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Microarray analysis identifies a set of CXCR3 and CCR2 ligand chemokines as early IFNbeta-responsive genes in peripheral blood lymphocytes in vitro: an implication for IFNbeta-related adverse effects in multiple sclerosis.

Satoh J, Nanri Y, Tabunoki H, Yamamura T - BMC Neurol (2006)

Bottom Line: At present, the molecular mechanism underlying IFNbeta-related adverse effects remains largely unknown.IFNbeta markedly upregulated CXCR3 ligand chemokines (SCYB11, SCYB10 and SCYB9) chiefly active on effector T helper type 1 (Th1) T cells, and CCR2 ligand chemokines (SCYA8 and SCYA2) effective on monocytes, whereas it downregulated CXCR2 ligand chemokines (SCYB2, SCYB1 and IL8) primarily active on neutrophils.IFNbeta immediately induces a burst of gene expression of proinflammatory chemokines in vitro that have potential relevance to IFNbeta-related early adverse effects in MS patients in vivo.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Bioinformatics, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan. satoj@ncnp.go.jp

ABSTRACT

Background: A substantial proportion of multiple sclerosis (MS) patients discontinue interferon-beta (IFNbeta) treatment due to various adverse effects, most of which emerge at the early phase after initiation of the treatment and then diminish with time. At present, the molecular mechanism underlying IFNbeta-related adverse effects remains largely unknown. The aim of this study is to identify a comprehensive list of early IFNbeta-responsive genes (IRGs) in peripheral blood mononuclear cells (PBMC) that may play a key role in induction of adverse effects.

Methods: Total RNA of PBMC exposed to 50 ng/ml recombinant human IFNbeta for 3 to 24 hours in vitro was processed for cDNA microarray analysis, followed by quantitative real-time RT-PCR analysis.

Results: Among 1,258 genes on the array, IFNbeta elevated the expression of 107 and 87 genes, while it reduced the expression of 22 and 23 genes at 3 and 24 hours, respectively. Upregulated IRGs were categorized into conventional IFN-response markers, components of IFN-signaling pathways, chemokines, cytokines, growth factors, and their receptors, regulators of apoptosis, DNA damage, and cell cycle, heat shock proteins, and costimulatory and adhesion molecules. IFNbeta markedly upregulated CXCR3 ligand chemokines (SCYB11, SCYB10 and SCYB9) chiefly active on effector T helper type 1 (Th1) T cells, and CCR2 ligand chemokines (SCYA8 and SCYA2) effective on monocytes, whereas it downregulated CXCR2 ligand chemokines (SCYB2, SCYB1 and IL8) primarily active on neutrophils.

Conclusion: IFNbeta immediately induces a burst of gene expression of proinflammatory chemokines in vitro that have potential relevance to IFNbeta-related early adverse effects in MS patients in vivo.

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Related in: MedlinePlus

Real-time RT-PCR analysis of ISG15 expression in PBMC. PBMC derived from three distinct healthy subjects numbered #1 (a 46 year-old man), #2 (a 28 year-old man), and #3 (a 42 year-old woman) were incubated for 3 hours or 24 hours in the culture medium with (+) or without (-) inclusion of recombinant human IFNβ, IFNγ, TNFα or IL-1β at a concentration of 50 ng/ml each. cDNA was processed for real-time PCR analysis using specific primers listed in Table 1. The levels of expression of ISG15 are standardized against those of the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene detected in identical cDNA samples. The assays were performed in triplicate measurements of the same sample, and the results were expressed as the average with standard error. The panels represent the expression of ISG15 in (a) #1, IFNβ; (b) #2, IFNβ; (c) #3, IFNβ; (d) #1, IFNγ; (e) #1, TNFα; and (f) #1, IL-1β.
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Figure 1: Real-time RT-PCR analysis of ISG15 expression in PBMC. PBMC derived from three distinct healthy subjects numbered #1 (a 46 year-old man), #2 (a 28 year-old man), and #3 (a 42 year-old woman) were incubated for 3 hours or 24 hours in the culture medium with (+) or without (-) inclusion of recombinant human IFNβ, IFNγ, TNFα or IL-1β at a concentration of 50 ng/ml each. cDNA was processed for real-time PCR analysis using specific primers listed in Table 1. The levels of expression of ISG15 are standardized against those of the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene detected in identical cDNA samples. The assays were performed in triplicate measurements of the same sample, and the results were expressed as the average with standard error. The panels represent the expression of ISG15 in (a) #1, IFNβ; (b) #2, IFNβ; (c) #3, IFNβ; (d) #1, IFNγ; (e) #1, TNFα; and (f) #1, IL-1β.

