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Transcription Factors Regulating Inflammatory Cytokine Production Are Differentially Expressed in Peripheral Blood Mononuclear Cells of Beh ç et Disease Depending on Disease Activity

View Article: PubMed Central - PubMed

ABSTRACT

Background: Behçet disease (BD) is a relapsing inflammatory disease with increased production of inflammatory cytokines in peripheral blood mononuclear cells (PBMCs); however, the underlying molecular mechanisms are not well known.

Objective: To analyze whether the differential expression of transcription factors is involved in the increased tumor necrosis factor (TNF)-α and interleukin (IL)-6 production by PBMCs of BD patients compared to healthy controls (HCs).

Methods: Expression of transcription factors was examined by real-time reverse transcriptase-polymerase chain reaction and western blotting. Cytokine production by CD11b+ cells transfected with siRNAs against transcription factors was measured by enzyme-linked immunosorbent assay.

Results: In the absence of lipopolysaccharide stimulation, the transcript level of CCAAT-enhancer-binding proteins (C/EBP) β was increased in PBMCs from patients with active BD compared to that in PBMCs from patients with stable BD. The C/EBPδ transcript level was higher in PBMCs from patients with active BD than in those from HCs. The activating transcription factor 3 (ATF3) transcript level was increased in PBMCs from patients with stable BD compared to that in PBMCs from HCs. siRNAs targeting C/EBPβ and C/EBPδ significantly reduced the production of IL-6 and TNF-α in lipopolysaccharide-stimulated CD11b+ cells from patients with BD as well as from HCs.

Conclusion: We found differential expression of C/EBPβ, C/EBPδ, and ATF3 in PBMCs from patients with BD depending on disease activity, indicating the involvement of these molecules in BD pathogenesis.

No MeSH data available.


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Differential mRNA expression of CCAAT-enhancer-binding proteins (C/EBP) β (A), C/EBPδ (B), and activating transcription factor 3 (ATF3) (C) in peripheral blood mononuclear cells (PBMCs) from Behçet disease (BD) patients. PBMCs isolated from 5 healthy controls (HCs), 4 to 6 stable BD patients (St) and 6 to 7 active BD patients (Ac) were cultured with or without lipopolysaccharide (LPS) for 3 hours. mRNA levels of C/EBPβ, C/EBPδ, and ATF3 were analyzed by real-time reverse transcription-polymerase chain reaction. Fold over HC(–): Relative mRNA level versus the average mRNA level in HC without LPS stimulation. Each symbol represents a single subject. Bars represent the mean of each group. The Kruskal-Wallis test with Dunn's procedure was conducted. *p <0.05, **p<0.01, ***p<0.005.
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Figure 2: Differential mRNA expression of CCAAT-enhancer-binding proteins (C/EBP) β (A), C/EBPδ (B), and activating transcription factor 3 (ATF3) (C) in peripheral blood mononuclear cells (PBMCs) from Behçet disease (BD) patients. PBMCs isolated from 5 healthy controls (HCs), 4 to 6 stable BD patients (St) and 6 to 7 active BD patients (Ac) were cultured with or without lipopolysaccharide (LPS) for 3 hours. mRNA levels of C/EBPβ, C/EBPδ, and ATF3 were analyzed by real-time reverse transcription-polymerase chain reaction. Fold over HC(–): Relative mRNA level versus the average mRNA level in HC without LPS stimulation. Each symbol represents a single subject. Bars represent the mean of each group. The Kruskal-Wallis test with Dunn's procedure was conducted. *p <0.05, **p<0.01, ***p<0.005.

Mentions: Given that transcription factors such as C/EBPβ, C/EBPδ, and ATF3 are induced 3 hours post-LPS stimulation and regulate the transcription of IL-6 and TNF-α5, we analyzed the mRNA levels of these transcription factors in PBMCs from BD patients (Fig. 2). C/EBPβ mRNA levels were approximately 2-fold higher in unstimulated PBMCs from patients with active BD compared to those in unstimulated PBMCs from patients with stable BD (p<.0.01); however, this increase was not observed following LPS stimulation. C/EBPδ mRNA levels in PBMCs from patients with active BD were significantly higher than those in PBMCs from HCs both in the absence (p<.0.005) and presence of LPS stimulation (p<.0.05). In contrast, ATF3 mRNA expression was increased in PBMCs from patients with stable BD compared to that in the PBMCs from HCs, and was further increased by LPS stimulation (p<.0.05). mRNA levels of these transcription factors did not show significant correlation with HLA-B51 genotype, ocular symptoms, or erythema nodosum (data not shown).


