Novel Expression of Vascular Endothelial Growth Factor Receptor (VEGFR)-3 and VEGF-C on Corneal Dendritic Cells
2003; Elsevier BV; Volume: 163; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)63630-9
ISSN1525-2191
AutoresPedram Hamrah, Lü Chen, Qiang Zhang, M. Reza Dana,
Tópico(s)Angiogenesis and VEGF in Cancer
ResumoVascular endothelial growth factor-3 (VEGFR-3) plays a critical role in embryonic cardiovascular development and is thought to be expressed exclusively on the lymphatic endothelium, high endothelial venules, and rarely on adult vascular endothelium. Recent evidence also suggests expression of VEGFR-3 on some tumor-associated macrophages. We have studied the expression of VEGFR-3, its ligand VEGF-C and the co-receptor neuropilin-2, in normal and inflamed corneas and characterized the phenotype and distribution of VEGFR-3+ cells. Our data demonstrate, for the first time, the expression of VEGFR-3 on corneal dendritic cells (DC) and its up-regulation in inflammation. VEGFR-3+ DC are CD11c+CD45+CD11b+, and are mostly major histocompatibility (MHC) class II−CD80−CD86−, indicating immature DC of a monocytic lineage. During inflammation, there is rapid increase in the number of VEGFR-3+ DC in the cornea associated with heightened membranous expression as compared to a mostly intracellular expression in uninflamed tissue. VEGFR-3+ DC in normal corneas are VEGF-C−neuropilin-2−, but express VEGF-C in inflammation. Interestingly, similar cells are absent both in the normal and inflamed skin. These data demonstrate, for the first time, the expression of VEGFR-3 and VEGF-C on tissue DC, which implicate a novel potential relationship between lymphangiogenesis and leukocyte trafficking in the eye. Vascular endothelial growth factor-3 (VEGFR-3) plays a critical role in embryonic cardiovascular development and is thought to be expressed exclusively on the lymphatic endothelium, high endothelial venules, and rarely on adult vascular endothelium. Recent evidence also suggests expression of VEGFR-3 on some tumor-associated macrophages. We have studied the expression of VEGFR-3, its ligand VEGF-C and the co-receptor neuropilin-2, in normal and inflamed corneas and characterized the phenotype and distribution of VEGFR-3+ cells. Our data demonstrate, for the first time, the expression of VEGFR-3 on corneal dendritic cells (DC) and its up-regulation in inflammation. VEGFR-3+ DC are CD11c+CD45+CD11b+, and are mostly major histocompatibility (MHC) class II−CD80−CD86−, indicating immature DC of a monocytic lineage. During inflammation, there is rapid increase in the number of VEGFR-3+ DC in the cornea associated with heightened membranous expression as compared to a mostly intracellular expression in uninflamed tissue. VEGFR-3+ DC in normal corneas are VEGF-C−neuropilin-2−, but express VEGF-C in inflammation. Interestingly, similar cells are absent both in the normal and inflamed skin. These data demonstrate, for the first time, the expression of VEGFR-3 and VEGF-C on tissue DC, which implicate a novel potential relationship between lymphangiogenesis and leukocyte trafficking in the eye. The development of blood vessels (angiogenesis) has been studied extensively, whereas the development of lymphatic vessels (lymphangiogenesis), despite its critical relevance, has gained relatively little attention until recently. 