Role of CD11b+ Macrophages in Intraperitoneal Lipopolysaccharide-Induced Aberrant Lymphangiogenesis and Lymphatic Function in the Diaphragm
2009; Elsevier BV; Volume: 175; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2009.090133
ISSN1525-2191
AutoresKyung-Eun Kim, Young-Jun Koh, Bong-Hyun Jeon, Cholsoon Jang, Jinah Han, Raghu P. Kataru, Reto A. Schwendener, Jin‐Man Kim, Gou-Young Koh,
Tópico(s)Sympathectomy and Hyperhidrosis Treatments
ResumoLymphatic vessels in the diaphragm are essential for draining peritoneal fluid, but little is known about their pathological changes during inflammation. Here we characterized diaphragmatic lymphatic vessels in a peritonitis model generated by daily i.p. administration of lipopolysaccharide (LPS) in mice. Intraperitoneal LPS increased lymphatic density, branching, sprouts, connections, and network formation in the diaphragm in time- and dose-dependent manners. These changes were reversible on discontinuation of LPS administration. The LPS-induced lymphatic density and remodeling occur mainly through proliferation of lymphatic endothelial cells. CD11b+ macrophages were massively accumulated and closely associated with the lymphatic vessels changed by i.p. LPS. Both RT-PCR assays and experiments with vascular endothelial growth factor-C/D blockade and macrophage-depletion indicated that the CD11b+ macrophage-derived lymphangiogenic factors vascular endothelial growth factor-C/D could be major mediators of LPS-induced lymphangiogenesis and lymphatic remodeling through paracrine activity. Functional assays with India ink and fluorescein isothiocyanate-microspheres indicated that impaired peritoneal fluid drainage in diaphragm of LPS-induced peritonitis mice was due to inflammatory fibrosis and massive attachment of CD11b+ macrophages on the peritoneal side of the diaphragmatic lymphatic vessels. These findings reveal that CD11b+ macrophages play an important role in i.p. LPS-induced aberrant lymphangiogenesis and lymphatic dysfunction in the diaphragm. Lymphatic vessels in the diaphragm are essential for draining peritoneal fluid, but little is known about their pathological changes during inflammation. Here we characterized diaphragmatic lymphatic vessels in a peritonitis model generated by daily i.p. administration of lipopolysaccharide (LPS) in mice. Intraperitoneal LPS increased lymphatic density, branching, sprouts, connections, and network formation in the diaphragm in time- and dose-dependent manners. These changes were reversible on discontinuation of LPS administration. The LPS-induced lymphatic density and remodeling occur mainly through proliferation of lymphatic endothelial cells. CD11b+ macrophages were massively accumulated and closely associated with the lymphatic vessels changed by i.p. LPS. Both RT-PCR assays and experiments with vascular endothelial growth factor-C/D blockade and macrophage-depletion indicated that the CD11b+ macrophage-derived lymphangiogenic factors vascular endothelial growth factor-C/D could be major mediators of LPS-induced lymphangiogenesis and lymphatic remodeling through paracrine activity. Functional assays with India ink and fluorescein isothiocyanate-microspheres indicated that impaired peritoneal fluid drainage in diaphragm of LPS-induced peritonitis mice was due to inflammatory fibrosis and massive attachment of CD11b+ macrophages on the peritoneal side of the diaphragmatic lymphatic vessels. These findings reveal that CD11b+ macrophages play an important role in i.p. LPS-induced aberrant lymphangiogenesis and lymphatic dysfunction in the diaphragm. The peritoneum provides the lining of the peritoneal cavity and is the most extensive serous membrane in the body.1Gandawidjaja L Hau T Anatomic, physiologic, bacteriologic and immunologic aspects of peritonitis.Acta Chirurgica Belgica. 