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Increased interleukin-17 production via a phosphoinositide 3-kinase/Akt and nuclear factor kappaB-dependent pathway in patients with rheumatoid arthritis.

Kim KW, Cho ML, Park MK, Yoon CH, Park SH, Lee SH, Kim HY - Arthritis Res. Ther. (2004)

Bottom Line: Anti-CD3 antibody activated the PI3K/Akt pathway; activation of this pathway resulted in a pronounced augmentation of nuclear factor kappaB (NF-kappaB) DNA-binding activity.However, inhibition of activator protein-1 and extracellular signal-regulated kinase 1/2 did not affect IL-17 production.These results suggest that signal transduction pathways dependent on PI3K/Akt and NF-kappaB are involved in the overproduction of the key inflammatory cytokine IL-17 in RA.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, Division of Rheumatology, The Center for Rheumatic Diseases, and The Rheumatism Research Center (RhRC), Catholic Research Institutes of Medical Sciences, Catholic University of Korea, Seoul, Korea. woon1212@catholic.ac.kr

ABSTRACT
Inflammatory mediators have been recognized as being important in the pathogenesis of rheumatoid arthritis (RA). Interleukin (IL)-17 is an important regulator of immune and inflammatory responses, including the induction of proinflammatory cytokines and osteoclastic bone resorption. Evidence for the expression and proinflammatory activity of IL-17 has been demonstrated in RA synovium and in animal models of RA. Although some cytokines (IL-15 and IL-23) have been reported to regulate IL-17 production, the intracellular signaling pathways that regulate IL-17 production remain unknown. In the present study, we investigated the role of the phosphoinositide 3-kinase (PI3K)/Akt pathway in the regulation of IL-17 production in RA. Peripheral blood mononuclear cells (PBMC) from patients with RA (n = 24) were separated, then stimulated with various agents including anti-CD3, anti-CD28, phytohemagglutinin (PHA) and several inflammatory cytokines and chemokines. IL-17 levels were determined by sandwich enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction. The production of IL-17 was significantly increased in cells treated with anti-CD3 antibody with or without anti-CD28 and PHA (P < 0.05). Among tested cytokines and chemokines, IL-15, monocyte chemoattractant protein-1 and IL-6 upregulated IL-17 production (P < 0.05), whereas tumor necrosis factor-alpha, IL-1beta, IL-18 or transforming growth factor-beta did not. IL-17 was also detected in the PBMC of patients with osteoarthritis, but their expression levels were much lower than those of RA PBMC. Anti-CD3 antibody activated the PI3K/Akt pathway; activation of this pathway resulted in a pronounced augmentation of nuclear factor kappaB (NF-kappaB) DNA-binding activity. IL-17 production by activated RA PBMC is completely or partly blocked in the presence of the NF-kappaB inhibitor pyrrolidine dithiocarbamate and the PI3K/Akt inhibitor wortmannin and LY294002, respectively. However, inhibition of activator protein-1 and extracellular signal-regulated kinase 1/2 did not affect IL-17 production. These results suggest that signal transduction pathways dependent on PI3K/Akt and NF-kappaB are involved in the overproduction of the key inflammatory cytokine IL-17 in RA.

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Effects of LY294002 or pyrrolidine dithiocarbamate (PDTC) on anti-CD3 antibody-triggered interleukin (IL)-17 mRNA expression by peripheral blood mononuclear cells (PBMC) from patients with rheumatoid arthritis. PBMC were cultured with medium only (lane 1), anti-CD3 antibody (1 μg/ml; lane 2), anti-CD3 antibody (10 μg/ml; lane 3), anti-CD3 antibody (10 μg/ml) plus LY294002 (20 μM; lane 4) or anti-CD3 antibody (10 μg/ml) plus PDTC (300 μM; lane 5) for 12 hours; lane 6 shows a negative control. Total RNA (2 μg) was used for cDNA synthesis in a volume of 20 μl; 1 μl of the synthesized cDNA was used for reverse transcription–polymerase chain reaction as described. PCR reaction product (25 μl) was separated on an agarose gel containing ethidium bromide. The relative intensities of the bands were revealed under UV radiation.
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Figure 5: Effects of LY294002 or pyrrolidine dithiocarbamate (PDTC) on anti-CD3 antibody-triggered interleukin (IL)-17 mRNA expression by peripheral blood mononuclear cells (PBMC) from patients with rheumatoid arthritis. PBMC were cultured with medium only (lane 1), anti-CD3 antibody (1 μg/ml; lane 2), anti-CD3 antibody (10 μg/ml; lane 3), anti-CD3 antibody (10 μg/ml) plus LY294002 (20 μM; lane 4) or anti-CD3 antibody (10 μg/ml) plus PDTC (300 μM; lane 5) for 12 hours; lane 6 shows a negative control. Total RNA (2 μg) was used for cDNA synthesis in a volume of 20 μl; 1 μl of the synthesized cDNA was used for reverse transcription–polymerase chain reaction as described. PCR reaction product (25 μl) was separated on an agarose gel containing ethidium bromide. The relative intensities of the bands were revealed under UV radiation.

Mentions: To see whether enhanced IL-17 production could be regulated at a transcriptional level, semi-quantatitive reverse transcription–polymerase chain reaction was performed. We observed a dose-dependent increase in IL-17 mRNA transcripts after stimulation with anti-CD3; this was inhibited by the PI3K inhibitor LY294002 and by the NF-κB inhibitor PDTC (Fig. 5).


