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Regular article Cardiovascular, pulmonary, and renal pathology| Volume 181, ISSUE 5, P1607-1620, November 2012

Critical Role of VEGF-C/VEGFR-3 Signaling in Innate and Adaptive Immune Responses in Experimental Obliterative Bronchiolitis

  • Rainer Krebs
    Correspondence
    Address reprint requests to Rainer Krebs, M.Sc., Transplantation Laboratory, Haartmaninkatu 3, P.O. Box 21, FIN-00014 Helsinki, Finland
    Affiliations
    Cardiopulmonary Research Group, Transplantation Laboratory, University of Helsinki, and Helsinki University Central Hospital, Helsinki, Finland
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  • Jussi M. Tikkanen
    Affiliations
    Cardiopulmonary Research Group, Transplantation Laboratory, University of Helsinki, and Helsinki University Central Hospital, Helsinki, Finland
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  • Jussi O. Ropponen
    Affiliations
    Cardiopulmonary Research Group, Transplantation Laboratory, University of Helsinki, and Helsinki University Central Hospital, Helsinki, Finland

    Department of Cardiothoracic Surgery, Helsinki University Central Hospital, Helsinki, Finland
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  • Michael Jeltsch
    Affiliations
    Molecular Cancer Biology Laboratory, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland
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  • Janne J. Jokinen
    Affiliations
    Cardiopulmonary Research Group, Transplantation Laboratory, University of Helsinki, and Helsinki University Central Hospital, Helsinki, Finland

    Department of Cardiothoracic Surgery, Helsinki University Central Hospital, Helsinki, Finland
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  • Seppo Ylä-Herttuala
    Affiliations
    A.I. Virtanen Institute for Molecular Sciences, University of Kuopio, Kuopio, Finland
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  • Antti I. Nykänen
    Affiliations
    Cardiopulmonary Research Group, Transplantation Laboratory, University of Helsinki, and Helsinki University Central Hospital, Helsinki, Finland
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  • Karl B. Lemström
    Affiliations
    Cardiopulmonary Research Group, Transplantation Laboratory, University of Helsinki, and Helsinki University Central Hospital, Helsinki, Finland

    Department of Cardiothoracic Surgery, Helsinki University Central Hospital, Helsinki, Finland
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Published:September 10, 2012DOI:https://doi.org/10.1016/j.ajpath.2012.07.021
      Chronic inflammation, a hallmark of obliterative bronchiolitis, is known to induce lymphangiogenesis. We therefore studied the role of lymphangiogenic vascular endothelial growth factor C (VEGF-C), its receptor VEGFR-3, and lymphangiogenesis during development of experimental obliterative bronchiolitis [ie, obliterative airway disease (OAD)] in rat tracheal allografts. The functional importance of VEGF-C was investigated by adenovirus-mediated overexpression of VEGF-C (AdVEGF-C), and by inhibition of VEGF-C activity with VEGFR-3-Ig (AdVEGFR-3-Ig). Analyses included histology, immunohistochemistry, and real-time RT-PCR 10 and 30 days after transplantation. In the course of OAD development, lymphangiogenesis was induced in the airway wall during the alloimmune response, which was reversed by cyclosporine A in a dose-dependent fashion. VEGF-C overexpression in tracheal allografts induced epithelial activation, neutrophil chemotaxis, and a shift toward a Th17 adaptive immune response, followed by enhanced lymphangiogenesis and the development of OAD. In contrast, inhibition of VEGF-C activity with VEGFR-3-Ig inhibited lymphangiogenesis and angiogenesis and reduced infiltration of CD4+ T cells and the development of OAD. Lymphangiogenesis was linked to T-cell responses during the development of OAD, and VEGF-C/VEGFR-3 signaling modulated innate and adaptive immune responses in the development of OAD in rat tracheal allografts. Our results thus suggest VEGFR-3-signaling as a novel strategy to regulate T-cell responses in the development of obliterative bronchiolitis after lung transplantation.
      Obliterative bronchiolitis (OB) is the pulmonary manifestation of chronic rejection and remains the leading cause of morbidity and mortality after lung transplantation.
      • Christie J.D.
      • Edwards L.B.
      • Kucheryavaya A.Y.
      • Aurora P.
      • Dobbels F.
      • Kirk R.
      • Rahmel A.O.
      • Stehlik J.
      • Hertz M.I.
      The Registry of the International Society for Heart and Lung Transplantation: twenty-seventh official adult lung and heart-lung transplant report–2010.
      • Stewart S.
      • Fishbein M.C.
      • Snell G.I.
      • Berry G.J.
      • Boehler A.
      • Burke M.M.
      • Glanville A.
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      • Studer S.M.
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      • Wallwork J.L.
      • Westall G.
      • Zamora M.R.
      • Zeevi A.
      • Yousem S.A.
      Revision of the 1996 working formulation for the standardization of nomenclature in the diagnosis of lung rejection.
      During transplantation, the lymphatic network is severed and lymphatic drainage from lung tissue is reduced until the lymphatic network has regenerated. Lymphatic disruption leads to congestion of lung tissue during the immediate postoperative course and may hinder recovery of the transplant recipients. Furthermore, in nontransplant situations, lymphatic vessels play a critical role in the resolution of inflammation.
      • Lawrence T.
      • Willoughby D.A.
      • Gilroy D.W.
      Anti-inflammatory lipid mediators and insights into the resolution of inflammation.
      On the other hand, the lymphatic network may have a central role in antigen presentation, a critical step in the rejection process.
      • Alitalo K.
      • Tammela T.
      • Petrova T.V.
      Lymphangiogenesis in development and human disease.
      Little is known about the role of lymphatic vessels in allograft inflammation and the development of OB.
      • El-Chemaly S.
      • Levine S.J.
      • Moss J.
      Lymphatics in lung disease.
      Vascular endothelial growth factor C (VEGF-C) is the most important molecule in lymphangiogenesis, with both alleles required for normal development of the lymphatic system.
      • Karkkainen M.J.
      • Haiko P.
      • Sainio K.
      • Partanen J.
      • Taipale J.
      • Petrova T.V.
      • Jeltsch M.
      • Jackson D.G.
      • Talikka M.
      • Rauvala H.
      • Betsholtz C.
      • Alitalo K.
      Vascular endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic veins.
      It is a member of the VEGF family and binds preferentially to the VEGF receptor 3 (VEGFR-3), but in its fully processed form it binds also to VEGFR-2.
      • Joukov V.
      • Pajusola K.
      • Kaipainen A.
      • Chilov D.
      • Lahtinen I.
      • Kukk E.
      • Saksela O.
      • Kalkkinen N.
      • Alitalo K.
      A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases [Erratum appeared in EMBO J 1996, 15:1751].
      Proinflammatory cytokines up-regulate VEGF-C expression,
      • Ristimäki A.
      • Narko K.
      • Enholm B.
      • Joukov V.
      • Alitalo K.
      Proinflammatory cytokines regulate expression of the lymphatic endothelial mitogen vascular endothelial growth factor-C.
      with macrophages as one of the central sources for VEGF-C in inflammatory settings.
      • Cursiefen C.
      • Chen L.
      • Borges L.P.
      • Jackson D.
      • Cao J.
      • Radziejewski C.
      • D'Amore P.A.
      • Dana M.R.
      • Wiegand S.J.
      • Streilein J.W.
      VEGF-A stimulates lymphangiogenesis and hemangiogenesis in inflammatory neovascularization via macrophage recruitment.
      Antigen-presenting cells leave the tissue via lymphatic vessels to reach secondary lymphoid organs.
      • Alitalo K.
      • Tammela T.
      • Petrova T.V.
      Lymphangiogenesis in development and human disease.
      Lymphatic vessels are not, however, merely passive conduit channels, but participate in inflammatory cell recruitment producing C-C motif chemokine 20 and 21 (CCL20 and CCL21), which are chemoattractants for C-C chemokine receptor type 6-positive (CCR6+) and CCR7+ antigen-presenting cells and T cells.
      • Förster R.
      • Davalos-Misslitz A.C.
      • Rot A.
      CCR7 and its ligands: balancing immunity and tolerance.
      • Hirota K.
      • Yoshitomi H.
      • Hashimoto M.
      • Maeda S.
      • Teradaira S.
      • Sugimoto N.
      • Yamaguchi T.
      • Nomura T.
      • Ito H.
      • Nakamura T.
      • Sakaguchi N.
      • Sakaguchi S.
      Preferential recruitment of CCR6-expressing Th17 cells to inflamed joints via CCL20 in rheumatoid arthritis and its animal model.
      • Greaves D.R.
      • Wang W.
      • Dairaghi D.J.
      • Dieu M.C.
      • Saint-Vis B.
      • Franz-Bacon K.
      • Rossi D.
      • Caux C.
      • McClanahan T.
      • Gordon S.
      • Zlotnik A.
      • Schall T.J.
      CCR6, a CC chemokine receptor that interacts with macrophage inflammatory protein 3alpha and is highly expressed in human dendritic cells.
      Growth of lymphatic vessels is seen in many situations with chronic inflammation. Persistent lymphangiogenesis is induced during chronic airway inflammation caused by Mycoplasma pulmonis infection and leads to a stronger and faster immune response on subsequent antigen challenge in the mouse.
      • Baluk P.
      • Tammela T.
      • Ator E.
      • Lyubynska N.
      • Achen M.G.
      • Hicklin D.J.
      • Jeltsch M.
      • Petrova T.V.
      • Pytowski B.
      • Stacker S.A.
      • Ylä-Herttuala S.
      • Jackson D.G.
      • Alitalo K.
      • McDonald D.M.
      Pathogenesis of persistent lymphatic vessel hyperplasia in chronic airway inflammation.
      In clinical renal transplantation, lymphangiogenesis is even thought to provide the basis for allograft rejection.
      • Kerjaschki D.
      • Regele H.M.
      • Moosberger I.
      • Nagy-Bojarski K.
      • Watschinger B.
      • Soleiman A.
      • Birner P.
      • Krieger S.
      • Hovorka A.
      • Silberhumer G.
      • Laakkonen P.
      • Petrova T.
      • Langer B.
      • Raab I.
      Lymphatic neoangiogenesis in human kidney transplants is associated with immunologically active lymphocytic infiltrates.
      On the other hand, VEGF-C treatment with a concomitant increase in lymphangiogenesis enhances resolution of acute skin inflammation.
      • Huggenberger R.
      • Ullmann S.
      • Proulx S.T.
      • Pytowski B.
      • Alitalo K.
      • Detmar M.
      Stimulation of lymphangiogenesis via VEGFR-3 inhibits chronic skin inflammation.
      In contrast to acute inflammation, after transplantation the offending antigen is not removed, which leads to continuous antigen presentation. We therefore hypothesized that enhanced lymphangiogenesis after transplantation may promote allograft rejection and that the VEGF-C/VEGFR-3 signaling pathway may have a significant effect in chronic lung allograft rejection. To test this hypothesis, we used a rat model of obliterative airway disease (OAD) and adenovirus-mediated gene transfection to induce overexpression of VEGF-C in the allograft and systemic VEGFR-3-Ig to block VEGF-C/VEGFR-3 signaling. Our results show that lymphangiogenesis was induced during the development of OAD and that VEGF-C/VEGFR-3 signaling modulated innate and adaptive immune responses in the development of OAD in rat tracheal allografts.

