Isolation and characterization of dermal lymphatic and blood endothelial cells reveal stable and functionally specialized cell lineages

Abstract

A plexus of lymphatic vessels guides interstitial fluid, passenger leukocytes, and tumor cells toward regional lymph nodes. Microvascular endothelial cells (ECs) of lymph channels (LECs) are difficult to distinguish from those of blood vessels (BECs) because both express a similar set of markers, such as CD31, CD34, podocalyxin, von Willebrand factor (vWF), etc. Analysis of the specific properties of LECs was hampered so far by lack of tools to isolate LECs. Recently, the 38-kD mucoprotein podoplanin was found to be expressed by microvascular LECs but not BECs in vivo. Here we isolated for the first time podoplanin(+) LECs and podoplanin(-) BECs from dermal cell suspensions by multicolor flow cytometry. Both EC types were propagated and stably expressed VE-cadherin, CD31, and vWF. Molecules selectively displayed by LECs in vivo, i.e., podoplanin, the hyaluronate receptor LYVE-1, and the vascular endothelial cell growth factor (VEGF)-C receptor, fms-like tyrosine kinase 4 (Flt-4)/VEGFR-3, were strongly expressed by expanded LECs, but not BECs. Conversely, BECs but not LECs expressed VEGF-C. LECs as well as BECs formed junctional contacts with similar molecular composition and ultrastructural features. Nevertheless, the two EC types assembled in vitro in vascular tubes in a strictly homotypic fashion. This EC specialization extends to the secretion of biologically relevant chemotactic factors: LECs, but not BECs, constitutively secrete the CC chemokine receptor (CCR)7 ligand secondary lymphoid tissue chemokine (SLC)/CCL21 at their basal side, while both subsets, upon activation, release macrophage inflammatory protein (MIP)-3alpha/CCL20 apically. These results demonstrate that LECs and BECs constitute stable and specialized EC lineages equipped with the potential to navigate leukocytes and, perhaps also, tumor cells into and out of the tissues.

Figure 1

Identification of dermal BECs and LECs in cryostat sections of human skin (A–C), and isolation by FACS^® (D–F). (A–C) Immunofluorescence double-labeling using Abs to podoplanin (TRITC, red), and CD34 (FITC, green). FITC and TRITC fluorescence images are shown in B and C. Panel A illustrates the double exposure with LECs (yellow-red) expressing both podoplanin and CD34. Microvascular tubes shown are ascending from the superficial vascular plexus (original magnification: ×400). (D–F) Isolation of BECs and LECs from dermal cell suspensions (approach A) prepared by enzymatic digestion. Cells were triple-labeled with anti-CD45 Cy5, anti-CD34-PE, and anti-podoplanin. CD45⁻ cells were gated electronically, and podoplanin ⁻/CD34^high (green, BECs) and podoplanin⁺/CD34^low ECs (red, LECs) were identified (D) and isolated (E and F) by FACS^®. The purity of the two sorted EC subsets was >98%.

Figure 2

BECs and LECs cannot be distinguished by conventional light microscopy when grown separate (A, BECs; B, LECs). (C) In mixed cultures, immunolabeling with anti-podoplanin (TRITC, red) and anti-vWF (FITC, green) reveal multicellular islands of podoplanin⁺/vWF⁺ LECs surrounded by podoplanin⁻/vWF⁺ BECs. BECs express more vWF than LECs (see also Fig. 3 A). Original magnification in A and B: ×300; C: 250. To confirm their phenotypic stability, cultured LECs and BECs were harvested, labeled with anti-podoplanin IgG, and analyzed by FACS ^® (insets in A–C; dotted line denotes the cut-off for podoplanin positivity). Cultured BECs (A) were homogeneously podoplanin-negative, cultured LECs (B) exclusively podoplanin-positive, while the mixed EC population (C) contained both qualities.