1997; 97: 163-167PubMed Google Scholar The peritoneal membrane is formed by a single layer of mesothelial cells. Beneath the mesothelial cells, there is a very thin and discontinuous layer of connective tissue and a layer of fenestrated lymphatic vessels.2Tsilibary E Wissig S Light and electron microscope observations of the lymphatic drainage units of the peritoneal cavity of rodents.Am J Anat. 1987; 180: 195-207Crossref PubMed Scopus (84) Google Scholar These three layers not only function as an absorptive surface for peritoneal fluid but also remove pathogens and prevent cells from leaking through damage in the gastrointestinal tract or ascending through the female genital tract.3Hall J Heel K Papadimitriou J Platell C The pathobiology of peritonitis.Gastroenterology. 1998; 114: 185-196Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 4Ho H Wu M Yang Y Peritoneal cellular immunity and endometriosis.Am J Reprod Immunol. 1997; 38: 400-412Crossref PubMed Scopus (155) Google Scholar The peritoneum also plays crucial roles in the local defensive response against bacterial invasion with appropriate activation of resident immune cells and the recruitment of circulating immune cells.5Broche F Tellado J Defense mechanisms of the peritoneal cavity.Curr Opin Crit Care. 2001; 7: 105-116Crossref PubMed Scopus (79) Google Scholar, 6Melichar B Freedman R Immunology of the peritoneal cavity: relevance for host-tumor relation.Int J Gynecol Cancer. 2002; 12: 3-17Crossref PubMed Scopus (66) Google Scholar Lymphatic vessels have distinctive morphologies and functions in different tissues and organs.7Ji R Lymphatic endothelial cells, lymphangiogenesis, and extracellular matrix.Lymphat Res Biol. 2006; 4: 83-100Crossref PubMed Scopus (99) Google Scholar, 8Pepper M Skobe M Lymphatic endothelium: morphological, molecular and functional properties.J Cell Biol. 2003; 163: 209-213Crossref PubMed Scopus (158) Google Scholar Lymphatic vessels play an essential role in the maintenance of tissue fluid homeostasis through regulated uptake of protein-rich interstitial fluid into draining lymphatic vessels and transport of the drained lymphatic fluid into the blood vasculature via collecting lymphatic vessels.9Saladin K Anatomy and physiology: the unity of form and function.in: Saladin K The Lymphatic and Immune System. McGraw Hill, Boston2004: 799Google Scholar In addition, lymphatic vessels have roles in lipid absorption, antigen presentation, tumor metastasis, and wound healing.10Alitalo K Tammela T Petrova T Lymphangiogenesis in development and human disease.Nature. 2005; 438: 946-953Crossref PubMed Scopus (1002) Google Scholar, 11Hong Y Shin J Detmar M Development of the lymphatic vascular system: a mystery unravels.Dev Dyn. 2004; 231: 462-473Crossref PubMed Scopus (81) Google Scholar, 12Baluk P Tammela T Ator E Lyubynska N Achen M Hicklin D Jeltsch M Petrova T Pytowski B Stacker S Pathogenesis of persistent lymphatic vessel hyperplasia in chronic airway inflammation.J Clin Invest. 2005; 115: 247-257Crossref PubMed Scopus (521) Google Scholar, 13Maruyama K Ii M Cursiefen C Jackson D Keino H Tomita M Van Rooijen N Takenaka H D'Amore P Stein-Streilein J Inflammation-induced lymphangiogenesis in the cornea arises from CD11b-positive macrophages.J Clin Invest. 2005; 115: 2363-2372Crossref PubMed Scopus (589) Google Scholar, 14Cursiefen C Chen L Borges L Jackson D Cao J Radziejewski C D'Amore P Dana M Wiegand S Streilein J VEGF-A stimulates lymphangiogenesis and hemangiogenesis in inflammatory neovascularization via macrophage recruitment.J Clin Invest. 2004; 113: 1040-1050Crossref PubMed Scopus (877) Google Scholar, 15Kerjaschki D The crucial role of macrophages in lymphangiogenesis.J Clin Invest. 