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The endothelial protein PLVAP in lymphatics controls the entry of lymphocytes and antigens into lymph nodes

Nature Immunology volume 16, pages 386–396 (2015)Cite this article

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An Erratum to this article was published on 21 April 2015

This article has been updated

Abstract

In the lymphatic sinuses of draining lymph nodes, soluble lymph-borne antigens enter the reticular conduits in a size-selective manner and lymphocytes transmigrate to the parenchyma. The molecular mechanisms that control these processes are unknown. Here we unexpectedly found that PLVAP, a prototypic endothelial protein of blood vessels, was synthesized in the sinus-lining lymphatic endothelial cells covering the distal conduits. In PLVAP-deficient mice, both small antigens and large antigens entered the conduit system, and the transmigration of lymphocytes through the sinus floor was augmented. Mechanistically, the filtering function of the lymphatic sinus endothelium was dependent on diaphragms formed by PLVAP fibrils in transendothelial channels. Thus, in the lymphatic sinus, PLVAP forms a physical sieve that regulates the parenchymal entry of lymphocytes and soluble antigens.

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Figure 1: Activation of B lymphocytes and T lymphocytes is induced in the lymph nodes of Plvap−/− mice.
Figure 2: PLVAP is expressed in LECs in lymph node sinus.
Figure 3: PLVAP diaphragms overlay the distal terminals of the conduit system in lymph nodes.
Figure 4: PLVAP determines the size-selective entry of antigens into conduits in the PLNs.
Figure 5: PLVAP diaphragms in transendothelial channels regulate the filling of conduits.
Figure 6: PLVAP-dependent migration of lymph-borne lymphocytes through the floor of lymphatic sinus to the PLN parenchyma.
Figure 7: PLVAP is concentrated in low-resistance areas of sinusoidal LECs that support transcellular lymphocyte migration.

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Change history

  • 20 March 2015

    In the version of this article initially published, the gray box in the key in the left graph in Figure 6a is labeled incorrectly. The correct label should be Plvap–/–. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank E. Butcher (Stanford University) for antibody MECA-367; The Cell Imaging Core at the Centre of Biotechnology and Laboratory of Electron Microscopy in University of Turku for help in imaging; E.-L. Väänänen, R. Sjöroos, S. Mäki and M. Pohjansalo for technical assistance; and A. Sovikoski-Georgieva for secretarial assistance. This work benefitted from data assembled by the Immunological Genome Project consortium. Supported by the Finnish Academy and Sigrid Juselius Foundation.

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  1. Pia Rantakari and Kaisa Auvinen: These authors contributed equally to this work.

Authors and Affiliations

  1. MediCity Research Laboratory, University of Turku, Turku, Finland

    Pia Rantakari, Kaisa Auvinen, Norma Jäppinen, Maria Kapraali, Joona Valtonen, Marika Karikoski, Heidi Gerke, Imtiaz Iftakhar-E-Khuda, Johannes Keuschnigg, Masayuki Miyasaka, Kati Elima, Sirpa Jalkanen & Marko Salmi

  2. WPI Immunology Frontier Research Center, Osaka University, Japan

    Eiji Umemoto & Masayuki Miyasaka

  3. Department of Anatomy, Kansai University of Health Sciences, Osaka, Japan

    Kazuo Tohya

  4. Department of Medical Biochemistry and Genetics, University of Turku, Turku, Finland

    Kati Elima

  5. Department of Medical Microbiology and Immunology, University of Turku, Turku, Finland

    Sirpa Jalkanen & Marko Salmi

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Contributions

P.R. and K.A. planned and performed experiments and analyzed data; N.J., M.Kap., J.V., M.Kar., H.G., I.I.-E.-K., J.K. and E.U. performed experiments; K.T. performed scanning electron microscopy; K.E. supervised the generation of Plvap−/− mice; M.M. and S.J. contributed to the preparation of the manuscript; and M.S. conceived of the project, directed the research and wrote the manuscript.

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Correspondence to Marko Salmi.