Increased interleukin-17 production via a phosphoinositide 3-kinase/Akt and nuclear factor kappaB-dependent pathway in patients with rheumatoid arthritis.

Kim KW, Cho ML, Park MK, Yoon CH, Park SH, Lee SH, Kim HY - Arthritis Res. Ther. (2004)

Effects of LY294002 or pyrrolidine dithiocarbamate (PDTC) on anti-CD3 antibody-triggered interleukin (IL)-17 mRNA expression by peripheral blood mononuclear cells (PBMC) from patients with rheumatoid arthritis. PBMC were cultured with medium only (lane 1), anti-CD3 antibody (1 μg/ml; lane 2), anti-CD3 antibody (10 μg/ml; lane 3), anti-CD3 antibody (10 μg/ml) plus LY294002 (20 μM; lane 4) or anti-CD3 antibody (10 μg/ml) plus PDTC (300 μM; lane 5) for 12 hours; lane 6 shows a negative control. Total RNA (2 μg) was used for cDNA synthesis in a volume of 20 μl; 1 μl of the synthesized cDNA was used for reverse transcription–polymerase chain reaction as described. PCR reaction product (25 μl) was separated on an agarose gel containing ethidium bromide. The relative intensities of the bands were revealed under UV radiation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1064895&req=5

Figure 5: Effects of LY294002 or pyrrolidine dithiocarbamate (PDTC) on anti-CD3 antibody-triggered interleukin (IL)-17 mRNA expression by peripheral blood mononuclear cells (PBMC) from patients with rheumatoid arthritis. PBMC were cultured with medium only (lane 1), anti-CD3 antibody (1 μg/ml; lane 2), anti-CD3 antibody (10 μg/ml; lane 3), anti-CD3 antibody (10 μg/ml) plus LY294002 (20 μM; lane 4) or anti-CD3 antibody (10 μg/ml) plus PDTC (300 μM; lane 5) for 12 hours; lane 6 shows a negative control. Total RNA (2 μg) was used for cDNA synthesis in a volume of 20 μl; 1 μl of the synthesized cDNA was used for reverse transcription–polymerase chain reaction as described. PCR reaction product (25 μl) was separated on an agarose gel containing ethidium bromide. The relative intensities of the bands were revealed under UV radiation.
Mentions: To see whether enhanced IL-17 production could be regulated at a transcriptional level, semi-quantatitive reverse transcription–polymerase chain reaction was performed. We observed a dose-dependent increase in IL-17 mRNA transcripts after stimulation with anti-CD3; this was inhibited by the PI3K inhibitor LY294002 and by the NF-κB inhibitor PDTC (Fig. 5).

Bottom Line: Anti-CD3 antibody activated the PI3K/Akt pathway; activation of this pathway resulted in a pronounced augmentation of nuclear factor kappaB (NF-kappaB) DNA-binding activity.However, inhibition of activator protein-1 and extracellular signal-regulated kinase 1/2 did not affect IL-17 production.These results suggest that signal transduction pathways dependent on PI3K/Akt and NF-kappaB are involved in the overproduction of the key inflammatory cytokine IL-17 in RA.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, Division of Rheumatology, The Center for Rheumatic Diseases, and The Rheumatism Research Center (RhRC), Catholic Research Institutes of Medical Sciences, Catholic University of Korea, Seoul, Korea. woon1212@catholic.ac.kr

ABSTRACT
Inflammatory mediators have been recognized as being important in the pathogenesis of rheumatoid arthritis (RA). Interleukin (IL)-17 is an important regulator of immune and inflammatory responses, including the induction of proinflammatory cytokines and osteoclastic bone resorption. Evidence for the expression and proinflammatory activity of IL-17 has been demonstrated in RA synovium and in animal models of RA. Although some cytokines (IL-15 and IL-23) have been reported to regulate IL-17 production, the intracellular signaling pathways that regulate IL-17 production remain unknown. In the present study, we investigated the role of the phosphoinositide 3-kinase (PI3K)/Akt pathway in the regulation of IL-17 production in RA. Peripheral blood mononuclear cells (PBMC) from patients with RA (n = 24) were separated, then stimulated with various agents including anti-CD3, anti-CD28, phytohemagglutinin (PHA) and several inflammatory cytokines and chemokines. IL-17 levels were determined by sandwich enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction. The production of IL-17 was significantly increased in cells treated with anti-CD3 antibody with or without anti-CD28 and PHA (P < 0.05). Among tested cytokines and chemokines, IL-15, monocyte chemoattractant protein-1 and IL-6 upregulated IL-17 production (P < 0.05), whereas tumor necrosis factor-alpha, IL-1beta, IL-18 or transforming growth factor-beta did not. IL-17 was also detected in the PBMC of patients with osteoarthritis, but their expression levels were much lower than those of RA PBMC. Anti-CD3 antibody activated the PI3K/Akt pathway; activation of this pathway resulted in a pronounced augmentation of nuclear factor kappaB (NF-kappaB) DNA-binding activity. IL-17 production by activated RA PBMC is completely or partly blocked in the presence of the NF-kappaB inhibitor pyrrolidine dithiocarbamate and the PI3K/Akt inhibitor wortmannin and LY294002, respectively. However, inhibition of activator protein-1 and extracellular signal-regulated kinase 1/2 did not affect IL-17 production. These results suggest that signal transduction pathways dependent on PI3K/Akt and NF-kappaB are involved in the overproduction of the key inflammatory cytokine IL-17 in RA.

Show MeSH
Related in: MedlinePlus