Authors
Charles Egwuagu, Cheng-Rong Yu, Ahjoku Amadi-Obi, Xuebin Liu, Rashid Mahdi, and Yun Sang Lee
Summary
This study implicates interleukin-17 (IL-17)-producing T-cells (TH17 cells) in etiology of human uveitis and scleritis. We show that blood of patients with uveitis or scleritis contained more TH17 cells than blood of healthy individuals. The numbers of TH17 cells increased during active uveitis/scleritis and decreased following treatment and in a mouse model, treatment with an IL-17-specific antibody reduced severity of ocular inflammation. These results suggest that TH17 cells may mediate eye diseases by inducing production of TNFa, as large amounts of this cytokine are found in retinal cells of mice with ocular inflammation. IL-2 was found to promote TH17 expansion while IFNg, produced by another T-cell type, inhibits TH17 proliferation by upregulating IL-27 expression. This study provides explanations for efficacy of IL-2R antibody therapy in uveitis and suggests that antagonism of TH17 by IFNg and IL-27 could be used for treatment of chronic inflammation.
In this protocol we described the detection of TH17 cells in human blood.
IL-17-expressing T helper cells are implicated as etiologic agents of several chronic inflammatory diseases of rodents. Peripheral blood mononuclear cells (PBMC) of normal human subjects and patients with uveitis (intraocular inflammatory diseases) or scleritis (sight-threatening idiopathic inflammatory ocular disease) were analyzed to examine whether TH17 cells are also etiologic agents of human chronic inflammatory diseases.
The steps of this protocol are:
- Isolation of human peripheral blood mononuclear cells (PBMC) and CD4+ T cells
- Expansion of Th17 cells in human PBMC by IL-2
- Intracellular cytokine staining assay
- RT-PCR and Quantitative RT-PCR (qRT-PCR)
- DNAse Digestion of RNA Samples
- First Strand cDNA Synthesis
Further details
The protocol was published on Protocol Exchange in 2007. To see the entire protocol, click on the source link.
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