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. 2023 Apr 28;220(8):e20220595. doi: 10.1084/jem.20220595

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© 2023 Zec et al.

This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).

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Figure 4. — Activation of microvasculature in the synovial lining niche at the onset of AIA. (A) Representative confocal image of the synovium demonstrating higher number of transferred CXCR2 OE T cells compared with the control T cells and the closer proximity of OE T cells to the lining (CD4⁺; CD8⁺ T cells, green; VSIG4, magenta; control T cells, cyan; CXCR2 OE T cells, orange; nuclei, gray; scale bar = 30 µm); single experiment with two animals. (B) Numbers ratio of CXCR2 OE T cells to control T cells measured by FACS in the blood and the synovium of mice 6 h p.c. (5 h after adoptive T cell transfer) shows preferential recruitment of CXCR2 OE T cells to the synovium. Significance value by paired t test: *, P < 0.05. Experiment performed two times. (C) CXCR2 OE T cells in the synovial cavity interacting with the lining macrophage (CD177, green; VSIG4, magenta; CD31, blue; control T cells, cyan; CXCR2 OE T cells, orange; nuclei, gray; scale bar = 10 µm). (D) Confocal image of the whole mouse knee cross-section immunostained for endothelial activation marker E-selectin (CD62E) reveals distinct position of activated vasculature close to the lining (CD62E, green; CD31, magenta; CD68, cyan; lining fibroblast Prg4, orange; nuclei, gray; scale bar = 200 µm). (E) Segmentation of the vasculature in the anterior synovium of the image in A using Imaris to classify highly activated (CD62E^high) and non-activated (CD62E^−/low) blood vessels. Inset shows representative distribution of activated microvasculature in regards to the synovial lining of the image mask in E; each point is one blood vessel surface of the respective image; median and interquartile range indicated. (F) Enlarged region of the synovial lining with CD62E^high blood vessel is shown in the left panel, whereas the right panel shows patellar region with CD62E^high blood vessels in direct contact with lining macrophages (arrowheads), as well as an activated larger-diameter blood vessel situated a few cell layers deeper (scale bar = 20 µm). B, bone; F, femur; FP, fat pad; M, meniscus; SC, synovial cavity; T, tibia. Images and quantification representative of four animals from two independent experiments.