develop these products demands continuous appraisal of safety concerns, even while the accumulated experience with these protein products has facilitated their safety evaluations. Because of the complex nature of these products and their inherent heterogeneity, a mechanistic understanding of the mode of action along with careful attention to product design and manufacture are critical to assuring a safe, effective and consistent product. Protein products may be highly species specific, thus pharmacologically relevant animal models are an important component in accurately assessing pre-clinical safety and establishing initial dosing. Furthermore, the immunogenicity of protein products can impact its safety profile, dose exposure, and efficacy. Mechanistic insight should form the basis of biological assays used for monitoring efficacy, safety, lot-to-lot consistency and manufacturing changes. The inherent uniqueness of each product necessitates a flexible case-by-case approach for biologics review that is based on a strong scientific understanding of relative risks. This review will provide an overview of approaches used in the development of antibody-based cancer therapeutics and the scientific basis of regulatory reviews.