      Materials and Methods

      Experimental Design

      First, we investigated the development of lymphangiogenesis and the kinetics and localization of VEGF-C and VEGFR-3 expression by peroxidase-based and fluorescent immunohistochemistry during the development of severe OAD. The severe OAD was induced by transplanting fully MHC-mismatched rat tracheal allografts from Dark Agouti (DA; AG-B4, RT1a) donors to nonimmunosuppressed Wistar Furth (WF; AG-B2, RT1u) recipients. Animals were sacrificed, and grafts were removed at 10 and 30 days after transplantation for analysis (n = 6 to 8 per group). Syngrafts and nontransplanted tracheas served as controls.
      Second, because cyclosporine A (CsA) dose-dependently inhibits acute rejection and the development of OAD,
      • Koskinen P.K.
      • Kallio E.A.
      • Krebs R.
      • Lemström K.B.
      A dose-dependent inhibitory effect of cyclosporine A on obliterative bronchiolitis of rat tracheal allografts.
      we investigated the effects of low and moderate doses of CsA (0, 1.0, and 1.5 mg/kg per day; Novartis, Basel, Switzerland) on lymphangiogenesis and luminal occlusion in tracheal allografts during the development of OAD at 10 and 30 days (n = 4 per group).
      Third, we investigated how overexpression of VEGF-C affects the development of OAD in allograft recipients that are kept on a moderate dose of CsA (1.5 mg/kg per day). At this dose, tracheal allografts develop approximately 20% airway occlusion at 30 days, allowing detection of any augmented development of OAD.
      • Tikkanen J.M.
      • Kallio E.A.
      • Bruggeman C.A.
      • Koskinen P.K.
      • Lemström K.B.
      Prevention of cytomegalovirus infection-enhanced experimental obliterative bronchiolitis by antiviral prophylaxis or immunosuppression in rat tracheal allografts.
      Tracheal allografts were transfected ex vivo with 0.2 × 109 plaque-forming units (PFU) of adenovirus encoding human VEGF-C (AdVEGF-C; n = 9), β-galactosidase (AdlacZ; n = 9), or luciferase (AdLuc; n = 3) under the control of cytomegalovirus promoter for 30 minutes before transplantation, as described previously.
      • Krebs R.
      • Tikkanen J.M.
      • Nykänen A.I.
      • Wood J.
      • Jeltsch M.
      • Ylä-Herttuala S.
      • Koskinen P.K.
      • LemströM K.B.
      Dual role of vascular endothelial growth factor in experimental obliterative bronchiolitis.
      Finally, we investigated the effect of inhibiting VEGF-C activity on the development of OAD in allograft recipients by adenoviral transfer of a gene encoding the soluble form of VEGFR-3 (AdVEGFR-3-Ig). CsA 1.0 mg/kg per day was used as background immunosuppression. With this CsA dose, tracheal allografts develop more than 70% airway occlusion 30 days after transplantation, allowing detection of improvement due to inhibition of VEGF-C signaling. Allograft recipients were injected intraportally with 0.2 × 109 PFU of adenovirus encoding the soluble form of VEGFR-3 (AdVEGFR-3-Ig; n = 7) or β-galactosidase (AdlacZ; n = 5) at the time of transplantation.

      Rat Tracheal Transplantation

      We used fully MHC-mismatched specific pathogen-free inbred male DA and WF rats (Scanbur, Sollentuna, Sweden), 2 to 3 months of age. Syngeneic tracheal grafts were heterotopically transplanted from DA donors to DA recipients and allografts from DA donors to WF recipients.
      • Koskinen P.K.
      • Kallio E.A.
      • Krebs R.
      • Lemström K.B.
      A dose-dependent inhibitory effect of cyclosporine A on obliterative bronchiolitis of rat tracheal allografts.
      The average length of the transplanted tracheal segments was 10 mm. Grafts were harvested 10 and 30 days after transplantation. Nontransplanted DA tracheas were used as normal controls. Upon harvest, grafts were transversely cut in half, with one half being used for RNA isolation and the other half for histological sampling and immunohistochemistry. Sectioning was started from the cut face (ie, the middle part of the graft). An average of three or four tracheal cross sections per animal were examined for histological analysis. Permission for animal experimentation was obtained from the Provincial State Office of Southern Finland (permission numbers STU 1041 A and STH 808 A). All rats received care in compliance with the 1996 edition of the Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington, DC).

      Production of Adenoviruses

      Adenoviruses encoding human VEGF-C, VEGFR-3-Ig, lacZ, and luciferase were produced as described previously.
      • Hiltunen M.O.
      • Turunen M.P.
      • Turunen A.M.
      • Rissanen T.T.
      • Laitinen M.
      • Kosma V.M.
      • Ylä-Herttuala S.
      Biodistribution of adenoviral vector to nontarget tissues after local in vivo gene transfer to arterial wall using intravascular and periadventitial gene delivery methods.
      Briefly, cDNAs were subcloned into pAdCMV plasmid, constructed by subcloning the human CMV immediate early promoter, multiple cloning site, and the bovine growth hormone polyA signal from pcDNA3 plasmid (Invitrogen, Groningen, Netherlands) into a pAdBglII vector. Replication-deficient E1- to E3-deleted clinical adenoviruses [good manufacturing practices (GMP) grade] were produced in 293T cells. Adenoviruses were analyzed to be free of replication-competent viruses, lipopolysaccharide, mycoplasma, and other microbiological contaminants. The functionality of adenoviruses has been demonstrated.
      • Krebs R.
      • Tikkanen J.M.
      • Nykänen A.I.
      • Wood J.
      • Jeltsch M.
      • Ylä-Herttuala S.
      • Koskinen P.K.
      • LemströM K.B.
      Dual role of vascular endothelial growth factor in experimental obliterative bronchiolitis.
      • Hiltunen M.O.
      • Turunen M.P.
      • Turunen A.M.
      • Rissanen T.T.
      • Laitinen M.
      • Kosma V.M.
      • Ylä-Herttuala S.
      Biodistribution of adenoviral vector to nontarget tissues after local in vivo gene transfer to arterial wall using intravascular and periadventitial gene delivery methods.