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These VEGFR-3+ monocytes or macrophages are present, however, in vascularized tissues and are thought to play a role in lymphangiogenesis in tissues that unlike the cornea have a significant endowment of vascular endothelial cells. Very recently we have reported the novel presence of a heterogeneous population of APC in the normal cornea that has profoundly revised the paradigm of corneal DC recruitment and function.41Hamrah P Zhang Q Liu Y Dana MR Novel characterization of MHC class II-negative population of resident corneal Langerhans cell-type dendritic cells.Invest Ophthalmol Vis Sci. 2002; 43: 639-646PubMed Google Scholar, 42Hamrah P Liu Y Zhang Q Dana MR The corneal stroma is endowed with a significant number of resident dendritic cells.Invest Ophthalmol Vis Sci. 2003; 44: 581-589Crossref PubMed Scopus (280) Google Scholar, 43Hamrah P, Liu Y, Zhang Q, Dana MR: Alterations in corneal stromal dendritic cell phenotype and distribution in inflammation. Arch Ophthalmol 2003, in pressGoogle Scholar, 44Hamrah P, Huq SO, Liu Y, Zhang Q, Dana MR: Corneal immunity is mediated by heterogeneous population of antigen-presenting cells. J Leukoc Biol 2003, in pressGoogle Scholar In the epithelium, we have characterized a population of resident major histocompatibility (MHC) class II-negative Langerhans cells (LC) in the corneal center that become activated after inflammation.41Hamrah P Zhang Q Liu Y Dana MR Novel characterization of MHC class II-negative population of resident corneal Langerhans cell-type dendritic cells.Invest Ophthalmol Vis Sci. 2002; 43: 639-646PubMed Google Scholar Another lab has recently identified a population of resident macrophages in the normal stroma,45Brissette-Storkus CS Reynolds SM Lepisto AJ Hendricks RL Identification of a novel macrophage population in the normal mouse corneal stroma.Invest Ophthalmol Vis Sci. 2002; 43: 2264-2271PubMed Google Scholar while we have demonstrated that bone marrow-derived cells in the stroma consist of several subsets: a largely immature population of resident myeloid (CD8α−) dendritic (CD45+CD11c+) cells (DC) in the anterior stroma, and a smaller population of monocytic (CD11b+CD11c−) macrophages in the posterior stroma,42Hamrah P Liu Y Zhang Q Dana MR The corneal stroma is endowed with a significant number of resident dendritic cells.Invest Ophthalmol Vis Sci. 2003; 44: 581-589Crossref PubMed Scopus (280) Google Scholar, 43Hamrah P, Liu Y, Zhang Q, Dana MR: Alterations in corneal stromal dendritic cell phenotype and distribution in inflammation. Arch Ophthalmol 2003, in pressGoogle Scholar, 44Hamrah P, Huq SO, Liu Y, Zhang Q, Dana MR: Corneal immunity is mediated by heterogeneous population of antigen-presenting cells. J Leukoc Biol 2003, in pressGoogle Scholar and have reported their maturation during inflammation.43Hamrah P, Liu Y, Zhang Q, Dana MR: Alterations in corneal stromal dendritic cell phenotype and distribution in inflammation. Arch Ophthalmol 2003, in pressGoogle Scholar, 44Hamrah P, Huq SO, Liu Y, Zhang Q, Dana MR: Corneal immunity is mediated by heterogeneous population of antigen-presenting cells. J Leukoc Biol 2003, in pressGoogle Scholar We have established that these DC are able to gain access to lymphatics and to migrate to draining LN, and stimulate T cells to corneal alloantigens.46Liu Y Hamrah P Zhang Q Taylor AW Dana MR Draining lymph nodes of corneal transplant hosts exhibit evidence for donor major histocompatibility complex (MHC) class II-positive dendritic cells derived from MHC class II-negative grafts.J Exp Med. 2002; 195: 259-268Crossref PubMed Scopus (187) Google Scholar, 47Liu Y Hamrah P Taylor AW Dana MR Resident corneal dendritic cells that migrate from corneal explants may mediate alloreactivity.Invest Ophthalmol Vis Sci. 2002; 43: S2264PubMed Google Scholar, 48Huq SO Liu Y Illigens B Qian Y Benichou G Dana MR Direct pathway of allosensitization plays a significant role in high risk corneal transplantation.Invest Ophthalmol Vis Sci. 2002; 43: S2275Google Scholar We report herein, for the first time, that DC residing in the normal uninflamed cornea express VEGFR-3 and that the expression of this receptor, and its ligand