Mentions: Although the microarray we utilized contains total 64 spots of the G3PDH gene (see Additional file 1), G3PDH was neither identified as a significantly upregulated nor a downregulated gene in the microarray analysis, suggesting that G3PDH represents a reliable housekeeping gene in gene expression analysis of PBMC following treatment with IFNβ. Therefore, quantitative real-time RT-PCR analysis was performed by evaluating the levels of expression of target genes standardized against those of G3PDH detected in the identical cDNA samples. It verified the key observations of microarray analysis, such as marked upregulation of ISG15, the prototype of IRGs (Figure 1a–c), and great elevation of SCYB10, SCYA8 and SCYA2 (Figures 2, 3, 4a–c) in PBMC at both 3 and 24 hours of IFNβ treatment. Furthermore, the quantitative analysis confirmed substantial downregulation of FOS at both time points (Figure 5a–c), and RGS14 and SCYB2 predominantly at 3 hours (Figures 6, 7a–c). Exposure of PBMC to IFNγ greatly elevated the expression of SCYB10 and SCYA2, and to a lessor extent, ISG15 and SCYA8 at both time points (Figures 1, 2, 3, 4d), suggesting a functional overlap in induction of CXCR3 ligand and CCR2 ligand chemokines between type I and type II IFN signaling pathways. In contrast, TNFα and IL-1β the prototype of proinflammatory cytokines, did not at all elevate the levels of expression of ISG15, SCYB10 or SCYA8 (Figures 1, 2, 3e, f), while IL-1β significantly (p = 0.041 at 3 hours and p = 0.004 at 24 hours by two-sided paired t-test) but TNFα only marginally (p = 0.2102 at 3 hours and p = 0.0825 at 24 hours by two-sided paired t-test) upregulated SCYA2 expression (Figure 4e, f). Treatment with IFNγ, TNFα or IL-1β reduced the levels of FOS and RGS14 substantially at 24 hours (Figures 5, 6d–f). IFNγ reduced the expression of SCYB2, whereas TNFα and IL-1β elevated its levels at both time points, suggesting differential regulation of SCYB2 gene expression in PBMC by exposure to distinct cytokines (Figure 7d–f). The IFNβ-regulated gene expression pattern was similar among PBMC derived from three distinct healthy subjects #1, #2 and #3, supporting the reproducibility of these observations (Figures 1, 2, 3, 4, 5, 6, 7a–c).


Microarray analysis identifies a set of CXCR3 and CCR2 ligand chemokines as early IFNbeta-responsive genes in peripheral blood lymphocytes in vitro: an implication for IFNbeta-related adverse effects in multiple sclerosis.

Satoh J, Nanri Y, Tabunoki H, Yamamura T - BMC Neurol (2006)

Real-time RT-PCR analysis of ISG15 expression in PBMC. PBMC derived from three distinct healthy subjects numbered #1 (a 46 year-old man), #2 (a 28 year-old man), and #3 (a 42 year-old woman) were incubated for 3 hours or 24 hours in the culture medium with (+) or without (-) inclusion of recombinant human IFNβ, IFNγ, TNFα or IL-1β at a concentration of 50 ng/ml each. cDNA was processed for real-time PCR analysis using specific primers listed in Table 1. The levels of expression of ISG15 are standardized against those of the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene detected in identical cDNA samples. The assays were performed in triplicate measurements of the same sample, and the results were expressed as the average with standard error. The panels represent the expression of ISG15 in (a) #1, IFNβ; (b) #2, IFNβ; (c) #3, IFNβ; (d) #1, IFNγ; (e) #1, TNFα; and (f) #1, IL-1β.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1483835&req=5

