IL-27 is constitutively expressed in retina and inhibits T cells that mediate EAU
IL-27 is predominantly expressed in antigen-presenting lymphoid cells such as dendritic cells. Using a combination of RT-PCR, Western blot analyses we demonstrate expression of IL-27 in the retina and retina cells. Expression of IL-27 was localized to the retinal ganglion and photoreceptor cells as well as the retina pigmented epithelium (RPE) by immunohistochemical analysis. Expression of IL27 in the retina was found to be positively regulated in retinal cells by the proinflammatory cytokine, IFN-γ and chromosome immunoprecipitation assay (Chip) revealed direct binding of STAT1 transcription factor to the IL27 promoter.
RT-PCR and Quantitative RT-PCR (qRT-PCR)
All RNA samples were DNA-free and cDNA was generated with SuperScript III Reverse Transcriptase (Invitrogen, Gaithersburg, MD) and oligo(dT)12–16.
Perform RT-PCR with AmpliTaq Gold DNA polymerase (ABI, Foster City, CA) as described 7. (For genes with introns, each gene-specific primer pair was designed to span at least an intron. All cDNA preparations used were found to be suitable substrates for PCR amplification on the basis of efficient amplification of a β-actin sequence.)
Real-time RT-PCR (qRT-PCR) analysis was performed on an ABI 7500 (Applied Biosystems, Foster City, CA) as previously described 8 using primers and probes obtained from Applied Biosystems. The mRNA expression levels were normalized to both β-Actin and GAPDH house keeping genes. (see above for step-by-step procedures)
Western blot analyses
ARPE-19 or human Muller cell line (MIO-M1 were starved for 2 hours and cultured in DMEM supplemented with IFNγ for 15, 30, 60, 90 minutes in the presence or absence of AG-490 or the protein synthesis inhibitor, cycloheximide. Blots were probed with rabbit polyclonal IL-27 antibody or pSTAT1, IRF-1, IRF-8, β-Actin specific antibodies. Pre-immune serum was used in parallel as controls and signals were detected with the ECL-PLUS system.
The steps described in this protocol are:
Stimulation of cultured cells with Cytokines and preparation of whole cell extracts
Semi-Dry Transfer Assembly
Detection with ECL-plus
Other protocols related to our Nature Medicine paper can be found here:
"Detection of TH17 cells in human blood":http://www.natureprotocols.com/2007/08/30/th17_cells_contribute_to_uveit.php
"Analysis by confocal microscopy":http://www.natureprotocols.com/2007/08/31/th17_cells_contribute_to_uveit_3.php
"Retinal cell isolation":http://www.natureprotocols.com/2007/08/31/th17_cells_contribute_to_uveit_5.php