VEGF-C, increase substantially in inflammation. Seven to 12-week-old male BALB/c, C57BL/6, and C3H mice (Taconic Farms, Germantown, NY or from our own breeding facility) were used in these experiments. Most experiments were performed on BALB/c mice and experiments on other strains were performed only when noted. All protocols were approved by the Schepens Eye Research Institute Animal Care and Use Committee, and all animals were treated according to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. For each antibody staining study in the cornea, unless specified otherwise, three to five corneas were examined. For skin experiments, multiple sections derived from the skin of three mice were used per each double-staining study. All studies were repeated at least twice for confirmation. Application of electric cautery to the ocular surface is a standard method of inducing corneal inflammation without associated neovascularization.49Schanzlin DJ Cryr RJ Friedlaender MH Histopathology of corneal neovascularization.Arch Ophthalmol. 1983; 101: 472-474Crossref PubMed Scopus (20) Google Scholar, 50Dekaris I Zhu SN Dana MR TNF-α regulates corneal Langerhans cell migration.J Immunol. 1999; 162: 4235-4239PubMed Google Scholar, 51Williamson JSP Dimarco S Streilein JW Immunobiology of Langerhans cells on the ocular surface:: I. Langerhans cells within the central cornea interfere with induction of anterior chamber associated immune deviation.Invest Ophthalmol Vis Sci. 1987; 21: 759-765Google Scholar Mice were deeply anesthetized with an intraperitoneal injection of 3 to 4 mg ketamine and 0.1 mg xylazine and placed under the operating microscope. Using the tip of a hand-held thermal cautery (Aaron Medical Industries Inc., St. Petersburg, FL), five light burns were applied to the central 50% of the cornea as an experimental model for corneal inflammation as previously described,51Williamson JSP Dimarco S Streilein JW Immunobiology of Langerhans cells on the ocular surface:: I. Langerhans cells within the central cornea interfere with induction of anterior chamber associated immune deviation.Invest Ophthalmol Vis Sci. 1987; 21: 759-765Google Scholar followed by application of antibiotic ophthalmic ointment. Corneas were excised at 3, 7, and 14 days after cautery application and assessed in immunohistochemical studies as described below. Application of 2,4-dinitro-1-fluorobenzene (DNFB) (0.2% dissolved in acetone:olive oil (4:1)) is a standard method of inducing inflammation in skin and was performed as described previously.52Bacci S Alard P Dai R Nakamura T Streilein JW High and low doses of haptens dictate whether dermal or epidermal antigen-presenting cells promote contact hypersensitivity.Eur J Immunol. 1997; 27: 442-448Crossref PubMed Scopus (48) Google Scholar, 53Kurimoto I Grammer SF Shimizu T Nakamura T Streilein JW Role of F4/80+ cells during induction of hapten-specific hypersensitivity.Immunology. 1995; 85: 621-629PubMed Google Scholar, 54Kurimoto I Streilein JW Studies of contact hypersensitivity induction in mice with optimal sensitizing doses of hapten.J Invest Dermatol. 1993; 101: 132-136Abstract Full Text PDF PubMed Google Scholar In brief, DNFB (Sigma Chemical Co., St. Louis, MO) was applied epicutaneously to shaved abdominal skin (50 μl). This induced cutaneous inflammation at the application site within 48 hours. The skin was excised after 48 hours and assessed in immunohistochemical studies as described below. The following antibodies (Abs) were used: purified rabbit anti-mouse FLT-4 (VEGFR-3), purified rabbit anti-human FLT-4, purified goat anti-mouse VEGF-C and purified rabbit anti-mouse neuropilin-2 (Santa Cruz Biotechnology, Santa Cruz, CA); purified rat anti-mouse VEGFR-3 (a kind gift from Dr. Hajime Kubo, University of