      Kinetics of Adenovirus-Mediated Transgene Expression

      To investigate the kinetics of adenovirus-mediated transgene expression, tracheal transplants were incubated with AdLuc (0.2 × 109 PFU per graft; n = 3). Recipients were imaged using a bioluminescent in vivo imaging system (Xenogen IVIS; Caliper Life Science, Hopkinton, MA) at 1, 3, 7, 14, and 21 days after grafting. Recipients received the luciferase substrate d-luciferin (bc219; SynChem, Kassel, Germany) 30 mg/kg i.v. 2 minutes before the imaging. The quantity of photons emitted by the luciferase was visualized and measured with 1 minute exposure under a charge-coupled detector (CCD) camera. The total signal intensity was measured as the sum of photons in the region of interest. To obtain the negative control group, to exclude false positive transgene expression and inflammatory response, transplantations (n = 4) were performed with the same imaging protocol but without virus injection.

      Histological Evaluation

      Tracheal allografts were excised, embedded in Tissue-Tek compound (Miles Laboratories, Elkhart, IN), snap-frozen in liquid nitrogen, and stored at −70°C until use. For histological evaluation, cryostat sections were stained with Mayer's H&E. Loss of epithelium was determined as a percentage of the tracheal lumen not lined by epithelium. Luminal occlusion was evaluated by determining the reduction in luminal area using ImageJ version 1.59 software (NIH, Bethesda, MD).

      Immunohistochemistry

      Serial cryostat sections (4 to 6 mm) were stained using the peroxidase ABC method (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA), and the reaction was revealed by 3-amino-9-ethylcarbazole (AEC, Vectastain). Immunofluorescence double staining was performed using a sequential approach with Alexa Fluor 488 (green) and Alexa Fluor 568 (red) (Promega, Madison, WI) conjugated secondary antibodies. The primary antibodies used are listed in Table 1. The sections were counterstained with Mayer's hematoxylin and mounted with Aquamount (BDH, Poole, UK).
      Table 1Primary Antibodies Used in Immunohistochemistry
      Target proteinSupplierCatalog no.Concentration
      Rat CCL20Abcam, Cambridge, UKab251235 μg/mL
      Mouse CCL21R&D Systems, Minneapolis, MNAF4571 μg/mL
      Rat CD4BD Pharmingen, San Diego, CA5548355 μg/mL
      Rat CD8BD Pharmingen5548545 μg/mL
      Rat CXCL1R&D SystemsAF-515-NA1 μg/mL
      Rat ED1BD Pharmingen5549545 μg/mL
      Human IL-17eBioscience, San Diego, CA16–717810 μg/mL
      Mouse LYVE-1produced by Dr. Michael Jeltsch1:1000
      Rat MHC class IIAbD Serotec, Planegg, GermanyMCA46R5 μg/mL
      Human MPOAbcamab953510 μg/mL
      Rat OX-62AbD SerotecMCA1029G5 μg/mL
      Human Prox-1Acris Antibodies, Herford, GermanyDP3501P10 μg/mL
      Rat RECA-1AbD SerotecMCA970R50 μg/mL
      Synthetic α-SMC actinSigma-Aldrich, St. Louis, MOA25474.5 μg/mL
      Mouse TNF-αSanta Cruz Biotechnology, Santa Cruz, CAsc-13512 μg/mL
      Rat VEGF-CAbcamab95465 μg/mL
      Mouse VEGFR-3Santa Cruz Biotechnologysc-6371 μg/mL
      For inflammatory cells, results are expressed as the number of positively staining cells per allograft cross section. Immunostaining of VEGFR-3 was scored semiquantitatively from 0 to 3, as follows: 0, no visible staining; 1, few cells with faint staining; 2, moderate intensity with multifocal staining; and 3, intense diffuse staining of the cells analyzed. Specificity controls were performed using the same immunoglobulin concentration of species- and isotype-matched antibodies or irrelevant primary antibodies. Additional specificity control for VEGFR-3 staining involved the use of a working dilution of the polyclonal antibody after overnight incubation with a 20-molar excess of control peptide [Flt-4 antibody (M-20); sc-637-P; Santa Cruz Biotechnology, Santa Cruz, CA). Analyses were done by two independent observers (R.K. and J.M.T.) in a blinded fashion.

      Angiogenesis and Lymphangiogenesis

      Allograft angiogenesis and lymphangiogenesis were determined by a mouse monoclonal antibody to rat vascular endothelial cell antigen-1 (RECA-1, corresponding to human CD31)
      • Duijvestijn A.M.
      • van Goor H.
      • Klatter F.
      • Majoor G.D.
      • van Bussel E.
      • van Breda Vriesman P.J.
      Antibodies defining rat endothelial cells: rECA-1, a pan-endothelial cell-specific monoclonal antibody.
      and a rabbit polyclonal antibody to mouse lymphatic endothelium-specific hyaluronan receptor-1 (LYVE-1; 1:1000),
      • Banerji S.
      • Ni J.
      • Wang S.X.
      • Clasper S.
      • Su J.
      • Tammi R.
      • Jones M.
      • Jackson D.G.
      LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan.
      respectively. Results are expressed as the number of positive vessels per tracheal cross section. To assert the lymphatic nature of the vessels stained with LYVE-1 antibody, consecutive sections were stained using antibodies against LYVE-1, lymphatic endothelial transcription factor Prox-1, and secondary lymphoid chemokine CCL21 with immunofluorescence.

      Real-Time RT-PCR

      Real-time RT-PCR reactions were performed in a LightCycler system using LightCycler FastStart DNA MasterPLUS SYBR Green I mix (Roche, Basel, Switzerland). Measurement of the PCR product was performed at the end of each extension period. The number of copies of the gene of interest was calculated from the corresponding standard curve using LightCycler software (Roche) and is reported in relation to β-actin molecule numbers. The primers used are listed in Table 2.
      Table 2Primer Sequences for Target Genes
      Protein (rat gene; accession no.)Forward primerReverse primer
      CCL20 (Ccl20; NM_019233)5′-GCAAGCATCTGCCCTTCCTG-3′5′-GCCATCTGTGTTGTGAAACCCAC-3′
      CCL21 (Ccl21; NM_001008513)5′-GGGACTGAACAGACAGACTCCAAG-3′5′-GGTTGAAGCAGACAAGGGTGTG-3′
      CCR6 (Ccr6; NM_001013145)5′-CACCTGCGAGAGGAAGCAAAG-3′5′-GATTGGCACGAACACCTTGG-3′
      CCR7 (Ccr7; BC089762)5′-TGCTGGCTATGAGTTTCTGCTACC-3′5′-GAAGACGACGAACACTACGACCAC-3′
      CD80 (Cd80; NM_012926)5′-TGCTGCTGGTTGGTCTTTTCC-3′5′-CGGTGTATGGACTGCTCTTCAGAAC-3′
      CD83 (Cd83; NM_001108410)5′-GCCTTATTCCTTGACAATCCAAAAC-3′5′-GACCAGAGAGAAGAGCAACACAGC-3′
      CD86 (Cd86; NM_020081)5′-TTGGGAATCCTTTTCTCGGTG-3′5′-TTTGAGCCTTTGTGAACGGG-3′
      CXCL1
      Corresponding to human IL-8.
      (Cxcl1; NM_030845)
      5′-GTCCAAAAGATGCTAAAGGGTGTC-3′5′-TGTTGTCAGAAGCCAGCGTTC-3′
      IFN-γ (Ifng; NM_138880)5′-GAGGTGAACAACCCACAGA-3′5′-TATTGGCACACTCTCTACCC-3′
      IL-1β (Il1b; NM_031512)5′-GCTATGGCAACTGTCCCTGAACTC-3′5′-CGAGATGCTGCTGTGAGATTTGAAG-3′
      IL-2 (Il2; NM_053836)5′-CTGAGAGGGATCGATAATTACAAGA-3′5′-ATTGGCACTCAAATTTGTTTTCAG-3′
      IL-4 (Il4; NM_201270)5′-ATGTTTGTACCAGACGTCCTTACG-3′5′-TGCGAAGCACCCTGGAA-3′
      IL-6 (Il6; NM_012589)5′-TTGTTGACAGCCACTGCCTTC-3′5′-GAATTGCCATTGCACAACTCTTTTC-3′
      IL-10 (Il10; NM_012854)5′-TAAGGGTTACTTGGGTTGCC-3′5′-TATCCAGAGGGTCTTCAGC-3′
      IL-12p35 (Il12a; NM_053390)5′-CCTTGGTAGCATCTATGAGGACTTG-3′5′-CGCCGCTGTGATTCAGAGAC-3′
      IL-12p40 (Il12b; NM_022611)5′-AACATCAAGAGCAGCAGCAGTTC-3′5′-CTTCTCGTAGTCCCTTTGGTTCAG-3′
      IL17A (Il17a; NM_001106897)5′-CTCAAAGTTCAGTGTGTCCAAACG-3′5′-TCTATCAGGGTCCTCATTGCGG-3′
      IL-17E (Il25
      Alias Il17e.
      ; NM_001192007)
      5′-CGAGGAGTGGCTGAAGTGGAAC-3′5′-TCTGTAGGCTGACGCAGTGTGG-3′
      IL-21 (Il21; NM_001108943)5′-CACCTTCTGATTAGACTTCGTCACC-3′5′-GCTCACATTGCCCCTTTACATC-3′
      IL-23p19 (Il23a; NM_130410)5′-CACCAGTGGGACAAATGGATCTAC-3′5′-GACCTTGGGGATCACAACCATC-3′
      IP-10 (Cxcl10
      Alias Ip10, Scyb10.
      ; NM_139089)
      5′-TGAGCCAAAGAAGGTCTAAAAGAGC-3′5′-AGCCGCACACTGGGTAAAGG-3′
      TGF-β1 (Tgfb1; X52498)5′-GCTAATGGTGGACCGCAACAAC-3′5′-TGGCACTGCTTCCCGAATGTC-3′
      TNF-α (Tnf; NM_012675)5′-CTGTGCCTCAGCCTCTTCTCATTC-3′5′-TTGGGAACTTCTCCTCCTTGTTGG-3′
      low asterisk Corresponding to human IL-8.
      Alias Il17e.
      Alias Ip10, Scyb10.