Figure 3

Isolated LECs and BECs maintain their lineage-restricted antigen expression profile during in vitro expansion. (A) FACS^® analysis. Primary LECs and BECs were isolated by FACS^®, and propagated in vitro. Single cell suspensions of BECs or LECs were exposed to anti-CD31, anti–VE-cadherin/cadherin-5, anti-CD44, anti–PAL-E or to isotype-matched control mAbs and to rabbit anti-vWF, anti-podoplanin, or to preimmune rabbit serum. Dead cells were excluded by propidium iodide counterstaining and appropriate gate settings. For the analysis of vWF expression, ECs were fixed and permeabilized before immunostaining. Cells were used after the sixth passage (expansion factor: ×2,500) with the exception of cells that were stained with PAL-E which were harvested after passage two. X-axis: log fluorescence intensity; y-axis: relative cell numbers. The reactivities of specific and control Abs are shown by open and closed histograms, respectively. (B) Northern and (C and D) Western blot analyses. Third passage bulk microvascular ECs were flow sorted into podoplanin⁻/CD31⁺/CD45⁻ BEC and podoplanin⁺/CD31⁺/CD45⁻ LEC populations. After two further passages cells were harvested and total cellular RNA and protein were isolated. (B) RNA transfers were sequentially hybridized with ³²P-labeled probes derived from the cDNA of podoplanin, Flt-4, LYVE-1, CD31, and VEGF-C. To ensure equal loading and transfer of RNA, membranes were finally hybridized with an actin probe. Positions of standards are shown on the right in kb. (C and D) Equal amounts of LEC- and BEC-derived proteins were blotted (D, left panel), membranes were probed with Abs against podoplanin, KDR, Flt-1, CD31, Tie-1, Tie-2 (C), or Flt-4 (D, right panel), and Ab reactivity was revealed by chemoluminescence. Positions of molecular weight markers are shown in kD.

Figure 3

Isolated LECs and BECs maintain their lineage-restricted antigen expression profile during in vitro expansion. (A) FACS^® analysis. Primary LECs and BECs were isolated by FACS^®, and propagated in vitro. Single cell suspensions of BECs or LECs were exposed to anti-CD31, anti–VE-cadherin/cadherin-5, anti-CD44, anti–PAL-E or to isotype-matched control mAbs and to rabbit anti-vWF, anti-podoplanin, or to preimmune rabbit serum. Dead cells were excluded by propidium iodide counterstaining and appropriate gate settings. For the analysis of vWF expression, ECs were fixed and permeabilized before immunostaining. Cells were used after the sixth passage (expansion factor: ×2,500) with the exception of cells that were stained with PAL-E which were harvested after passage two. X-axis: log fluorescence intensity; y-axis: relative cell numbers. The reactivities of specific and control Abs are shown by open and closed histograms, respectively. (B) Northern and (C and D) Western blot analyses. Third passage bulk microvascular ECs were flow sorted into podoplanin⁻/CD31⁺/CD45⁻ BEC and podoplanin⁺/CD31⁺/CD45⁻ LEC populations. After two further passages cells were harvested and total cellular RNA and protein were isolated. (B) RNA transfers were sequentially hybridized with ³²P-labeled probes derived from the cDNA of podoplanin, Flt-4, LYVE-1, CD31, and VEGF-C. To ensure equal loading and transfer of RNA, membranes were finally hybridized with an actin probe. Positions of standards are shown on the right in kb. (C and D) Equal amounts of LEC- and BEC-derived proteins were blotted (D, left panel), membranes were probed with Abs against podoplanin, KDR, Flt-1, CD31, Tie-1, Tie-2 (C), or Flt-4 (D, right panel), and Ab reactivity was revealed by chemoluminescence. Positions of molecular weight markers are shown in kD.

Figure 4

In vitro–cultured LECs contain caveolin and caveolae, and WP bodies. ECs were grown to confluence, fixed, labeled by indirect immunofluorescence for caveolin (A), or by a preembedding immunogold protocol for podoplanin (B). ECs express uniformly caveolin in a granular pattern (A) that corresponds in LECs to typical endothelial caveolae by electron microscopy (B). (C and D) Electron microscopy also reveals WP bodies in podoplanin-expressing LECs. Original magnification in A: ×700; B: ×12.000; C and D: ×45.000.