2005; 115: 2316-2319Crossref PubMed Scopus (192) Google Scholar, 16Kataru R Jung K Jang C Yang H Schwendener R Baik J Han S Alitalo K Koh G Critical role of CD11b+ macrophages and VEGF in inflammatory lymphangiogenesis, antigen clearance, and inflammation resolution.Blood. 2009; 113: 5650-5659Crossref PubMed Scopus (313) Google Scholar Lymphatic vessels beneath the peritoneum, particularly lymphatic vessels on the peritoneal side of the muscular region of diaphragm, provide the central route for draining peritoneal fluid.2Tsilibary E Wissig S Light and electron microscope observations of the lymphatic drainage units of the peritoneal cavity of rodents.Am J Anat. 1987; 180: 195-207Crossref PubMed Scopus (84) Google Scholar, 17Nagy J Lymphatic and nonlymphatic pathways of peritoneal absorption in mice: physiology versus pathology.Blood Purif. 1992; 10: 148-162Crossref PubMed Scopus (30) Google Scholar, 18Abu-Hijleh M Habbal O Moqattash S The role of the diaphragm in lymphatic absorption from the peritoneal cavity.J Anat. 1995; 186: 453-467PubMed Google Scholar Lymphatic vessels on the peritoneal side of the diaphragm are largely attenuated but well designed for fluid absorption with extremely flattened and broad lumina (also called lacunae), which are connected with openings between mesothelial cells covering the peritoneal surface.2Tsilibary E Wissig S Light and electron microscope observations of the lymphatic drainage units of the peritoneal cavity of rodents.Am J Anat. 1987; 180: 195-207Crossref PubMed Scopus (84) Google Scholar, 19Tsilibary E Wissig S Lymphatic absorption from the peritoneal cavity: regulation of patency of mesothelial stomata.Microv Res. 1983; 25: 22-39Crossref PubMed Scopus (100) Google Scholar In comparison, lymphatic vessels on the pleural side of diaphragm are tubular, like other lymphatic vessels.20Azzali G The lymphatic vessels and the so-called “lymphatic stomata” of the diaphragm: a morphologic ultrastructural and three-dimensional study.Microvasc Res. 1999; 57: 30-43Crossref PubMed Scopus (42) Google Scholar, 21Shao X Ohtani O Saitoh M Ohtani Y Development of diaphragmatic lymphatics: the process of their direct connection to the peritoneal cavity.Arch Histol Cytol. 1998; 61: 137-149Crossref PubMed Scopus (19) Google Scholar There are seven to nine parallel lymphatic strips on each hemisphere (sterno-costal muscular region) of the peritoneal side of diaphragm, and these lymphatic vessels are directly connected to the tubular lymphatic vessels on the pleural side by transmural lymphatic branches.20Azzali G The lymphatic vessels and the so-called “lymphatic stomata” of the diaphragm: a morphologic ultrastructural and three-dimensional study.Microvasc Res. 1999; 57: 30-43Crossref PubMed Scopus (42) Google Scholar, 21Shao X Ohtani O Saitoh M Ohtani Y Development of diaphragmatic lymphatics: the process of their direct connection to the peritoneal cavity.Arch Histol Cytol. 1998; 61: 137-149Crossref PubMed Scopus (19) Google Scholar, 22Shinohara H Lymphatic system of the mouse diaphragm: morphology and function of the lymphatic sieve.Anat Rec. 1997; 249: 6-15Crossref PubMed Scopus (18) Google Scholar Therefore, the peritoneal fluid absorbed by lymphatic lacunae is directly transported into the lymphatic vessels on the pleural side. In contrast, there are few lymphatic vessels in the central tendon region of the diaphragm. However, little is known about relationship between the structural and functional changes of diaphragmatic lymphatic vessels and peritoneal illnesses. Our understanding of the molecular and cellular regulation of new lymphatic vessel formation, “lymphangiogenesis,” has greatly advanced in recent years.10Alitalo K Tammela T Petrova T Lymphangiogenesis in development and human disease.Nature. 