      Statistical Analysis

      All data are expressed as means ± SEM. Student's t-test was used for parametric comparisons. Because the data did not meet the requirements for parametric analysis, the numbers of VEGF-C+ mononuclear cells in normal DA tracheas, syngrafts, and allografts in nonimmunosuppressed recipients 10 and 30 days after transplantation were compared using the Mann-Whitney test. Epithelial expression of VEGFR-3 in the same samples was analyzed using Mann-Whitney test, because of the nonparametric scoring method. Linear regression analysis was applied to evaluate a possible relation of increasing CsA doses to lymphangiogenesis and development of luminal occlusion. All analyses were performed using Abacus Concepts StatView version 4.1 software (SAS Institute, Cary, NC). P < 0.05 was regarded as statistically significant.

      Results

      Lymphangiogenesis Is Induced in the Airway Wall during the Development of OAD

      Lymphangiogenesis is generally induced during chronic inflammation.
      • Baluk P.
      • Tammela T.
      • Ator E.
      • Lyubynska N.
      • Achen M.G.
      • Hicklin D.J.
      • Jeltsch M.
      • Petrova T.V.
      • Pytowski B.
      • Stacker S.A.
      • Ylä-Herttuala S.
      • Jackson D.G.
      • Alitalo K.
      • McDonald D.M.
      Pathogenesis of persistent lymphatic vessel hyperplasia in chronic airway inflammation.
      • Kerjaschki D.
      • Regele H.M.
      • Moosberger I.
      • Nagy-Bojarski K.
      • Watschinger B.
      • Soleiman A.
      • Birner P.
      • Krieger S.
      • Hovorka A.
      • Silberhumer G.
      • Laakkonen P.
      • Petrova T.
      • Langer B.
      • Raab I.
      Lymphatic neoangiogenesis in human kidney transplants is associated with immunologically active lymphocytic infiltrates.
      We investigated whether it also occurs during the development of OAD in rat tracheal allografts. We first analyzed lymphangiogenesis and the kinetics of VEGF-C and VEGFR-3 expression during the development of OAD in nonimmunosuppressed normal DA tracheas, syngrafts (DA→DA), and allografts (DA→WF, fully MHC-mismatched) by immunohistochemistry at 10 and 30 days. Comparison of normal tracheas versus syngrafts permitted us to distinguish between changes in lymphatic vessel density and in VEGF-C and VEGFR-3 expression induced by the transplantation procedure or by transplantation-linked ischemia and innate immune response; comparison of syngrafts versus allografts allowed us to detect changes caused by the alloimmune response without interference of immunosuppression. In brief, in nonimmunosuppressed syngrafts, the epithelium showed ischemic injury at 3 days (data not shown) but recovered 10 days after transplantation (Figure 1, A and C), and no myofibroproliferation was observed at either time point (Figure 1, E and G). In nonimmunosuppressed allografts, progressive loss of epithelium was seen at 10 days (Figure 1, A and D), and intense myofibroproliferation totally occluding the airway lumen was seen at 30 days (Figure 1, E and H). Immunohistochemical staining for α-SMC actin confirmed the fibroproliferative character of the occlusion area (Figure 1H, inset).
      Figure thumbnail gr1
      Figure 1Chronic rejection induced lymphangiogenesis in the airway wall of tracheal allografts. Histological changes and lymphatic endothelial cell marker LYVE-1 immunoreactivity were analyzed in normal DA rat tracheas, syngrafts (DA→DA), and fully MHC-mismatched heterotopic allografts (DA→WF) in nonimmunosuppressed recipients at 10 and 30 days after transplantation. In contrast to syngrafts with fully regenerated epithelium at 10 days (A, C), allografts suffered from progressive to complete loss of epithelium (A, D). At 30 days, the tracheal lumen of syngrafts remained completely open (E, G), whereas the lumina of allografts were totally occluded (E, H). Immunohistochemical staining for α-SMC actin confirmed the fibroproliferative character of the occlusion area (H, inset). Syngrafts showed transient lymphangiogenesis at 10 days, whereas allografts showed increased lymphangiogenesis at both time points (I). In normal tracheas and syngrafts, LYVE-1+ vessels were localized mostly to the subepithelial space (J, K). In allografts, only few LYVE-1+ vessels were seen in the subepithelial space, because most of them had been destroyed by the alloimmune response. Instead, lymphangiogenesis is induced in the allograft airway wall during chronic alloimmune response at 30 days (L). Immunofluorescence double staining for blood vessel endothelial cell marker RECA-1 (red) and for lymphatic vessel endothelial cell marker LYVE-1 (green) showed a clear separation between the two signals (M). Further evidence for the lymphatic phenotype of LYVE-1+ vessels (N) was provided by the expression of lymphatic endothelial cell transcription factor Prox-1 (O), and secondary lymphoid chemokine CCL21 (P) in consecutive sections of the same vessels. Histological cryostat sections (B–D and F–H) were counterstained with H&E. Immunohistochemical sections (H, inset, J–L) were developed with the avidin-biotin system (red) and were counterstained with hematoxylin (blue). AW, airway wall; C, cartilage; EL, epithelial layer; L, lumen; SEL, subepithelial layer. Data are expressed as means ± SEM. n = 6 to 8 per group. P < 0.0001 Student's t-test versus syngrafts. Scale bar = 100 μm.
      We used lymphatic endothelium-specific hyaluronan receptor (LYVE-1) antibody as a marker for lymphatic vessels.
      • Banerji S.
      • Ni J.
      • Wang S.X.
      • Clasper S.
      • Su J.
      • Tammi R.
      • Jones M.
      • Jackson D.G.
      LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan.
      Compared with syngrafts, immunohistochemical staining showed a more than threefold number of LYVE-1+ lymphatic vessels in allografts at 10 days (P < 0.0001) (Figure 1I). At 30 days, the number of LYVE-1+ vessels in syngrafts had returned to the level seen in normal tracheas, whereas in allografts the number of LYVE-1+ vessels remained elevated (Figure 1I). In normal tracheas and syngrafts, LYVE-1+ vessels were localized mostly to the subepithelial space (Figure 1, J and K). In allografts, the subepithelial LYVE-1+ vessels were only few in number, and showed signs of endothelial disruption and damage. Instead, significant lymphangiogenesis was induced in the allograft airway wall during chronic alloimmune response at 30 days (Figure 1L). At 30 days, the nonimmunosuppressed tracheal allografts were totally occluded by fibrotic tissue and the inflammation had subsided. This partly explains the relative reduction in lymph vessels, compared with the 10-day time point, when allograft inflammation was at its peak level (Figure 1I).
      Immunofluorescence staining of consecutive tracheal allograft sections showed that LYVE-1 and blood vessel endothelial cell marker RECA-1 were not expressed by the same vessels (Figure 1M). The lymphatic phenotype of LYVE-1+ vessels was further supported by the observation that lymphatic endothelial cell transcription factor Prox-1 was expressed in the nuclei of the LYVE-1+ cells (Figure 1, N and O). Additionally, secondary lymphoid chemokine CCL21 was localized to the same lymphatic vessels (Figure 1P).