Figure 1: Real-time RT-PCR analysis of ISG15 expression in PBMC. PBMC derived from three distinct healthy subjects numbered #1 (a 46 year-old man), #2 (a 28 year-old man), and #3 (a 42 year-old woman) were incubated for 3 hours or 24 hours in the culture medium with (+) or without (-) inclusion of recombinant human IFNβ, IFNγ, TNFα or IL-1β at a concentration of 50 ng/ml each. cDNA was processed for real-time PCR analysis using specific primers listed in Table 1. The levels of expression of ISG15 are standardized against those of the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene detected in identical cDNA samples. The assays were performed in triplicate measurements of the same sample, and the results were expressed as the average with standard error. The panels represent the expression of ISG15 in (a) #1, IFNβ; (b) #2, IFNβ; (c) #3, IFNβ; (d) #1, IFNγ; (e) #1, TNFα; and (f) #1, IL-1β.
Mentions: Although the microarray we utilized contains total 64 spots of the G3PDH gene (see Additional file 1), G3PDH was neither identified as a significantly upregulated nor a downregulated gene in the microarray analysis, suggesting that G3PDH represents a reliable housekeeping gene in gene expression analysis of PBMC following treatment with IFNβ. Therefore, quantitative real-time RT-PCR analysis was performed by evaluating the levels of expression of target genes standardized against those of G3PDH detected in the identical cDNA samples. It verified the key observations of microarray analysis, such as marked upregulation of ISG15, the prototype of IRGs (Figure 1a–c), and great elevation of SCYB10, SCYA8 and SCYA2 (Figures 2, 3, 4a–c) in PBMC at both 3 and 24 hours of IFNβ treatment. Furthermore, the quantitative analysis confirmed substantial downregulation of FOS at both time points (Figure 5a–c), and RGS14 and SCYB2 predominantly at 3 hours (Figures 6, 7a–c). Exposure of PBMC to IFNγ greatly elevated the expression of SCYB10 and SCYA2, and to a lessor extent, ISG15 and SCYA8 at both time points (Figures 1, 2, 3, 4d), suggesting a functional overlap in induction of CXCR3 ligand and CCR2 ligand chemokines between type I and type II IFN signaling pathways. In contrast, TNFα and IL-1β the prototype of proinflammatory cytokines, did not at all elevate the levels of expression of ISG15, SCYB10 or SCYA8 (Figures 1, 2, 3e, f), while IL-1β significantly (p = 0.041 at 3 hours and p = 0.004 at 24 hours by two-sided paired t-test) but TNFα only marginally (p = 0.2102 at 3 hours and p = 0.0825 at 24 hours by two-sided paired t-test) upregulated SCYA2 expression (Figure 4e, f). Treatment with IFNγ, TNFα or IL-1β reduced the levels of FOS and RGS14 substantially at 24 hours (Figures 5, 6d–f). IFNγ reduced the expression of SCYB2, whereas TNFα and IL-1β elevated its levels at both time points, suggesting differential regulation of SCYB2 gene expression in PBMC by exposure to distinct cytokines (Figure 7d–f). The IFNβ-regulated gene expression pattern was similar among PBMC derived from three distinct healthy subjects #1, #2 and #3, supporting the reproducibility of these observations (Figures 1, 2, 3, 4, 5, 6, 7a–c).

Bottom Line: At present, the molecular mechanism underlying IFNbeta-related adverse effects remains largely unknown.IFNbeta markedly upregulated CXCR3 ligand chemokines (SCYB11, SCYB10 and SCYB9) chiefly active on effector T helper type 1 (Th1) T cells, and CCR2 ligand chemokines (SCYA8 and SCYA2) effective on monocytes, whereas it downregulated CXCR2 ligand chemokines (SCYB2, SCYB1 and IL8) primarily active on neutrophils.IFNbeta immediately induces a burst of gene expression of proinflammatory chemokines in vitro that have potential relevance to IFNbeta-related early adverse effects in MS patients in vivo.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Bioinformatics, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan. satoj@ncnp.go.jp

ABSTRACT

Background: A substantial proportion of multiple sclerosis (MS) patients discontinue interferon-beta (IFNbeta) treatment due to various adverse effects, most of which emerge at the early phase after initiation of the treatment and then diminish with time. At present, the molecular mechanism underlying IFNbeta-related adverse effects remains largely unknown. The aim of this study is to identify a comprehensive list of early IFNbeta-responsive genes (IRGs) in peripheral blood mononuclear cells (PBMC) that may play a key role in induction of adverse effects.

Methods: Total RNA of PBMC exposed to 50 ng/ml recombinant human IFNbeta for 3 to 24 hours in vitro was processed for cDNA microarray analysis, followed by quantitative real-time RT-PCR analysis.

Results: Among 1,258 genes on the array, IFNbeta elevated the expression of 107 and 87 genes, while it reduced the expression of 22 and 23 genes at 3 and 24 hours, respectively. Upregulated IRGs were categorized into conventional IFN-response markers, components of IFN-signaling pathways, chemokines, cytokines, growth factors, and their receptors, regulators of apoptosis, DNA damage, and cell cycle, heat shock proteins, and costimulatory and adhesion molecules. IFNbeta markedly upregulated CXCR3 ligand chemokines (SCYB11, SCYB10 and SCYB9) chiefly active on effector T helper type 1 (Th1) T cells, and CCR2 ligand chemokines (SCYA8 and SCYA2) effective on monocytes, whereas it downregulated CXCR2 ligand chemokines (SCYB2, SCYB1 and IL8) primarily active on neutrophils.

Conclusion: IFNbeta immediately induces a burst of gene expression of proinflammatory chemokines in vitro that have potential relevance to IFNbeta-related early adverse effects in MS patients in vivo.

Show MeSH
Related in: MedlinePlus