Transcription Factors Regulating Inflammatory Cytokine Production Are Differentially Expressed in Peripheral Blood Mononuclear Cells of Beh ç et Disease Depending on Disease Activity
Differential mRNA expression of CCAAT-enhancer-binding proteins (C/EBP) β (A), C/EBPδ (B), and activating transcription factor 3 (ATF3) (C) in peripheral blood mononuclear cells (PBMCs) from Behçet disease (BD) patients. PBMCs isolated from 5 healthy controls (HCs), 4 to 6 stable BD patients (St) and 6 to 7 active BD patients (Ac) were cultured with or without lipopolysaccharide (LPS) for 3 hours. mRNA levels of C/EBPβ, C/EBPδ, and ATF3 were analyzed by real-time reverse transcription-polymerase chain reaction. Fold over HC(–): Relative mRNA level versus the average mRNA level in HC without LPS stimulation. Each symbol represents a single subject. Bars represent the mean of each group. The Kruskal-Wallis test with Dunn's procedure was conducted. *p <0.05, **p<0.01, ***p<0.005.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5383742&req=5

Figure 2: Differential mRNA expression of CCAAT-enhancer-binding proteins (C/EBP) β (A), C/EBPδ (B), and activating transcription factor 3 (ATF3) (C) in peripheral blood mononuclear cells (PBMCs) from Behçet disease (BD) patients. PBMCs isolated from 5 healthy controls (HCs), 4 to 6 stable BD patients (St) and 6 to 7 active BD patients (Ac) were cultured with or without lipopolysaccharide (LPS) for 3 hours. mRNA levels of C/EBPβ, C/EBPδ, and ATF3 were analyzed by real-time reverse transcription-polymerase chain reaction. Fold over HC(–): Relative mRNA level versus the average mRNA level in HC without LPS stimulation. Each symbol represents a single subject. Bars represent the mean of each group. The Kruskal-Wallis test with Dunn's procedure was conducted. *p <0.05, **p<0.01, ***p<0.005.
Mentions: Given that transcription factors such as C/EBPβ, C/EBPδ, and ATF3 are induced 3 hours post-LPS stimulation and regulate the transcription of IL-6 and TNF-α5, we analyzed the mRNA levels of these transcription factors in PBMCs from BD patients (Fig. 2). C/EBPβ mRNA levels were approximately 2-fold higher in unstimulated PBMCs from patients with active BD compared to those in unstimulated PBMCs from patients with stable BD (p<.0.01); however, this increase was not observed following LPS stimulation. C/EBPδ mRNA levels in PBMCs from patients with active BD were significantly higher than those in PBMCs from HCs both in the absence (p<.0.005) and presence of LPS stimulation (p<.0.05). In contrast, ATF3 mRNA expression was increased in PBMCs from patients with stable BD compared to that in the PBMCs from HCs, and was further increased by LPS stimulation (p<.0.05). mRNA levels of these transcription factors did not show significant correlation with HLA-B51 genotype, ocular symptoms, or erythema nodosum (data not shown).

View Article: PubMed Central - PubMed

ABSTRACT

Background: Beh&ccedil;et disease (BD) is a relapsing inflammatory disease with increased production of inflammatory cytokines in peripheral blood mononuclear cells (PBMCs); however, the underlying molecular mechanisms are not well known.

Objective: To analyze whether the differential expression of transcription factors is involved in the increased tumor necrosis factor (TNF)-&alpha; and interleukin (IL)-6 production by PBMCs of BD patients compared to healthy controls (HCs).

Methods: Expression of transcription factors was examined by real-time reverse transcriptase-polymerase chain reaction and western blotting. Cytokine production by CD11b+ cells transfected with siRNAs against transcription factors was measured by enzyme-linked immunosorbent assay.

Results: In the absence of lipopolysaccharide stimulation, the transcript level of CCAAT-enhancer-binding proteins (C/EBP) &beta; was increased in PBMCs from patients with active BD compared to that in PBMCs from patients with stable BD. The C/EBP&delta; transcript level was higher in PBMCs from patients with active BD than in those from HCs. The activating transcription factor 3 (ATF3) transcript level was increased in PBMCs from patients with stable BD compared to that in PBMCs from HCs. siRNAs targeting C/EBP&beta; and C/EBP&delta; significantly reduced the production of IL-6 and TNF-&alpha; in lipopolysaccharide-stimulated CD11b+ cells from patients with BD as well as from HCs.

Conclusion: We found differential expression of C/EBP&beta;, C/EBP&delta;, and ATF3 in PBMCs from patients with BD depending on disease activity, indicating the involvement of these molecules in BD pathogenesis.

No MeSH data available.


Related in: MedlinePlus