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Integrated supplementary information

Supplementary Figure 1 Generation of Plvap−/− mice.

(a) Schematic presentation of the targeting construct as well as the wild-type and targeted Plvap alleles]. PuroR, puromycin resistance. The location of the probes (5ext1, 3ext2, CAG) used in hybridizations, and the PCR primers (A,B,C) are depicted. (b) Southern blot analysis of Ssp1-digested genomic DNA isolated from wild-type (Plvap+/+) and heterozygous (Plvap+/−) embryonic stem cell clones (G7, G11, C9 and H4) with the indicated probes. The sizes of the hybridization bands corresponding wild-type (WT) and targeted (Targ) alleles are indicated. (c) Genotyping of the WT (Plvap+/+), Plvap+/− and Plvap−/− mice from heterozygous matings by PCR with the primers A-C. The sizes of the expected WT and targeted products are indicated. Primers A-B: lanes1,3,5,7; Primers B-C: lanes 2,4,6,8. (d) Immunoblotting of spleen, kidney and PLN lysates from littermate WT (Plvap+/+) and Plvap−/− mice with anti-PLVAP (MECA-32) mAb. The specific PLVAP band (105 kDa) is indicated by an arrow. GAPDH is a loading control. Data are representative of three or more independent experiments.

Supplementary Figure 2 The phenotype of lymphocytes and subcapsular sinus in lymph nodes of wild-type and Plvap−/− mice.

(a-c) Flow cytometry of (a) surface immunoglobulin receptors IgM and IgD, (b) chemokine receptor CXCR5 and (c) chemoattractant receptor SIP1R on B220+ B-cells in PLNs of WT and Plvap−/− mice. Representative flow cytometry plots and quantification of data are shown. Grey histograms in (b,c) represent stainings with isotype controls. MFI, mean fluorescence intensity. (d-g) Immunoreactivity of PLVAP (MECA-32) and (d) Prox-1, (e,f) podoplanin or (g) Lyve-1 in PLNs. C, ceiling; F, floor. The red arrow in f and g points to a sinus-traversing strand, and the white arrow to a blood vessel in d. (h) Immunoreactivity of PLVAP (MECA-32) and Prox-1 in WT mice of pure BALB/c, C57BL/6N and NMRI strains and in F1 hybrids of C57BL/6;129. White arrows point to blood vessels. C, ceiling; F, floor. Scale bars, 20 µm (d,e: tile scans), 10 µm (d,e (insets), f-h). Data in a-d are from littermate WT and Plvap−/− mice. Data are representative of or are pooled (all quantifications, mean ± s.e.m.) from 3 independent experiments (n=6 mice/genotype (a), 3-4 mice/genotype (b,c,d,e,f,g) or 2 mice/genotype (h)).

Supplementary Figure 3 Endothelial phenotypes of wild-type and Plvap−/− mice and humans.

(a) Immunoreactivity of mouse mesenteric lymph nodes for PLVAP (MECA-32) and Prox-1. C, ceiling; F, floor. White arrowheads point to representative blood vessels. (b) Plvap mRNA expression in blood endothelial cells (BEC) and lymphatic endothelial cells (LEC) in mesenteric lymph nodes of WT mouse (from the ImmGen data). Expression values >120 (red dashed line) indicate positivity. (c) Immunoreactivity of human PLNs for PLVAP (PAL-E) and Lyve-1. In control stainings an isotype-matched negative control antibody was used instead of PAL-E. C, ceiling, F, floor, white arrows point to HEVs. (d) Immunoreactivity of PLN subcapsular sinus of WT and Plvap−/− mice for CD31, VCAM-1, ICAM-1, VE-cadherin, CCRL1 and Lyve-1. C, ceiling, F, floor. White arrowheads point to blood vessels. (e) Immunoreactivity of skin vasculature for PLVAP (MECA-32) and CD31 (blood vessels, arrowheads) in whole-mount split ear preparations. (f) Immunoreactivity afferent lymphatic vasculature of skin for PLVAP (MECA-32) and Lyve-1 determined by confocal microscopy in ear sections. White arrowheads point to blood vessels, and red arrowheads to a lymphatic vessel. Blue is DAPI. Scale bars, 10 µm (a,b), 20 µm (c,d,f), 100 µm (e). Data in a and d-f are from littermate WT and Plvap−/− mice. Data are representative of three or more independent experiments with a total of 3-5 mice/genotype (a,d,e,f), are pooled (mean) from the ImmGen database (b) and of 3 different individuals (c).