2005; 438: 946-953Crossref PubMed Scopus (1002) Google Scholar Among lymphangiogenic growth factors, the roles of vascular endothelial growth factor (VEGF)-C and VEGF-D (VEGF-C/D) and their lymphatic vessel-specific receptor VEGF receptor-3 (VEGFR-3) are specific and essential in lymphangiogenesis.10Alitalo K Tammela T Petrova T Lymphangiogenesis in development and human disease.Nature. 2005; 438: 946-953Crossref PubMed Scopus (1002) Google Scholar In addition, VEGF-A and its receptors play additional roles in lymphangiogenesis in certain pathological conditions.10Alitalo K Tammela T Petrova T Lymphangiogenesis in development and human disease.Nature. 2005; 438: 946-953Crossref PubMed Scopus (1002) Google Scholar, 11Hong Y Shin J Detmar M Development of the lymphatic vascular system: a mystery unravels.Dev Dyn. 2004; 231: 462-473Crossref PubMed Scopus (81) Google Scholar Moreover, proinflammatory cytokine-induced activation of macrophages is closely involved in pathological lymphangiogenesis in tracheal mucosa and cornea by reciprocal interactions with the VEGF-C/D-VEGFR-3 system.12Baluk P Tammela T Ator E Lyubynska N Achen M Hicklin D Jeltsch M Petrova T Pytowski B Stacker S Pathogenesis of persistent lymphatic vessel hyperplasia in chronic airway inflammation.J Clin Invest. 2005; 115: 247-257Crossref PubMed Scopus (521) Google Scholar, 13Maruyama K Ii M Cursiefen C Jackson D Keino H Tomita M Van Rooijen N Takenaka H D'Amore P Stein-Streilein J Inflammation-induced lymphangiogenesis in the cornea arises from CD11b-positive macrophages.J Clin Invest. 2005; 115: 2363-2372Crossref PubMed Scopus (589) Google Scholar, 14Cursiefen C Chen L Borges L Jackson D Cao J Radziejewski C D'Amore P Dana M Wiegand S Streilein J VEGF-A stimulates lymphangiogenesis and hemangiogenesis in inflammatory neovascularization via macrophage recruitment.J Clin Invest. 2004; 113: 1040-1050Crossref PubMed Scopus (877) Google Scholar, 15Kerjaschki D The crucial role of macrophages in lymphangiogenesis.J Clin Invest. 2005; 115: 2316-2319Crossref PubMed Scopus (192) Google Scholar However, the relationship between proinflammatory cytokine-induced activation of macrophages and pathological changes of diaphragmatic lymphatic vessels during peritonitis is unknown. In this study, we investigated how acute inflammatory peritonitis affects the diaphragmatic lymphatic vessels structurally and functionally and what roles are played by activated macrophages in this situation. To generate a peritonitis model, we administered lipopolysaccharide (LPS; endotoxin) directly into the peritoneal cavity of mouse. LPS is a well-known cell wall component of most Gram-negative bacteria and acts as a potent initiator of inflammation.23Holst O Ulmer A Brade H Flad H Rietschel E Biochemistry and cell biology of bacterial endotoxins.FEMS Immunol Med Microbiol. 1996; 16: 83-104Crossref PubMed Google Scholar, 24Tobias P Tapping R Gegner J Endotoxin interactions with lipopolysaccharide-responsive cells.Clin Infect Dis. 1999; 28: 476-481Crossref PubMed Scopus (76) Google Scholar Interestingly, mice with LPS-induced peritonitis displayed aberrant lymphangiogenesis, lymphatic remodeling, and lymphatic dysfunction in the diaphragm. We have defined the underlying mechanisms and the responsible molecules by using specific blocking agents to reveal the roles of critical effector cells and molecules. Our results show that CD11b+ macrophages have an important role in LPS-induced aberrant lymphangiogenesis and lymphatic dysfunction in the diaphragm. Specific pathogen-free FVB/N and C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and bred in our pathogen-free animal facility. GFP+ mice (C57BL/6J genetic background) were a gift from Dr. Masaru Okabe (Osaka