Figure 6

BECs and LECs form separate homotypic layers in mixed EC cultures. Mixed BECs and LECs were allowed to reach confluence and then were cultured for four additional days. (A–D) Cells were fixed and stained with anti-podoplanin Abs (FITC) and anti-CD31 (TRITC). In the double exposure shown in A, podoplanin⁺/CD31⁺ ECs (yellow) appear to cover the monolayer of podoplanin⁻/CD31⁺ ECs (red). In B–D, vertical optical sectioning by confocal microscopy directly shows that podoplanin⁺/CD31⁺/LECs (green; C and D) are positioned on top of podoplanin⁻CD31⁺ BECs (red; B and D). The blue line indicates the position of the culture dish. In E, EC bilayers were fixed and processed for anti-podoplanin immunogold labeling and vertical sections were analyzed by electron microscopy. LECs as identified by their intense labeling with 10-nm gold particles are found on top of nonlabeled BECs. Original magnification in A–D: ×1,000; E: ×40.000.

Figure 5

LECs form continuous homotypic cell junctions. (A–D) Cell contacts formed by LECs and those by BECs differ in the extent of junctional protein recruitment. Confluent LECs (A and C) and BECs (B and D) were fixed, and stained with anti-CD31 (A and B) or anti–VE-cadherin (C and D). LECs display a thin rim of CD31 and VE-cadherin in their junctional areas while BECs form ruffled, indented homotypic junctions with a broad overlap of the VE-cadherin- and CD31-bearing cell membranes. (E-I) Podoplanin⁺ LECs were identified by 10 nm immunogold labeling. LECs form typical junctional complexes consisting of adherent junctions (E, double-headed arrow) and tight junctions (arrowheads in E–H). (I) Occasionally, gap junctions were observed. Similar junctional structures were also found on cultured, immunogold-negative BECs (J–L). Asterisk in L indicates a Weibel-Palade body, gap junctions are marked (GJ). Original magnification in A–D: ×700; E, G, I, K, and L: ×75.000; F and J: ×40.000; H: ×120.000.

Figure 8

Chemokine secretion of isolated LECs and BECs. (A and B) LECs, but not BECs, secrete SLC/CCL21, but both EC types produce MIP-3α/CCL20 upon activation. EC subsets grown to confluence were exposed to EGF- and hydrocortisone-deficient medium (non-stim.) or to the same medium supplemented with TNFα or IL-1β. 24 h supernatants were analyzed by CCL21- (A) and CCL20-specific ELISAs (B). The concentrations of chemokines produced by BECs (hatched bars) and LECs (black bars) are shown (mean values (±SD) obtained in two independent experiments). (C) LECs secrete CCL21 but not CCL20 basolaterally. LECs grown to confluence on 0.4-μm poresize Transwell^TM filters, were nonstimulated (non-stim.) or stimulated with TNFα- or IL-1β for 24 h. CCL21 (black bars) and CCL20 (hatched bars) secreted into the upper (apical) and the lower (basolateral) chamber of the Transwells™ were measured. The percentage of chemokine recovered from the lower chamber relative to the total amount of secreted chemokine (i.e., secreted into the upper and lower chamber) is shown. Mean values (±SD) obtained from triplicate cultures; n.d.; not done. (D) LEC monolayers form a tight barrier for exogenous CCL19 used as a tracer. To control for leakage of chemokines through the LEC monolayer, MIP-3β/CCL19 was added to the upper chamber. Data are given as the percentage of the amount of chemokine recovered from the lower chamber relative to the amount of chemokine retrieved from the upper and lower chamber. Approximately 80% of initially added CCL19 was recovered after 24 h. Mean values (±SD) obtained from triplicate cultures.

Figure 7

Lymph and blood ECs form independent capillary tubes that wind around each other. Flow-sorted primary BECs or LECs were expanded through six passages, harvested, and labeled with cell-permeant dyes emitting red and green fluorescence, respectively. Labeled BECs (A and B; red), LECs (C and D; green), or BECs and LECs mixed in a 1:1 ratio (E–H) were seeded onto Matrigel, cultured in EGF- and hydrocortisone-deficient medium for 24 h, fixed, and analyzed by confocal microscopy. A, C, and E are phase contrast images corresponding to the fluorescence images B (red), D (green), and F–H (green, red), respectively. F and G show the single red and single green fluorescence signals of the double exposure in H. (F and G) In mixed cultures, BECs and LECs form tubes that are built up by one EC subtype only. (H) Homotypic BEC and LEC tubes are closely juxtaposed in a double helical pattern. Original magnification in A–G: ×100; H: ×400.