      Macrophages and CD4+ T Cells Are a Major Source of VEGF-C during the Development of OAD

      Because our results indicated induction of lymphangiogenesis during the development of OAD, we next investigated the expression of the primary lymphangiogenic factor VEGF-C in the same experimental setting. Immunohistochemical staining showed no marked difference in VEGF-C expression in mononuclear inflammatory cells between normal and syngeneic tracheas (Figure 2, A–C). In tracheal allografts, however, the number of VEGF-C+ mononuclear inflammatory cells was markedly increased both at 10 days (P < 0.05) (Figure 2, A and D) and at 30 days (P < 0.05) (Figure 2A), compared with syngrafts. Immunofluorescence double staining revealed that VEGF-C was expressed mainly by subsets of ED1+ macrophages (Figure 2E) and occasional CD4+ T cells (Figure 2F), but not by CD8+ T cells (Figure 2G).
      Figure thumbnail gr2
      Figure 2VEGF-C and VEGFR-3 expression was up-regulated in chronically rejecting tracheal allografts. Vascular endothelial growth factor C (VEGF-C) and VEGF receptor 3 (VEGFR-3) immunoreactivity in normal DA tracheas, syngrafts, and allografts in nonimmunosuppressed recipients 10 and 30 days after transplantation. A–D: The number of VEGF-C+ mononuclear inflammatory cells was significantly increased in allografts. E: Immunofluorescence double staining showed that ED-1+ macrophages were the major source of VEGF-C (arrows). F: Occasional CD4+ T cells expressed VEGF-C (arrows). G: CD8+ T cells, however, did not express VEGF-C. H–K: In normal DA tracheas and syngrafts, VEGFR-3 expression was localized to the ciliated epithelium, whereas the damaged epithelium of nonimmunosuppressed allografts showed only faint expression. VEGFR-3 immunoreactivity in the epithelium was scored from nonexistent (0) to strong (3). L–O: Compared with normal DA tracheas, VEGFR-3 expression in small vessels (arrows) was enhanced in both syngrafts and allografts at 10 days. Allografts showed enhanced VEGFR-3 expression at 10 and 30 days, compared with syngrafts. P–S: The number of airway wall-infiltrating VEGFR-3+ mononuclear cells (MNC) was significantly higher in allografts at 10 days. Immunohistochemical sections (B–D, I–K, M–O, Q–S) were developed with the avidin-biotin system (red) and were counterstained with hematoxylin (blue). AW, airway wall; C, cartilage; EL, epithelial layer; L, lumen; SEL, subepithelial layer. Data are expressed as means ± SEM. n = 3 to 5 per group (A); n = 6 to 8 per group (H, L, P). *P < 0.05, **P < 0.01, and P < 0.0001 Student's t-test (L and P) or Mann-Whitney (A and H) versus syngrafts. Scale bar = 100 μm.

      VEGFR-3 Is Expressed in Graft-Infiltrating Mononuclear Inflammatory Cells during the Development of OAD

      Next, we investigated the expression of VEGFR-3, which is the main receptor for VEGF-C. Normal nontransplanted tracheas showed strong VEGFR-3 expression in the epithelium (Figure 2, H and I), but weak expression both in small vessels (Figure 2, L and M) and in mononuclear inflammatory cells (Figure 2, P and Q). In syngrafts, the epithelium strongly expressed VEGFR-3 after recovery from ischemic injury (Figure 2, H and J), whereas the numbers of VEGFR-3+ small vessels (Figure 2, L and N) and inflammatory cells (Figure 2, P and R) were transiently elevated. In chronically rejecting tracheal allografts, the damaged epithelium showed a gradual reduction of VEGFR-3 immunoreactivity (Figure 2, H and K), but the number of VEGFR-3+ small vessels was markedly increased both at 10 days (P < 0.01) and at 30 days (P < 0.05) (Figure 2, L and O), compared with syngrafts. We performed immunofluorescence double staining to characterize the VEGFR-3+ small vessels and identified them to be mostly LYVE-1+ lymphatic vessels (see Supplemental Figure S1A at http://ajp.amjpathol.org). VEGFR-3+ blood vessels were also present, but in clearly lower numbers and with weaker expression. Furthermore, the number of VEGFR-3+ graft-infiltrating mononuclear inflammatory cells was significantly higher at 10 days (P < 0.0001) (Figure 2, P and S), compared with syngrafts. To identify the VEGFR-3+ inflammatory cells, we performed immunofluorescence double staining for VEGFR-3 and inflammatory cell markers. The VEGFR-3+ cells were mostly ED1+ macrophages (see Supplemental Figure S1B at http://ajp.amjpathol.org), with occasional CD4+ T cells among them (see Supplemental Figure S1C at http://ajp.amjpathol.org).

      Cyclosporine A Down-Regulates Lymphangiogenesis in a Dose-Dependent Fashion

      Because CsA inhibits allograft inflammation and OAD development dose-dependently,
      • Koskinen P.K.
      • Kallio E.A.
      • Krebs R.
      • Lemström K.B.
      A dose-dependent inhibitory effect of cyclosporine A on obliterative bronchiolitis of rat tracheal allografts.
      we investigated the anti-inflammatory effects of low and moderate doses of CsA (0, 1.0, and 1.5 mg/kg per day) on lymphangiogenesis at 10 days and on luminal occlusion at 30 days. Linear regression analysis revealed a clear correlation of increasing doses of CsA with reduced numbers of LYVE-1+ lymphatic vessels in tracheal allografts at 10 days (Figure 3, A and C–E) and with decreasing development of luminal occlusion 30 days after transplantation (Figure 3B). Additionally, the numbers of graft-infiltrating VEGF-C+ and VEGFR-3+ inflammatory cells and of VEGFR-3+ small vessels were significantly decreased 10 days after transplantation by administration of 1.5 mg/kg per day CsA (see Supplementary Figure S2 at http://ajp.amjpathol.org).
      Figure thumbnail gr3
      Figure 3Cyclosporine A (CsA) dose-dependently reduced lymphangiogenesis and luminal occlusion in chronically rejecting tracheal allografts. A and B: Correlation of lymphangiogenesis and luminal occlusion in rat tracheal allografts with increasing doses of CsA (0, 1.0, and 1.5 mg/kg per day) was analyzed using linear regression. CsA dose-dependently reduced the number of LYVE-1+ lymphatic vessels (A) and the development of luminal occlusion (B) at 10 and 30 days after transplantation, respectively. C–E: LYVE-1+ vessels in representative sections of grafts in recipients with increasing doses of CsA (0, 1.0, and 1.5 mg/kg per day). Immunohistochemical sections (C–E) were developed with the avidin-biotin system (red) and were counterstained with hematoxylin (blue). Linear regression analysis was applied to evaluate a possible relation of increasing CsA doses to lymphangiogenesis and development of luminal occlusion. Data are expressed as means ± SEM. n = 4 per group. *P < 0.05. Scale bar = 100 μm.

