Figure 7
Figure 7. TREM-1 expression in vivo in hypoxic mDCs from JIA-SF. SF was collected from children affected by JIA, and SFMCs were purified as detailed in “SFMC isolation.” (A) sTREM-1 concentrations were quantified by ELISA in paired plasma and SF from 8 JIA patients and plasma from 5 age-matched control subjects. Individual samples were run in triplicate. Boxes represent the values falling between the 25th and 75th percentiles; whiskers, the highest and lowest values for each subgroup. Bold horizontal lines represent median values. P value by the Wilcoxon rank test is indicated: *P < .01 relative to plasma controls; **P < .001 relative to plasma from JIA patients. (B) HIF-1α expression was assessed by Western blot analysis on whole protein extracts from 3 pairs of fresh PBMCs and SFMCs purified from a subset of the JIA patients shown in panel A. Protein (100 μg) was resolved on 8% SDS-PAGE, and the blots were hybridized with anti-HIF-1α mAb. β-actin was evaluated as a loading control. (C) Flow cytometric analysis of TREM-1 expression in SF-mDCs. SFMCs from the 3 JIA patients shown in panel B were stained with anti-CD1a-allophycocyanin, CD83-FITC, and-TREM-1-PE Abs and analyzed on a FACScan. Cells were electronically gated according to their light scatter properties to exclude cell debris. The gated population was analyzed for CD1a positivity (left panels, region R1), and CD1a+ cells were then examined for CD83 and TREM-1 expression (right panels). Nonspecific staining was corrected using isotype-matched Abs. Numbers represent the percentage of single- and double-positive cells within the CD1a-gated population and are indicated for each patient: TREM-1/CD83 double-positive cells are contained in the top right quadrant, whereas CD83 and TREM-1 single-positive cells are contained in the bottom right and top left quadrants, respectively. Cells stained with control Abs are in the bottom left quadrant. The percentage of TREM-1+ cells relative to the total number of CD83+ cells (representing TREM-1-expressing mDCs) was 88%, 60%, and 80% in patients 1, 3, and 6, respectively, whereas the percentage of TREM-1+ cells relative to the total number of CD83− cells (representing TREM-1-expressing iDCs) was 74%, 80%, and 80% in patients 1, 3, and 6, respectively.

TREM-1 expression in vivo in hypoxic mDCs from JIA-SF. SF was collected from children affected by JIA, and SFMCs were purified as detailed in “SFMC isolation.” (A) sTREM-1 concentrations were quantified by ELISA in paired plasma and SF from 8 JIA patients and plasma from 5 age-matched control subjects. Individual samples were run in triplicate. Boxes represent the values falling between the 25th and 75th percentiles; whiskers, the highest and lowest values for each subgroup. Bold horizontal lines represent median values. P value by the Wilcoxon rank test is indicated: *P < .01 relative to plasma controls; **P < .001 relative to plasma from JIA patients. (B) HIF-1α expression was assessed by Western blot analysis on whole protein extracts from 3 pairs of fresh PBMCs and SFMCs purified from a subset of the JIA patients shown in panel A. Protein (100 μg) was resolved on 8% SDS-PAGE, and the blots were hybridized with anti-HIF-1α mAb. β-actin was evaluated as a loading control. (C) Flow cytometric analysis of TREM-1 expression in SF-mDCs. SFMCs from the 3 JIA patients shown in panel B were stained with anti-CD1a-allophycocyanin, CD83-FITC, and-TREM-1-PE Abs and analyzed on a FACScan. Cells were electronically gated according to their light scatter properties to exclude cell debris. The gated population was analyzed for CD1a positivity (left panels, region R1), and CD1a+ cells were then examined for CD83 and TREM-1 expression (right panels). Nonspecific staining was corrected using isotype-matched Abs. Numbers represent the percentage of single- and double-positive cells within the CD1a-gated population and are indicated for each patient: TREM-1/CD83 double-positive cells are contained in the top right quadrant, whereas CD83 and TREM-1 single-positive cells are contained in the bottom right and top left quadrants, respectively. Cells stained with control Abs are in the bottom left quadrant. The percentage of TREM-1+ cells relative to the total number of CD83+ cells (representing TREM-1-expressing mDCs) was 88%, 60%, and 80% in patients 1, 3, and 6, respectively, whereas the percentage of TREM-1+ cells relative to the total number of CD83− cells (representing TREM-1-expressing iDCs) was 74%, 80%, and 80% in patients 1, 3, and 6, respectively.

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