University, Osaka, Japan). Animal care and experimental procedures were performed under approval from the Animal Care Committees of Korea Advanced Institute of Science and Technology. All experiments were conducted in FVB/N mice, otherwise specifically indicated. Five or 25 μg of LPS (from Escherichia coli 0111:B4; Sigma-Aldrich, St. Louis, MO) in 200 μl of PBS was injected daily into the peritoneal cavity of 8-week-old male mice. As a control, 200 μl of PBS was injected in the same manner. To block VEGF-C and VEGF-D, mice were treated with a single i.v. injection of 1 × 109 plaque-forming units of adenovirus-encoding soluble VEGFR-3 (Ad-sVEGFR-3)25Makinen T Jussila L Veikkola T Karpanen T Kettunen M Pulkkanen K Kauppinen R Jackson D Kubo H Nishikawa S Inhibition of lymphangiogenesis with resulting lymphedema in transgenic mice expressing soluble VEGF receptor-3.Nat Med. 2001; 7: 199-205Crossref PubMed Scopus (650) Google Scholar one day before the LPS (5 μg/day for 7 days) administration. As a control, 1 × 109 plaque-forming units of Ad-LacZ was injected in the same manner. On the indicated days after the treatments, mice were anesthetized by intramuscular injection of a combination of anesthetics (80 mg/kg ketamine and 12 mg/kg xylazine). The indicated tissues were fixed by 1% paraformaldehyde in PBS and whole-mounted or cryoembedded and sectioned. Whole-mounted tissues and cryosections were incubated for 1 hour at room temperature with blocking solution containing 5% goat serum (Jackson ImmunoResearch Laboratories, West Grove, PA) in PBST (0.3% Triton X-100 in PBS). After blocking, the samples were incubated overnight at 4°C with one or more of the following primary antibodies: (a) for lymphatic vessels, rabbit anti-lymph vessel endothelial hyaluronan receptor (LYVE)-1 polyclonal antibody (1:1000; Upstate Biotechnology, Lake Placid, NY) and rat anti-LYVE-1 monoclonal antibody (clone Han-1, 1:1000; Aprogen, Daejeon, Korea); rabbit anti-Prox1 polyclonal antibody (1:1000; ReliaTech, Braunschweig, Germany); (b) for blood vessels, hamster anti-PECAM-1 antibody (clone 2H8, 1:1000; Chemicon International, Temecula, CA); (c) for macrophages, rat anti-CD11b antibody (clone M1/70, 1:1000; BD Pharmingen, San Diego, CA); rat anti-mouse F4/80 antibody (clone Cl:A3-1, 1:1000; Serotec, Oxford, UK), rat anti-mouse Gr-1 antibody (clone RB6-8C5, 1:1000; BD Pharmingen), hamster anti-mouse CD11c antibody (clone N418, 1:1000; Serotec), and rat anti-mouse CD45R/B220 antibody (clone RA3-6B2a, 1:1000; BD Pharmingen); and (d) for an assay of proliferation, rabbit anti-phosphohistone H3 polyclonal antibody (1:500; Upstate Biotechnology). After several washes in PBST, the samples were incubated for 3 hours at room temperature with the following secondary antibodies. For 3,3′-diaminobenzidine (DAB) immunostaining, samples were incubated with horseradish peroxidase-conjugated anti-rat IgG antibody (Jackson ImmunoResearch Laboratories) or horseradish peroxidase-conjugated anti-rabbit IgG antibody (Amersham, Piscataway, NJ) and developed with DAB substrate kit (Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions. For immunofluorescent staining, the samples were incubated with the following antibodies: fluorescein isothiocyanate (FITC)- or Cy5-conjugated anti-rat IgG antibody (1:1000; Jackson ImmunoResearch Laboratories); Cy3- or Cy5-conjugated anti-hamster IgG antibody (1:1000; Jackson ImmunoResearch Laboratories); and FITC- or Cy3-conjugated anti-rabbit IgG antibody (1:1000; Jackson ImmunoResearch Laboratories). For control experiments, the primary antibody was omitted or substituted with preimmune serum. DAB and fluorescent signals were visualized, and digital images were obtained using a Zeiss inverted microscope, a Zeiss ApoTome microscope, or