      VEGF-C Overexpression Preserves the Epithelium but Enhances Luminal Occlusion in Tracheal Allografts

      Based on the above findings and our previous results with VEGF-A,
      • Krebs R.
      • Tikkanen J.M.
      • Nykänen A.I.
      • Wood J.
      • Jeltsch M.
      • Ylä-Herttuala S.
      • Koskinen P.K.
      • LemströM K.B.
      Dual role of vascular endothelial growth factor in experimental obliterative bronchiolitis.
      we hypothesized that VEGF-C may regulate the inflammatory response linked to the development of OAD. We therefore performed ex vivo epithelium-targeted gene transfer into tracheal allografts using adenoviruses encoding either human VEGF-C (AdVEGF-C) or β-galactosidase (AdlacZ). Allograft recipients received CsA 1.5 mg/kg per day subcutaneously. With this CsA dose, control allografts show delayed loss of epithelium and a luminal occlusion of approximately 20% to 30% at 30 days,
      • Tikkanen J.M.
      • Kallio E.A.
      • Bruggeman C.A.
      • Koskinen P.K.
      • Lemström K.B.
      Prevention of cytomegalovirus infection-enhanced experimental obliterative bronchiolitis by antiviral prophylaxis or immunosuppression in rat tracheal allografts.
      allowing detection of enhanced chronic rejection due to VEGF-C overexpression. Adenoviral infection with control AdlacZ gene transfer does not induce OAD in this setting,
      • Krebs R.
      • Tikkanen J.M.
      • Nykänen A.I.
      • Wood J.
      • Jeltsch M.
      • Ylä-Herttuala S.
      • Koskinen P.K.
      • LemströM K.B.
      Dual role of vascular endothelial growth factor in experimental obliterative bronchiolitis.
      and neither did adenoviral gene transfer in itself promote the development of OAD in the present study, because infection with AdlacZ resulted in an occlusion level (17 ± 2%) at 30 days similar to that of noninfected tracheal recipients receiving the same immunosuppression (data not shown). To determine the transgene expression kinetics after adenoviral gene transfer, tracheal transplants were infected with AdLuc. Noninvasive bioluminescent imaging of AdLuc-transfected recipients (Figure 4A) revealed that adenovirus-mediated transgene expression peaked 3 days after transplantation and gradually decreased during the following 3 weeks (Figure 4B).
      Figure thumbnail gr4
      Figure 4VEGF-C overexpression preserved the epithelium, but enhanced the development of OAD in tracheal allografts. A and B: To show the efficacy of adenoviral gene transfer, tracheal transplants were ex vivo infected with AdLuc, and the transgene expression was visualized by noninvasive bioluminescent imaging. Luciferase expression in the transplants (A, arrow) peaked at 3 days and gradually decreased thereafter (B); the framed area in B indicates base-level emittance from control transplantations with equal imaging protocol, but without virus injection. C–S: The effects of VEGF-C overexpression on the loss of epithelium (C–E), luminal occlusion (F–H), lymphangiogenesis (I–K), angiogenesis (L–N), and inflammatory cell influx (O–S) in rat tracheal allografts in immunosuppressed recipients receiving CsA 1.5 mg/kg per day. C–E: AdVEGF-C gene transfer reduced the loss of epithelium at 10 days. F–H: Importantly, AdVEGF-C gene transfer more than tripled the development of luminal occlusion at 30 days. I–N: VEGF-C overexpression significantly elevated the numbers of LYVE-1+ vessels at 30 days (I–K), but had no effect on the numbers of RECA-1+ blood vessels per tracheal allograft section (L–N). AdVEGF-C gene transfer increased the number of MPO+ neutrophils at 10 days (O), with most of the neutrophils located in the subepithelial space, and the number of CD8+ T cells at 30 days (R). Histological sections (D, E, G, H) were stained with H&E. Immunohistochemical sections (J, K, M, N) were developed with the biotin-avidin system (red) and were counterstained with hematoxylin (blue). Data are expressed as means ± SEM. n = 3 (B); n = 6 to 9 per group (C, F, I, L, O–S). *P < 0.05, **P < 0.01 Student's t-test versus AdlacZ. Scale bar = 100 μm.
      Overexpression of VEGF-C markedly attenuated the loss of epithelial cells at 10 days, compared with controls (P < 0.05) (Figure 4, C–E). However, AdVEGF-C gene transfer significantly increased the development of OAD at 30 days, compared with the AdlacZ group (P < 0.01) (Figure 4, F–H). In the AdVEGF-C group, there was a significant increase in the number of LYVE-1+ lymphatic vessels in the airway wall of tracheal allografts at 30 days, compared with the AdlacZ group (P < 0.05) (Figure 4, I–K). No angiogenic response, as analyzed by the number of RECA-1+ blood vessels in the airway wall, was observed in the AdVEGF-C group at 10 or 30 days, compared with the AdlacZ group (Figure 4, L–N). The number of VEGFR-3+ small vessels was significantly higher in the AdVEGF-C-perfused group, compared with AdLacZ-perfused control group (P < 0.005) (see Supplemental Figure S3 at http://ajp.amjpathol.org).

      VEGF-C Overexpression Enhances Neutrophil Chemotaxis and Activation of Epithelial Cells in Tracheal Allografts

      Because early loss of epithelium usually correlates with early allograft inflammation and later development of OAD in this model,
      • Koskinen P.K.
      • Kallio E.A.
      • Krebs R.
      • Lemström K.B.
      A dose-dependent inhibitory effect of cyclosporine A on obliterative bronchiolitis of rat tracheal allografts.
      • King M.B.
      • Pedtke A.C.
      • Levrey-Hadden H.L.
      • Hertz M.I.
      Obliterative airway disease progresses in heterotopic airway allografts without persistent alloimmune stimulus.
      our findings were somewhat contradictory. However, immunohistochemistry revealed that AdVEGF-C gene transfer was associated with a significantly increased number of allograft-infiltrating myeloperoxidase-positive (MPO+) neutrophils 10 days after transplantation, compared with AdlacZ-treated allografts (P < 0.05) (Figure 4O), but had no effect on the number of OX62+ dendritic cells, CD4+ T cells, CD8+ T cells, or ED1+ macrophages (Figure 4, P–S). At 30 days, AdVEGF-C significantly increased the number of graft-infiltrating CD8+ T cells, compared with AdlacZ-infected allografts (P < 0.05) (Figure 4R).
      Because neutrophils and the neutrophil chemoattractant human IL-8 play an important role in the development of OB,
      • DiGiovine B.
      • Lynch 3rd, J.P.
      • Martinez F.J.
      • Flint A.
      • Whyte R.I.
      • Iannettoni M.D.
      • Arenberg D.A.
      • Burdick M.D.
      • Glass M.C.
      • Wilke C.A.
      • Morris S.B.
      • Kunkel S.L.
      • Strieter R.M.
      Bronchoalveolar lavage neutrophilia is associated with obliterative bronchiolitis after lung transplantation: role of IL-8.
      • Verleden G.M.
      • Vanaudenaerde B.M.
      • Dupont L.J.
      • Van Raemdonck D.E.
      Azithromycin reduces airway neutrophilia and interleukin-8 in patients with bronchiolitis obliterans syndrome.
      we next investigated by immunohistochemistry whether the preserved epithelium is responsible for neutrophil chemotaxis. In the VEGF-C overexpressing epithelium, we found strong expression of the proinflammatory molecules MHC class II and TNF-α, as well as of the neutrophil, lymphocyte, and dendritic cell chemoattractant CCL20 and the neutrophil chemoattractant CXCL1 (which corresponds to human IL-8) (Figure 5).
      Figure thumbnail gr5
      Figure 5VEGF-C overexpression enhanced proinflammatory activation in the airway epithelium of tracheal allografts. The effects of VEGF-C overexpression on epithelial expression of MHC class II and inflammatory cytokines were analyzed 10 days after transplantation. In the AdVEGF-C group, immunohistochemical staining revealed enhanced epithelial expression of the proinflammatory molecules MHC class II (A, B) and TNF-α (C, D), the neutrophil, lymphocyte, and dendritic cell chemoattractant CCL20 (E, F), and the neutrophil chemoattractant CXCL1 (G, H) 10 days after transplantation. Immunohistochemical sections were developed with the biotin-avidin system (red) and were counterstained with hematoxylin (blue).

