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. 2008 Oct;173(4):1202-9.
doi: 10.2353/ajpath.2008.080101. Epub 2008 Sep 4.

Lymphatic Precollectors Contain a Novel, Specialized Subpopulation of Podoplanin Low, CCL27-expressing Lymphatic Endothelial Cells

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Free PMC article

Lymphatic Precollectors Contain a Novel, Specialized Subpopulation of Podoplanin Low, CCL27-expressing Lymphatic Endothelial Cells

Nikolaus Wick et al. Am J Pathol. .
Free PMC article

Abstract

Expression of the lymphoendothelial marker membrane mucoprotein podoplanin (podo) distinguishes endothelial cells of both blood and lymphatic lineages. We have previously discovered two distinct subpopulations of lymphatic endothelial cells (LECs) in human skin that were defined by their cell surface densities of podoplanin and were designated LEC podo-low and LEC podo-high. LEC podo-low is restricted to lymphatic precollector vessels that originate from initial LEC podo-high-containing lymphatic capillaries and selectively express several pro-inflammatory factors. In addition to the chemokine receptor protein Duffy blood group antigen receptor for chemokines, these factors include the constitutively expressed chemokine CCL27, which is responsible for the accumulation of pathogenic CCR10+ T lymphocytes in human inflammatory skin diseases. In this study, we report that CCR10+ T cells accumulate preferentially both around and within CCL27+ LEC podo-low precollector vessels in skin biopsies of human inflammatory disease. In transmigration assays, isolated CCR10+ T lymphocytes are chemotactically attracted by LEC podo-low in a CCL27-dependent fashion, but not by LEC podo-high. These observations indicate that LEC podo-low-containing precollector vessels constitute a specialized segment of the initial lymphatic microvasculature, and we hypothesize that these LEC podo-low-containing vessels are involved in the trafficking of CCR10+ T cells during skin inflammation.

Figures

Figure 1
Figure 1
Ex vivo isolation, in vitro expansion, and characterization of LECpodo-low and LECpodo-high. A: BECs, LECs, LECpodo-low, and LECpodo-high were separated by fluorescence-activated cell sorting ex vivo from CD45-gated human dermatome skin cell suspensions, using CD31 and podoplanin antibodies. CD31+ podoplanin BECs (top left), CD31+ podoplanin+ total LECs (top right, the red bar separates two overlapping subpopulations), CD31+ podoplaninhigh LECs (bottom left, red circle), and CD31+podoplaninlow LECs (bottom right, red circle) are resolved. B: Cytospin preparations of ex vivo isolated LECpodo-low and LECpodo-high show differential podoplanin expression by immunofluorescence. C: Nonquantitative RT-PCR reveals the expression of the LEC marker genes Prox1 and LYVE-1, and the pan-endothelial marker von Willebrand factor (vWF) in LECpodo-low and LECpodo-high. D: After two rounds of expansion in tissue culture of LECpodo-low and LECpodo-high, the differential expression of podoplanin protein is confirmed by immunoblotting and related to CD31 expression. E: Quantitative RT-PCR confirmed the different levels of podoplanin mRNA expression (P < 0.001). F: Prox1 is stably expressed in cultured LECpodo-low and more abundant in LECpodo-high, whereas expression of the LECpodo-low specific marker CCL27, indicated here by red immunofluorescence in cultured LECpodo-low (G, left) is lost after the third passage in cell culture. LECpodo-high fail to express CCL27 (G, right). All bars = 10 μm.
Figure 2
Figure 2
Segmentation of the dermal lymphatic microvasculature into LECpodo-low precollectors and LECpodo-high initial lymphatic capillaries, revealed by expression of distinct marker proteins. A: Localization of DARC (left, red), podoplanin (middle, green), and a merged picture with 4′,6′-diamidino-2-phenylindole hydrochloride nuclear staining (right, blue). Endothelial cells of the LECpodo-low DARC+ type are restricted to a small vessel and do not intermix with the larger LECpodo-high DARC vessel. B: Immunoelectron microscopy supports the differential expression of podoplanin on the luminal cell surfaces of both LEC subpopulations. The density of gold particles that indicate podoplanin differ three- to fourfold (P < 0.05). C: Three-dimensional reconstitution of the initial lymphatic microvessels in human skin resolved by confocal laser scanning microscopy and subsequent three-dimensional reconstruction of 100-μm-thick sections, using antibodies specific for podoplanin (green) and CCL27 (red). The LECpodo-low indicators CCL27 and podoplaninlow are restricted to a distinct vessel (Low) that branches out of a larger, LECpodo-high CCL27 initial lymphatic capillary (High). Merged versions of rotated presentations of the anastomosing vessels are shown on the left, and single channels on the right. D: Triple immunofluorescence showing coexpression of high levels of podoplanin (blue) with CCL21 (red) in LECpodo-high (High) and of lower levels of podoplanin with CCL27 (green) in LECpodo-low (Low). Merge of channels is on the bottom panel. Bars = 10 μm in A, C, and D. Magnification: ×70,000 (D).
Figure 3
Figure 3
Segregation of LECpodo-low and LECpodo-high vessels in human kidney transplant rejection (A) and chronic bronchiolitis (B). Sections are immunolabeled with antibodies specific for podoplanin (green, left) and DARC (red, middle), and merged (right). DARC expression is restricted to vessels containing LECpodo-low (Low) and absent from those containing LECpodo-high (High). Nuclear 4′,6′-diamidino-2-phenylindole hydrochloride stain is blue. Bar = 10 μm.
Figure 4
Figure 4
Association of CCR10+ T lymphocytes with LECpodo-low-containing lymphatic precollector vessels. A: Determination of the blood and lymphatic microvessel densities in normal (n = 14) and inflamed human skin samples (n = 15) expressed as percentage of the total vessel number counted (all P values <0.001; error bars are SD). The ratio of vessel densities remain constant in normal and inflamed skin. B: Sections of a case with atopic dermatitis are immunolabeled for the localization of CCL27 (top left, green), podoplanin (bottom left, blue), and CCR10 (top right, red) and a merged picture on the bottom right. CCL27 and podoplanin differentially mark a LECpodo-high initial lymphatic capillary (High) and a LECpodo-low precollector (Low). CCR10 is expressed in inflammatory cells that are centered around the precollector, and it is also expressed in small amounts in LECpodo-low (Low). Bar = 20 μm. C: Histogram showing the preferential accumulation of CCR10+ T lymphocytes around LECpodo-low precollectors (dark green columns) and LECpodo-high lymphatic capillaries (light green columns) and in the vascular lumen (Lum). R1, R2, and R3 indicate concentric perivascular areas with increasing radius (10 μm) in which the quantitation is performed. T lymphocytes are encountered up to three times more frequently around LECpodo-low precollectors than around LECpodo-high lymphatic capillaries (all P values <0.05; error bars = SD) and are found exclusively within the lumina of LECpodo-low precollectors (Lum). D: Chemotactic transmigration assay of isolated human CCR10+ CLA+ CD3+ T lymphocytes toward LECpodo-low (dark green columns), LECpodo-high (light green column), and BECs (red column) grown in the lower compartment of Transwell chambers. Transmigration of T lymphocytes is observed toward LECpodo-low and is reduced by a blocking antibody against CCL27 (Blocking IgG, dark green column, red-shaded) but not by a control IgG (Control, dark green column). Attraction by LECpodo-high and BECs are considered as baseline values (all P values <0.05; error bars = SD).

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