Also shown (Figure 2D), which demonstrates a remarkable overlap
Zoomed photos are shown under. Scale bars are 100 m, above, and 40 m, below. (D) Tiled confocal microscopy pictures of a spleen section immunostained with CD169 (red) and SIGN-R1 (cyan) and overlaid with fluorescent gp120 (green). As indicated the photos show CD169 alone, CD169 and SIGN-R1; CD169 and gp120; and overlay of all three. Scale bar is 300 m. DOI: 10.7554/eLife.06467.007 The following figure supplements are obtainable for figure two: Figure supplement 1. The IFC network macrophages make contact with CD11c PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/21231855 constructive cells. DOI: 10.7554/eLife.06467.008 Figure supplement 2. Overlay of gp120 visualizes DC-SIGN+/CD163+ macrophages inside a human LN section. DOI: ten.7554/eLife.06467.Park et al. eLife 2015;four:e06467. DOI: 10.7554/eLife.six ofResearch articleImmunologywe immunostained one particular for CD163, a human macrophage marker (Martens et al., 2006), CD11c, and DC-SIGN; and also the other for gp120 and DC-SIGN. This permitted the identification of a group of CD163+, DC-SIGN+, and CD11c- cells close to the LN follicle that bound the overlaid gp120 (Figure 2--figure supplement 2). These benefits indicate that in human LN DC-SIGN expressing macrophage near the follicle may uptake gp120 similar to the mouse IFC SIGN-R1+ macrophages.The SIGN-R1+ subcapsular macrophages uptake gp120 before the SIGN-R1+ IFC macrophagesTo ascertain the kinetics of gp120 uptake by SIGN-R1+ subcapsular macrophages plus the underlying SIGN-R1+ IFC macrophages, we intravitally imaged for three.five hr following gp120 injection. The volume of gp120 associated with the SIGN-R1+ subcapsular macrophages TG-101348 gradually increased after which declined, when the underlying network cells incrementally improved their gp120 binding ultimately surpassing the sinus macrophages (Figure 3A,B, Videos 1, 2).Also shown (Figure 2D), which demonstrates a outstanding overlap between the Also shown (Figure 2D), which demonstrates a outstanding overlap involving the SIGN-R1 and gp120 signals. These final results indicate that there is a network of SIGN-R1 good macrophages inside the LN IFC that offer a platform for nearby B cells and DCs to acquire gp120 and that a subset of SIGN-R1+ cells within the marginal zone in the spleen are also poised to provide gp120 to splenic marginal zone and trafficking follicular B cells. The ability to specifically detect gp120 binding cells using an overlay assay prompted us to figure out no matter if we could detect related macrophages in human LN. To recognize the LN follicles and T cell zone we immunostained a LN section for CD19 and CD4 expression. Employing two adjacent sectionsPark et al. eLife 2015;four:e06467. DOI: 10.7554/eLife.5 ofResearch articleImmunologyFigure 2. IFC cell processes bearing gp120 directly make contact with B cells and also a subset of splenic marginal zone cells also bind gp120. (A) Confocal microscopy of a thick LN section from a mouse previously injected with fluorescent gp120 and immunostained for B220 and CD3. Scale bar is 30 m. Boxed places in left image had been enlarged and shown in the right panels. Scale bars are 10 m. (B) Confocal microscopy of a thick splenic section from a mouse previously injected intravenously with fluorescent gp120 and immunostained with the indicated markers.