      VEGF-C Overexpression Modulates Adaptive Immune Response toward Th17 Response in Tracheal Allografts

      Because our results suggested that VEGF-C overexpression promoted activation of epithelial cells and neutrophil chemotaxis, we performed real-time RT-PCR analysis of tracheal allografts for the expression of inflammatory cell chemokines, cytokines, and growth factors 10 and 30 days after transplantation. In recipients treated with a moderate dose (1.5 mg/kg per day) of CsA, AdVEGF-C increased the intragraft mRNA expression of the proinflammatory cytokine TNF-α (P < 0.05), neutrophil, lymphocyte, and dendritic cell chemoattractant CCL20 (P < 0.0001), mature dendritic cell marker CD83 (P < 0.05), Th-17 response driving molecule IL-23 (P < 0.05), and Th17 cytokines IL-17A and IL-21 (both P < 0.05) 10 days after transplantation. The mRNA levels of Th2 cytokines IL-4 (P < 0.001), IL-10 (P < 0.05), and IL-17E (P < 0.05) were decreased 10 days after transplantation (Figure 6). Because mRNA levels of several Th17-related factors were significantly up-regulated, we investigated the cellular source of IL-17A. Immunofluorescence staining revealed that intragraft IL-17A was produced by MPO-expressing neutrophils and CD4+ T cells (Figure 6, I and J).
      Figure thumbnail gr6
      Figure 6VEGF-C overexpression promoted the Th17 adaptive immune response. The effects of VEGF-C overexpression on mRNA expression levels of innate and adaptive immune response-related molecules and cytokines in rat tracheal allografts were analyzed in immunosuppressed recipients receiving CsA 1.5 mg/kg per day. AdVEGF-C gene transfer up-regulated intragraft mRNA levels of CD83 (A), TNF-α (B), IL-17A and IL-21 (E), CCL20 (G), and IL-23 (H), but decreased the expression of IL-4, IL-10, and IL-17E (D) at 10 days after transplantation. At 30 days, VEGF-C overexpression enhanced the mRNA levels of TGF-β1 (E) and secondary lymphoid chemokine CCL21 and its receptor CCR7 (F), whereas it decreased the expression of IL-10 mRNA (D). Immunofluorescence double stainings showed that intragraft IL-17A was produced by MPO-expressing neutrophils and CD4+ T cells (I and J). Data are expressed as means ± SEM. n = 5 to 9 per group. *P < 0.05, P < 0.005, ***P < 0.001, and P < 0.0001 Student's t-test versus AdlacZ.
      At 30 days, at the time of increased lymphangiogenic response, AdVEGF-C increased the mRNA levels of the secondary lymphoid chemokine CCL21 (P < 0.05), its receptor CCR7 (P < 0.05), and the profibrotic growth factor TGF-β1 (P < 0.005), whereas it decreased IL-10 mRNA expression (P < 0.05), compared with the AdlacZ group (Figure 6).

      VEGFR-3-Ig Prevents the Development of OAD and Lymphangiogenesis

      We next investigated the effect of inhibition of VEGF-C signaling on the development of OAD in rat tracheal allografts at 10 and 30 days. Allograft recipients were injected intraportally either with adenovirus vector encoding the soluble form of VEGFR-3 (AdVEGFR-3-Ig) or with control adenovirus encoding β-galactosidase (AdlacZ). Allograft recipients received CsA 1.0 mg/kg per day as low-dose immunosuppression. With this CsA dose, control allografts show an average luminal occlusion of more than 70% at 30 days,
      • Koskinen P.K.
      • Kallio E.A.
      • Krebs R.
      • Lemström K.B.
      A dose-dependent inhibitory effect of cyclosporine A on obliterative bronchiolitis of rat tracheal allografts.
      allowing detection of OAD attenuation due to inhibition of VEGF-C signaling. The functionality of the adenovirus gene transfer was confirmed in our preliminary studies, which showed that the serum level of VEGFR-3-Ig reached 1 μg/mL at 5 days and remained expressed for at least 21 days.
      • Nykänen A.I.
      • Sandelin H.
      • Krebs R.
      • Keränen M.A.
      • Tuuminen R.
      • Kärpänen T.
      • Wu Y.
      • Pytowski B.
      • Koskinen P.K.
      • Ylä-Herttuala S.
      • Alitalo K.
      • Lemström K.B.
      Targeting lymphatic vessel activation and CCL21 production by vascular endothelial growth factor receptor-3 inhibition has novel immunomodulatory and antiarteriosclerotic effects in cardiac allografts.
      In immunosuppressed recipients, VEGFR-3-Ig significantly reduced airway occlusion in tracheal allografts at 30 days, compared with the AdlacZ group (P < 0.05) (Figure 7, D–F). VEGFR-3-Ig also markedly reduced the number of LYVE-1+ lymphatic vessels in the airway wall 10 days (P < 0.01) and 30 days (P < 0.05) (Figure 7, G–I) after transplantation, as well as the angiogenic response, as analyzed by the number of RECA-1+ vessels 10 days after transplantation (P < 0.05) (Figure 7, J–L).
      Figure thumbnail gr7
      Figure 7VEGFR-3-Ig prevented the development of OAD and lymphangiogenesis and halted dendritic cells in tracheal allografts. The effects of VEGFR-3-Ig on the loss of epithelium (A–C), luminal occlusion (D–F), lymphangiogenesis (G–I), angiogenesis (J–L), inflammatory cell influx (M–Q), and CCL21 mRNA levels (R) in rat tracheal allografts were analyzed in immunosuppressed recipients receiving CsA 1.0 mg/kg per day. D–F: VEGFR-3-Ig reduced the development of luminal occlusion by more than 50% 30 days after transplantation. G–L: VEGFR-3-Ig significantly reduced the number of LYVE-1+ lymphatic vessels (G–I) per tracheal allograft section 10 and 30 days after transplantation, and the number of RECA-1+ blood vessels 10 days after transplantation (J–L). VEGFR-3-Ig decreased the number of intragraft CD4+ T cells 10 days after transplantation (O) and increased the number of intragraft OX62+ dendritic cells 30 days after transplantation (N). Real-time RT-PCR showed that VEGFR-3-Ig significantly reduced the expression of secondary lymphoid chemokine CCL21 mRNA 30 days after transplantation (R). VEGFR-3-Ig had no effect on the number of graft-infiltrating MPO+ neutrophils (M), CD8+ T cells (P), or ED1+ macrophages (Q). Immunofluorescence double staining showed that LYVE-1+ lymphatic vessels were the source of CCL21 protein production (S). Histological sections (B, C, E, F) were counterstained with H&E. Immunohistochemical sections (H, I, K, L) were developed with the avidin-biotin system (red) and were counterstained with hematoxylin (blue). Data are expressed as means ± SEM. n = 5 to 7 per group. *P < 0.05, **P < 0.01 Student's t-test versus AdlacZ. Scale bar = 100 μm.

      Inhibition of VEGF-C Signaling with VEGFR-3-Ig Reduces Early Influx of CD4+ T-Cells and Eventually Traps Dendritic Cells in the Allograft

      The number of graft-infiltrating CD4+ T cells was significantly decreased at 10 days, and the number of OX62+ dendritic cells significantly increased at 30 days in the VEGFR-3-Ig group, compared with the AdlacZ group (both P < 0.05) (Figure 7, N and O). Real-time RT-PCR analysis showed that VEGFR-3-Ig significantly decreased mRNA levels of the secondary lymphoid chemokine CCL21 (P < 0.05) (Figure 7R) 30 days after transplantation. VEGFR-3-Ig had no effect on the number of graft-infiltrating MPO+ neutrophils (Figure 7M), CD8+ T cells (Figure 7P), or ED1+ macrophages (Figure 7Q). We investigated the same panel of genes (those included in Figure 6), but none of the other investigated factors showed any significant differences in expression between the groups (data not shown). We then performed immunofluorescence double staining to determine the source of CCL21 in the grafts. Staining on AdLacZ samples at 30 days revealed that lymphatic vessels of various sizes produced CCL21 (Figure 7S).