a Zeiss LSM 510 confocal microscope equipped with argon and helium-neon lasers (Carl Zeiss). Morphometric analyses on the lymphatic vessels of diaphragm were made by ImageJ software (http://rsb.info.nih.gov/ij) or using Zeiss ApoTome microscope image analysis software (AxioVision; Carl Zeiss). Measurements of the density of the lymphatic vessels and the areas of CD11b+ cells in diaphragms were made at three regions at peritoneal and pleural surfaces of muscle regions, or central tendon region, each 35.18 or 0.21 mm2 in the area, with three to four mice per group. Numbers of Prox1 or PH3 immunopositivelymphatic endothelial cells (LECs) were counted at 3 regions, each 0.21 mm2 in the immunopositive area, of the central tendon regions or muscular regions of diaphragms. Measurements of the density of the lymphatic and blood vessels in lymph nodes (LNs) were made on a whole field, each 0.17 mm2 in the immunopositive area. Values were obtained per mm2 and expressed as relative densities. In some instances, the sectioned tissues were stained with H&E or Masson’s trichrome according to standard methods. To estimate the size of the LNs, we measured the length (L) and width (W) of each LN, then calculated the volume according to following equation: 4/3π × L/2 × W/2 × (L + W)/4. After anesthesia, diaphragms were harvested and dissected into small pieces by a microscissor, and the pieces were incubated with 5 ml of Hanks’ balanced salt solution (Sigma-Aldrich) containing 0.2% collagenase type-II (Worthington) for 1 hour at 37°C. After inactivation of collagenase activity with bovine serum, the cell suspension was filtered through a 70-μm nylon filter (BD Biosciences) and centrifuged at 400 × g for 5 minutes. The filtered cells were incubated with Red Blood Cell lysing buffer (Sigma-Aldrich) for 5 minutes and washed with a FACS buffer (0.5% BSA and 0.01 M EDTA in PBS) and double-stained with perCP-cy5.5-conjugated rat anti-mouse CD11b and FITC-conjugated rat anti-mouse F4/80, FITC-conjugated rat anti-mouse Gr-1, PE-conjugated rat anti-mouse CD11c, or FITC-conjugated rat anti-mouse B220 (BD Biosciences) for 15 minutes at 4°C. Then the cells were analyzed by flow cytometry (FACSCalibur; BD Biosciences) using a Cell Quest software. A total of ∼50,000 cells were counted per sample. Data were analyzed by using FlowJo software (Tree Star, Ashland, OR). Bone marrow cells (2 × 106) were harvested from the femurs and tibias of GFP+ mice (C57BL/6J genetic background) by flushing with ice-cold Dulbecco’s PBS (Sigma-Aldrich). The recipient mice (8-week-old C57BL/6J) were sublethally irradiated at a dose of 4.5 Gy with a gamma irradiator (Gammacell 3000; MDS Nordion, Canada). Bone marrow cells were then injected i.v. into the recipient mice 16 hours after irradiation. At 8 weeks after transplant, 5 μg of LPS was injected daily into the peritoneal cavity for 1 week. Flow cytometric analysis revealed that more than 90% of peripheral blood mononuclear cells were GFP+ cells at this time point. At the indicated days after treatment, mice were anesthetized and diaphragms were harvested. To collect CD11b+ macrophages from diaphragms, the attached cells were collected following the method as described in the flow cytometric analysis. After several washes of collected cells in cold PBS, the CD11b+ macrophages were enriched by using anti-mouse CD11b antibody-coupled MicroBeads (Miltenyi Biotec) and a Magnetic Cell Sorter (MACS, Miltenyi Biotec) according to the manufacturer’s instructions. Total RNA from the enriched CD11b+ macrophages was extracted by using Total RNA Isolation System (Promega, Madison, WI) according to the manufacturer’s instructions. Each cDNA was made with Reverse Transcription System (Promega), and semiquantitative PCR was performed