Supplementary Figure 4 Ultrastructure of subcapsular sinus of PLNs.

(a-g) Electron micrographs of WT sinus. (a) The overall structure of the sinus showing highly elongated ceiling and floor LECs (indicated with a 20 % opacity magenta overlay), intrasinusoidal leukocytes (indicated with a 20 % opacity turquoise overlay) and a subcapsular sinus macrophage (M) partially extending to the lumen. Higher magnifications show (b) a transendothelial channel (outlined by arrows) closed by diaphragms (arrowheads), (c) vesicular bodies composed of multiple caveolae with diaphragms (arrowheads), (d) a tight junction (arrows) between 2 subcapsular sinus LECs, (e) a transection of a conduit ensheated by a floor (F) LEC, (f) highly abundant caveolae and vesicular bodies in the attenuated part of floor LECs (arrowheads point to diaphragms), and (g) tubular and canalicular structures (arrows) of LECs. (h,i) Electron micrographs of subcapsular sinus in Plvap−/− mice showing (h) a tight LEC junction (arrows), and (i) tubular and canalicular structures (arrows) within a LEC cytoplasm. SCS, lumen of the subcapsular sinus; N, nucleus. Scale bars, 5 µm (a), 100 nm (b,c), 200 nm (d,f,g,h), and 500 nm (e,i). Data are representative of three or more independent experiments with a total of 3-5 mice/genotype.

Supplementary Figure 5 The conduit network and sinus permeability to different antigens in wild-type and Plvap−/− mice.

(a) Co-localization of subcutaneously administered fluorescent dextrans and perlecan (a conduit marker) immunoreactivity in the conduits of WT and Plvap−/− mice. (b) Localization of a 20 nm quantum dots (Qtracker) in the subcapsular sinus (SCS) and medulla in PLNs of WT and Plvap−/− mice 10 min after subcutaneous injections. Quantitation of the fluorescence signal in the cortical area was determined using image analyses. (c) Schematic drawing showing the main components and molecular markers of the conduits. (d-f) Localization of (d) ER-TR7 and collagen type I, (e) perlecan, and (f) smooth muscle actin (SMA) in the conduits in the interfollicular parenchymal area of PLNs in WT and Plvap−/− mice. Scale bars, 50 µm (a,b) and 10 µm (d-f). All data are from littermate WT and Plvap−/− mice. Data are representative of 2 independent experiments with a total of 4-5 (a,b) and 3 (c-f) and mice/genotype.

Supplementary Figure 6 Conduit filtering function is intact in Cav1−/− mice and in the absence of subcapsular macrophages.

(a-c) Electron micrographs of subcapsular sinus in Cav1−/− mice showing (a) tubular and canalicular structures (arrows), (b) a transendothelial channel (white arrows) guarded by luminal and abluminal diaphragms (arrowheads) and (c) a conduit covered by LEC cytoplasm. SCS, lumen of the subcapsular sinus. (d) Flow cytometry of distribution of CD8+, CD4+ and B220+ lymphocytes in WT and Cav1−/− mice. (e) Immunostainings and quantitative analyses of ER-TR7 reactivity in the follicles (outlined by the white dashed line) and interfollicular areas of WT and Cav1−/− mice. (f) Microscopic analyses of the filling of the conduits 2 min after a subcutaneous injection of Pacific-Blue-labeled 10 kDa, TRITC-labeled 70 kDa and Alexa488-labeled 500 kDa dextrans in WT and Cav1−/− mice. (g,h) Immunoreactivity of cells in the subcapsular sinus (SCS) and medulla for (g) CD169 and Lyve-1 and (h) MAdCAM-1, Lyve and PLVAP (MECA-32) in WT and Plvap−/− mice. C, ceiling; F, floor. (i) Filling of the conduits with the fluorescent dextrans of different molecular weights after 1 min of subcutaneous injections in WT mice pretreated with chlodronate or control liposomes 7 d earlier. White arrows point to medullary macrophages. Scale bars, Scale bars, 500 nm (a), 200 nm (b), 2 µm (c), 100 µm (e,f), 20 µm (g), 10 µm (h) and 50 µm (i). Data are representative of or are pooled (all quantifications, mean ± s.e.m.) from experiments with a total of 3-5 mice/genotype.

