Immunnohistochemical analysis of CD200R expression in macrophages, the role of CD200-CD200R axis in hMSCs mediated immunosuppression.

<p>The immunofluorescent staining with PE-conjugated anti-human CD200R (red) against nuclear marker Hoechst (blue) revealed interesting differences in CD200R expression between <b>A,D</b>) un-stimulated (24 h culture without LPS or IFN-γ) THP-1 macrophage-like cells (magnification 400×) <b>B,E</b>) LPS stimulated (100 ng/ml for 24 h) THP-1 macrophage-like cells (magnification 400×) and <b>C,F</b>) IFN-γ stimulated (100 ng/ml for 24 h) THP-1 macrophage-like cells (magnification 400×). CD200R expression was clearly evident only after IFN-γ stimulation whereas LPS stimulated and unstimulated THP-1 macrophage-like cells did not revealed clear CD200R expression. <b>G</b>) CD200Hi, CD200Me and C200Lo BMMSC lines decreased significantly TNF-α secretion from THP-1 macrophage-like cells in presence of IFN-γ whereas UCBMSC lines did not. There were also significant differences between CD200Hi, CD200Me and CD200Lo BMMSC lines in their ability to inhibit TNF-α secretion of IFN-γ (100 ng/ml for 24 h) activated THP-1 macrophage-like cells whereas <b>H</b>) there were no correlation between CD200 expression and the ability of hMSCs to decrease TNF-α secretion from THP-1 macrophage-like cells in presence of LPS (100 ng/ml for 24 h). Only CD200Me 1, CD200Lo 2 and UCBMSC 2 lines had significant reduction in TNF-α secretion. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031671#s3" target="_blank">Results</a> are represented as a mean±SD of three independent replicates. TNF-α concentration in supernatants from LPS or IFN-γ activated THP-1 macrophage-like cells alone were indicated as 100% and TNF-α concentration in supernatants from co-cultures with different hMSC lines were compared to that. Before co-cultures, THP-1 macrophage-like cells were differentiated 48 hours in PMA (100 ng/ml) supplemented medium as described in materials and methods. * and ** indicates two-sample t-test p-values p<0.05 and p<0.01. !!! indicates one way analysis of variance (ANOVA) between CD200Hi, CD200Me and CD200Lo BMMSCs p-value p<0.001.