      Discussion

      This is the first study to show that VEGF-C/VEGFR-3 signaling actively participates in innate and adaptive immune responses in the development of OAD. Importantly, we show that adenovirus-mediated up-regulation of VEGF-C/VEGFR-3 signaling induced epithelial activation, neutrophil chemotaxis, and Th17-like alloimmune activation and enhanced the development of OAD in tracheal allografts. Moreover, inhibition of VEGF-C signaling by VEGFR-3-Ig decreased the infiltration of CD4+ T cells and inhibited the development of luminal occlusion, findings that further emphasize the importance of VEGF-C/VEGFR-3 signaling in the development of OAD.
      Injury to airway epithelium is known to be a prerequisite for the development of OAD.
      • King M.B.
      • Pedtke A.C.
      • Levrey-Hadden H.L.
      • Hertz M.I.
      Obliterative airway disease progresses in heterotopic airway allografts without persistent alloimmune stimulus.
      In the present study, we found that overexpression of VEGF-C was associated with a reduced loss of epithelium at 10 days but also with increased development of OAD. Rat tracheal allograft epithelium expresses VEGFR-2
      • Krebs R.
      • Tikkanen J.M.
      • Nykänen A.I.
      • Wood J.
      • Jeltsch M.
      • Ylä-Herttuala S.
      • Koskinen P.K.
      • LemströM K.B.
      Dual role of vascular endothelial growth factor in experimental obliterative bronchiolitis.
      and VEGFR-3, to both of which VEGF-C can bind. To repair epithelial injury, VEGF-C could exert direct protective effects on epithelial cells, either by inducing cell proliferation or protein expression. However, VEGF-C gene transfer increased the expression of proinflammatory molecules and neutrophil chemoattractant molecules in the epithelium and infiltration of neutrophils at 10 days. In the allograft microenvironment, this activation of innate immunity probably enhanced adaptive immune responses and the development of OAD.
      One of the most striking observations from the present study was the significant neutrophilia in the VEGF-C-overexpressing group 10 days after transplantation. Neutrophils and the neutrophil chemoattractant human IL-8 play an important role in the development of OB.
      • Verleden G.M.
      • Vanaudenaerde B.M.
      • Dupont L.J.
      • Van Raemdonck D.E.
      Azithromycin reduces airway neutrophilia and interleukin-8 in patients with bronchiolitis obliterans syndrome.
      • Meyer K.C.
      • Nunley D.R.
      • Dauber J.H.
      • Iacono A.T.
      • Keenan R.J.
      • Cornwell R.D.
      • Love R.B.
      Neutrophils, unopposed neutrophil elastase, and alpha1-antiprotease defenses following human lung transplantation.
      Neutrophils cause inflammation in epithelial and subepithelial structures,
      • Meyer K.C.
      • Nunley D.R.
      • Dauber J.H.
      • Iacono A.T.
      • Keenan R.J.
      • Cornwell R.D.
      • Love R.B.
      Neutrophils, unopposed neutrophil elastase, and alpha1-antiprotease defenses following human lung transplantation.
      • De Andrade J.A.
      • Crow J.P.
      • Viera L.
      • Alexander C.B.
      • Young R.K.
      • McGiffin D.C.
      • Zorn G.L.
      • Zhu S.
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      Considering that TGF-β is associated with Th17 skewing as well as with increased development of OB and fibrosis, it is interesting to notice that mRNA levels of TGF-β were up-regulated in the VEGF-C overexpressing allografts.
      We also used VEGFR-3-Ig to test whether inhibition of VEGF-C/VEGFR-3 signaling would affect the development of OAD. VEGFR-3-Ig significantly inhibited CD4+ T-cell infiltration, lymphangiogenesis, angiogenesis, and the development of OAD. However, VEGFR-3-Ig did not inhibit the infiltration of neutrophils, nor did it reduce Th17-mediated alloimmune activation. The mRNA expression levels of CCL21 mRNA were not reduced until 30 days after transplantation. This down-regulation of CCL21 was mirrored by the increased number of intragraft OX62+ dendritic cells. These cells express the CCL21 receptor CCR7 on activation and, under normal conditions, are recruited to the lymphatic system, following a CCL21 gradient that is generated by lymphatic endothelial cells. The elevated number of OX62+ dendritic cells at 30 days in the AdVEGFR-3 Ig group may therefore reflect dendritic cells that are unable to migrate to the secondary lymphoid organs. A reduced flow of activated dendritic cells to the lymph nodes could imply reduced levels of antigen presentation, and therefore an attenuated alloimmune response. This result is in accord with a recent study on corneal transplant rejection, which showed that inhibition of VEGFR-3 signaling suppresses dendritic cell trafficking from the cornea to draining lymph nodes and corneal transplant rejection.
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      In the present study, a transient increase in lymphangiogenesis was observed after transplantation, which subsided as the injury healed in the syngrafts. In the allograft microenvironment, however, with persistent and continuous presentation of alloantigens, the effect of lymphatic vessel growth may be different.
      In our nonimmunosuppressed rat tracheal allograft model, the alloimmune response was associated with prominent lymphangiogenesis in the allograft airway wall, which was reduced by the inhibition of the calcineurin activation pathway with CsA. This pathway is responsible for the activation of NFAT transcription factors in lineage specification during Th1/Th2 responses. Furthermore, NFAT1c regulates lymphangiogenesis.
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      In the present study, overexpression of lymphatic growth factor VEGF-C did not increase the number of LYVE-1+ lymphatic vessels in the allograft airway wall until at 30 days. It must be taken into consideration, however, that these allograft recipients received CsA 1.5 mg/kg per day, which may have inhibited early lymphangiogenic responses. On the other hand, suppression of VEGF-C/VEGFR-3 signaling with VEGFR-3-Ig markedly reduced the number of LYVE-1+ lymphatic vessels in the airway wall 10 and 30 days after transplantation. These observations suggest that lymphangiogenesis may have a role in regulation of T-cell responses in the development of OAD.
      Although the heterotopic rat tracheal model reproduces the obliterative changes seen in human transplants, there are some intrinsic limitations of the model. First of all, the tissue under investigation is the trachea, whereas the changes of OB in human grafts develop in the bronchioles, which are small, cartilage-free airways. Second, the tracheal graft does not experience airflow, which may alter its pathological response. Third, the tracheal allograft is not primarily vascularized, but is revascularized by the capillary network. This might affect the initial influx of inflammatory cells. Finally, the trachea contains less lymphoid tissue than a lung allograft. Allorecognition might be impaired until connection to the lymphatic network has been established. Nonetheless, the obliterative changes in tracheal allografts reproducibly mimic the lesions seen in human allograft bronchioles. The rat heterotopic allograft model is therefore suitable for proof-of-concept studies in the investigation of lung allograft rejection.
      In conclusion, our results suggest that VEGF-C/VEGFR-3 signaling plays an important role in regulating innate and adaptive immune responses after lung transplantation. Although VEGF-C favored epithelial recovery after ischemic injury, concomitant activation of the epithelium enhanced proinflammatory responses and increased the development of OAD in our rat tracheal model. Importantly, blocking of VEGF-C/VEGFR-3 signaling inhibited lymphangiogenesis, angiogenesis, infiltration of CD4+ T cells, and the development of OAD, but did not affect T-cell cytokine responses. These results may suggest a new immunomodulatory strategy to regulate rejection and pathological responses in the development of OB.

      Acknowledgments

      We thank Eeva Rouvinen and Eriika Wasenius for excellent technical assistance.

      Supplementary data

      • Supplemental Figure S1

        Characterization of VEGFR-3+ small vessels and mononuclear inflammatory cells in rat tracheal allografts at 10 days in recipients without immunosuppression. A: VEGFR-3 expression in small vessels was enhanced in both syngrafts and allografts at 10 days. To characterize these vessels, we performed double immunofluorescence immunohistochemistry for VEGFR-3 with lymphatic endothelial cell marker LYVE-1 and blood vessel endothelium marker RECA-1. The VEGFR-3+ small vessels were identified as mostly LYVE-1+ lymphatic structures. VEGFR-3+ blood vessels were also present, but in considerably smaller numbers and stained with lower intensity. B and C: We performed immunofluorescence double staining for VEGFR-3 and several inflammatory cell markers, to identify the VEGFR-3+ mononuclear inflammatory cells. The VEGFR-3+ cells were mostly ED1+ macrophages (B), with occasional CD4+ cells among them (C). Immunohistochemical section (A) was developed with the avidin-biotin system (red) and counterstained with hematoxylin (blue). Scale bars: 100 μm (A, black bar); 50 μm (B and C); 5 μm (A, white bar).

      • Supplemental Figure S2

        Effect of immunosuppression with CsA on VEGF-C+ mononuclear cell numbers, VEGFR-3+ mononuclear cell (MNC) numbers, and VEGFR-3+ small vessel numbers in 10 day allografts. The numbers of VEGF-C+ (A) and VEGFR-3+ (B) mononuclear inflammatory cells, as well as the number of VEGFR-3+ small vessels (C) were all significantly decreased in allografts with 1.5 mg/kg per day CsA as immunosuppression, compared with nonimmunosuppressed allografts 10 days after transplantation. Data are expressed as means ± SEM. n = 5 to 8 per group (A); n = 7 to 8 per group (B and C). Student's t-test was used for parametric comparisons.

      • Supplemental Figure S3

        The effect of VEGF-C overexpression on the number of VEGFR-3+ small vessels in rat tracheal allografts in recipients receiving CsA 1.5 mg/kg per day. VEGF-C overexpression significantly elevated the numbers of VEGFR-3+ small vessels at 30 days. Data are expressed as means ± SEM. n = 5 to 9 per group. Student's t-test was used for parametric comparisons.

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