with the appropriate primers (Table 1) with 30 cycles used for the PCR. Quantitative RT-PCR was performed with SYBR Premix Ex Taq (Takara, Japan) using the iCycler iQ5 Real-time PCR system (Bio-Rad, Hercules, CA) with the appropriate primers (Table 1) for 40 cycles.Table 1Primers for Semiquantitative RP-PCR and Quantitative Real-Time RT-PCRVEGF-A*Primers for semiquantitative RT-PCR.Forward5′-GAGAGCAGAAGTCCCATGAAGTG-3′Reverse5′-CTTTCCGGTGAGAGGTCTGG-3′VEGF-A**Primers for quantitative real-time RT-PCR.Forward5′-GCTGTACCTCCACCATGCCAAG-3′Reverse5′-CGCACTCCAGGGCTTCATCG-3′VEGF-C*Primers for semiquantitative RT-PCR.Forward5′-GACATGTCCAACAAACTATGTGTGG-3′Reverse5′-CTGTTACCATGGTCCCACAGAG-3′VEGF-D*Primers for semiquantitative RT-PCR.Forward5′-CCGGGAGATCTCATTCAGCACC-3′Reverse5′-GCACAATAACTCATGAGCATTGCCC-3′Angiopoietin-1*Primers for semiquantitative RT-PCR.Forward5′-CAGTGGCTGCAAAAACTTGA-3′Reverse5′-TCCGCACAGTCTCGAAATGG-3′Angiopoietin-2*Primers for semiquantitative RT-PCR.Forward5′-CCGCTATGAAGTTCCTCTCTGC-3′Reverse5′-CTGCTATGCAATGGTGTCTCTC-3′Interleukin-6*Primers for semiquantitative RT-PCR.Forward5′-GTCCAAGCAGAGCTCTGTCATTG-3′Reverse5′-GATGGTCTTGGTCCTTAGCCAC-3′Interleukin-1β*Primers for semiquantitative RT-PCR.Forward5′-GAAGAGCCCATCCTCTGTGACTC-3′Reverse5′-GTCCTGACCACTGTTGTTTCCCAG-3′TNF-α*Primers for semiquantitative RT-PCR.Forward5′-CCCACGTCGTAGCAAACCAC-3′Reverse5′-CACAGAGCAATGACTCCAAAGTAG-3′β-Actin*Primers for semiquantitative RT-PCR.Forward5′-CCCGCCACCAGTTCGCC-3′Reverse5′-GAGGGAGAGCATAGCCCTCG-3′GAPDH**Primers for quantitative real-time RT-PCR.Forward5′-AGGTCGGTGTGAACGGATTTG-3′Reverse5′-TGTAGACCATGTAGTTGAGGTCA-3′TNF, tumor necrosis factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.* Primers for semiquantitative RT-PCR.** Primers for quantitative real-time RT-PCR. Open table in a new tab TNF, tumor necrosis factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. On the indicated days after the LPS or PBS treatment, the mice were anesthetized, 300 μl of India ink (Pelikan, catalog no. 4001, Hannover, Germany) was injected into the peritoneal cavity, and the mice were kept on a warm pad in a supine position. At 15 minutes after injecting India ink, diaphragms were harvested, fixed with 1% paraformaldehyde in PBS and photographed. To determine a functional rate of lymphatic drainage of peritoneal large molecules into the sentinel LNs (SLNs) through diaphragmatic lymphatic vessels, 50 μl of Fluoresbrite (plain fluorescent YG microspheres, diameter, 2.0 μm; Polysciences, Washington, PA) diluted with 250 μl of PBS was injected into the peritoneal cavity of mice, and the mice were kept on a warm pad in a supine position. At 15 and 60 minutes after injecting the microspheres, two SLNs and diaphragms were harvested and fixed with 1% paraformaldehyde in PBS or embedded with tissue freezing medium. The fluorescent microspheres were visualized in the midsectioned SLNs and diaphragm after immunostaining for lymphatic vessels. For systemic depletion of macrophages, mice were treated with i.p. injections of clodronate liposome (25 mg/kg for every 3 days) as described previously.26Zeisberger S Odermatt B Marty C Zehnder-Fjällman A Ballmer-Hofer K Schwendener R Clodronate-liposome-mediated depletion of tumour-associated macrophages: a new and highly effective antiangiogenic therapy approach.Br J Cancer. 2006; 95: 272-281Crossref PubMed Scopus (509) Google Scholar, 27Seiler P Aichele P Odermatt B Hengartner H Zinkernagel R Schwendener R Crucial role of marginal zone macrophages and marginal zone metallophils in the clearance of lymphocytic choriomeningitis virus infection.Eur J Immunol. 