Supplementary Figure 7 PLVAP-dependent lymphocyte migration through the LECs at sinus-cortex interface in PLNs.

(a) Immunofluorescence imaging of a representative CFSE+ cell (green arrow; injected subcutaneously 4 h earlier) that is partially positioned within the Lyve-1+ LEC floor in PLN sinus. (b-d) CFSE-labeled lymphocytes (isolated from WT spleens) and non-conjugated MECA-32 or isotype-matched control antibodies were co-administered subcutaneously to WT mice and the cells were allowed to migrate for 8 h. The phenotype of CD8+ T lymphocytes among the injected cells and among the donor cells recovered from the draining PLNs was determined using flow cytometry. Shown are representative flow cytometry dot plots and quantification of the data. Naive T cells are CD44loCD62Lhi, activated T-lymphocytes CD44hiCD62Llo, and central memory T cells CD44hiCD62Lhi. (e) Immunoreactivity of cultured LECs isolated from human PLNs for VE-cadherin and an isotype-matched negative control antibody for PLVAP. (f,g) Immunostainings of an endogenous lymphocyte (CD45+) that is attached to the sinusoidal LEC floor for F-actin, Lyve-1 and PLVAP in WT mice. C, ceiling; F, floor. The correlation of F-actin and PLVAP (MECA-32) staining intensities along the sinusoidal floor (arrow line) was measured using image analyses. Scale bars, 10 µm (a,e), and 5 µm (f,g). Data are representative of or are pooled (all quantifications, mean ± s.e.m.) from 2-3 independent experiments (n=8 WT mice (a), injected cells as a pool from 10 mice and migrated cells from 3 WT mice/ treatment (b-d), 4 stainings (e), 2 WT mice (f) or from 3 WT mice (g)). * P < 0.05 (Mann-Whitney U-test).

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Plvap is expressed in the Prox-1 positive floor and ceiling LECs in subcapsular sinus of WT mice.

Immunoreactivity of Plvap (MECA-32, green) and Prox-1 (white) in co-stained whole mount popliteal lymph node preparations after optical clearing. Note that Plvap is present in the LECs at the floor (F) and ceiling (C) of the subcapsular sinus (SCS), and in the cortical blood vessels (BECs). Nuclear Prox-1 staining defines LECs. Scale bar, 30 μm (at the beginning of the video). (MOV 23986 kb)

Plvap is expressed in the podoplanin positive floor and ceiling LECs in subcapsular sinus of WT mice.

Immunoreactivity of Plvap (MECA-32, green) and podoplanin (white) in co-stained whole mount popliteal lymph node preparations after optical clearing. Plvap and podoplanin signals are shown on individual channels (left video: Plvap channel only; right video: podoplanin channel only). Note that podoplanin stains both the LECs and FRCs. FRC staining is strongest above the ceiling LECs in the capsule of the lymph node. Scale bar, 30 μm (at the beginning of the video) (MOV 16037 kb)

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Rantakari, P., Auvinen, K., Jäppinen, N. et al. The endothelial protein PLVAP in lymphatics controls the entry of lymphocytes and antigens into lymph nodes. Nat Immunol 16, 386–396 (2015). https://doi.org/10.1038/ni.3101

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