Helsinki, Helsinki, Finland); FITC-conjugated rat anti-mouse DEC-205 (Cedarlane Laboratories Limited, Ontario, Canada); purified rabbit anti-mouse LYVE-1 (a kind gift from Dr. David G. Jackson, Institute of Molecular Medicine Oxford, Oxford, UK); FITC-conjugated hamster anti-mouse CD3 (T cell marker), FITC-conjugated rat anti-mouse CD14 (immature myeloid marker), FITC-conjugated rat anti-mouse CD11b (monocyte/macrophage marker); purified (immunohistochemistry) and PE-conjugated (flow cytometry) hamster anti-mouse CD11c (dendritic cell marker); purified rat anti-mouse CD45 (leukocyte common marker); FITC-conjugated hamster anti-mouse CD80 (costimulatory molecule; B7–1); PE-conjugated rat anti-mouse CD86 (costimulatory molecule; B7–2); FITC-conjugated rat anti-mouse GR-1 (neutrophil marker); FITC-conjugated rat anti-mouse CD8α (lymphoid DC marker); FITC-conjugated mouse anti-mouse IAd (major histocompatibility (MHC) class II) and FITC-conjugated mouse anti-human HLA-DR, DP, DQ. Secondary Abs were rhodamine-conjugated goat anti-rat IgG, rhodamine-conjugated donkey anti-rabbit IgG, rhodamine-conjugated goat anti-rabbit IgG, rhodamine-conjugated donkey anti-goat IgG, FITC-conjugated donkey anti-rabbit IgG, FITC-conjugated goat anti-rabbit IgG, FITC-conjugated donkey anti-goat IgG (Santa Cruz) and Cy5-conjugated goat anti-hamster IgG. Isotype-matched control Abs were FITC-conjugated mouse IgG3, FITC-conjugated rat IgG1, FITC-conjugated rat IgG2a, FITC-conjugated rat IgG2b, FITC-conjugated hamster IgG, FITC-conjugated mouse IgG2a, PE-conjugated rat IgG2a, purified hamster IgG, purified rat IgG2b, purified goat IgG, and purified rabbit IgG. All primary and secondary Abs (except where noted) and isotype matched controls were purchased from BD PharMingen (San Diego, CA). Anti-Flt-4 blocking peptide was obtained from Santa Cruz. A green-fluorescent dye for nuclear acids, YOYO-1 (DNA staining), was a kind gift from the Ksander Laboratory at the Schepens Eye Research Institute, Boston. Corneal and limbal (intervening area between cornea and conjunctiva) tissue were excised, immersed in phosphate-buffered saline (PBS) and used as whole-mounts; skin was excised from the ear and abdomen of mice and 8-μm frozen sections were prepared. Twenty-μm horizontal sections of human eye bank corneas were also prepared. The tissues were fixed in acetone for 15 minutes at room temperature (RT) or used unfixed for staining where noted. Whole-mount corneas or tissue sections were then incubated in 2% bovine serum albumin (BSA) diluted in PBS (PBS-BSA) for 15 minutes. To block non-specific staining, whole-mounts or sections were blocked with anti-FcR mAb (CD16/CD32) for 30 minutes before they were immunostained with primary antibodies or isotype-matched control antibodies for 2 hours. Afterward, a second FITC- or PE-conjugated primary antibody, or secondary antibodies were added and incubated for 60 minutes (all diluted for optimal concentrations in PBS-BSA). All staining procedures were performed at room temperature. Whole-mounts or sections were covered with mounting medium (Vector, Burlingame, CA) and examined by a confocal microscope (Leica TCS 4D, Heidelberg, Germany). The YOYO-1 anti-nuclear dye, was added just before covering slides with mounting medium. To ensure specificity, negative controls were performed by omitting the primary antibody, or by using irrelevant primary antibodies of the same isotype or by incubating VEGFR-3 Ab with different concentrations of its blocking peptide for 2 hours at room temperature before staining. At least three to five different corneas were examined per each double staining experiment; representative data are presented below. All studies were repeated at least twice for confirmat
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