1997; 27: 2626-2633Crossref PubMed Scopus (137) Google Scholar As a control, empty control liposome was injected in the same manner. Mice were anesthetized by intramuscular injection of the combination of anesthetics as described above, and a 1-cm midline incision was made to expose the cecum. The cecum was ligated with a 4-0 black silk suture and punctured with an 18-gauge needle. The cecum was squeezed to push a small amount of cecal contents from the punctured site into the peritoneal cavity, then was returned to the peritoneal cavity.28Corral J Yelamos J Hernandez-Espinosa D Monreal Y Mota R Arcas I Minano A Parrilla P Vicente V Role of lipopolysaccharide and cecal ligation and puncture on blood coagulation and inflammation in sensitive and resistant mice models.Am J Pathol. 2005; 166: 1089-1098Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar Sham operation without cecal ligation-puncture (CLP) was performed as a control. Values are presented as mean ± SD. Significant differences between means were determined by analysis of variance followed by the Student-Newman-Keuls test. Statistical significance was set at P < 0.05. Because lymphatic vessels in the diaphragm play a primary role in absorbing peritoneal fluid and in removing pathogens,2Tsilibary E Wissig S Light and electron microscope observations of the lymphatic drainage units of the peritoneal cavity of rodents.Am J Anat. 1987; 180: 195-207Crossref PubMed Scopus (84) Google Scholar, 3Hall J Heel K Papadimitriou J Platell C The pathobiology of peritonitis.Gastroenterology. 1998; 114: 185-196Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 20Azzali G The lymphatic vessels and the so-called “lymphatic stomata” of the diaphragm: a morphologic ultrastructural and three-dimensional study.Microvasc Res. 1999; 57: 30-43Crossref PubMed Scopus (42) Google Scholar, 21Shao X Ohtani O Saitoh M Ohtani Y Development of diaphragmatic lymphatics: the process of their direct connection to the peritoneal cavity.Arch Histol Cytol. 1998; 61: 137-149Crossref PubMed Scopus (19) Google Scholar, 22Shinohara H Lymphatic system of the mouse diaphragm: morphology and function of the lymphatic sieve.Anat Rec. 1997; 249: 6-15Crossref PubMed Scopus (18) Google Scholar we focused our experiments on characterizing the LPS-induced changes to lymphatic vessels in the diaphragm. LPS (5 μg) was administered daily into the i.p. cavity of 8-week-old mice. At 1 week after the LPS treatment, whole-mounted diaphragms were immunostained for LECs by using a specific marker, LYVE-1.29Banerji S Ni J Wang S Clasper S Su J Tammi R Jones M Jackson D LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan.J Cell Biol. 1999; 144: 789-801Crossref PubMed Scopus (1329) Google Scholar The daily administration of LPS gradually increased lymphatic densities on the peritoneal side muscular region, the pleural side muscular region, and the pleural side central tendon region in a time-dependent manner (Figure 1, Figure 2). In the peritoneal side muscular region, the lymphatic strips were widened and enlarged, and typical lymphatic patterning was disrupted over time (Figure 2A). In the pleural side muscular region, randomly oriented lymphatic branching increased over time (Figure 2A). In the pleural side, central tendon region, lymphatic connections, and networks increased over time (Figure 2A). Daily administration of 25 μg of LPS for 1 week did not further increase the lymphatic density on the pleural side muscular region and pleural side central tendon regions, but it did further widen and enlarge the lymphatic strips on the peritoneal side muscular region (Figure 2, A and B). Immunofluorescent staining of sagittal sections of the diaphragm in the mice treated with 5 μg of LPS for 1 week displayed enlarged lymphatic vessels in the peritoneal side muscular region and enlarged and connected lymphatic ves
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