The principal function of the lymphatic system is to transport lymph from the interstitium to the nodes and then from the nodes to the blood. In doing so lymphatics play important roles in fluid homeostasis, macromolecular/antigen transport and immune cell trafficking. To better understand the genes that contribute to their unique physiology, we compared the transcriptional profile of muscular lymphatics (prenodal mesenteric microlymphatics and large, postnodal thoracic duct) to axillary and mesenteric arteries and veins isolated from rats. Clustering of the differentially expressed genes demonstrated that the lymph versus blood vessel differences were more profound than between blood vessels, particularly the microvessels. Gene ontology functional category analysis indicated that microlymphatics were enriched in antigen processing/presentation, IgE receptor signaling, catabolic processes, translation and ribosome; while they were diminished in oxygen transport, regulation of cell proliferation, glycolysis and inhibition of adenylate cyclase activity by G-proteins. We evaluated the differentially expressed microarray genes/products by qPCR and/or immunofluorescence. Immunofluorescence documented that multiple MHC class II antigen presentation proteins were highly expressed by an antigen-presenting cell (APC) type found resident within the lymphatic wall. These APCs also expressed CD86, a co-stimulatory protein necessary for T-cell activation. We evaluated the distribution and phenotype of APCs within the pre and postnodal lymphatic network. This study documents a novel population of APCs resident within the walls of muscular, prenodal lymphatics that indicates novel roles in antigen sampling and immune responses. In conclusion, these prenodal lymphatics exhibit a unique profile that distinguishes them from blood vessels and highlights the role of the lymphatic system as an immunovascular system linking the parenchymal interstitium, lymph nodes and the blood.
BanerjiS, NiJ, WangSX, ClasperS, SuJ, TammiR, JonesM, JacksonDG. LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan. J Cell Biol, 1999; 144:789–801.
4.
BatesMD, ErwinCR, SanfordLP, WigintonD, BezerraJA, SchatzmanLC, JeggaAG, Ley-EbertC, WilliamsSS, SteinbrecherKA, WarnerBW, CohenMB, AronowBJ. Novel genes and functional relationships in the adult mouse gastrointestinal tract identified by microarray analysis. Gastroenterology, 2002; 122:1467–1482.
5.
BeilhackA, RocksonS. Immune traffic: A functional overview. Lymphat Res Biol, 2003; 1:219–234.
6.
BenjaminiY, HochbergY. Controlling the false discovery rate: A practical and powerful approach to multiple testing. J R Statist Soc B, 1995; 57:289–300.
7.
BohlenHG, WangW, GashevAA, GashevaOY, ZawiejaDC. Phasic contractions of rat mesenteric lymphatics increase basal and phasic nitric oxide generation in vivo. Am J Physiol Heart Circ Physiol, 2009; 297:H1319–H1328.
8.
BridenbaughEA, GashevAA, ZawiejaDC. Lymphatic muscle: A review of contractile function. Lymphat Res Biol, 2003; 1:147–158.
9.
BustinSA. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol, 2000; 25:169–193.
10.
DellingerM, HunterR, BernasM, GaleN, YancopoulosG, EricksonR, WitteM. Defective remodeling and maturation of the lymphatic vasculature in Angiopoietin-2 deficient mice. Dev Biol, 2008; 319:309–320.
11.
EdgarR, DomrachevM, LashAE. Gene expression omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res, 2002; 30:207–210.
12.
GashevAA, DavisMJ, DelpMD, ZawiejaDC. Regional variations of contractile activity in isolated rat lymphatics. Microcirculation, 2004; 11:477–492.
13.
GashevAA, DavisMJ, ZawiejaDC. Inhibition of the active lymph pump by flow in rat mesenteric lymphatics and thoracic duct. J Physiol, 2002; 540:1023–1037.
14.
GashevaOY, ZawiejaDC, GashevAA. Contraction-initiated NO-dependent lymphatic relaxation: a self-regulatory mechanism in rat thoracic duct. J Physiol, 2006; 575:821–832.
KaipainenA, KorhonenJ, MustonenT, van HinsberghVW, FangGH, DumontD, BreitmanM, AlitaloK. Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development. Proc Natl Acad Sci USA, 1995; 92:3566–3570.
19.
KataruRP, JungK, JangC, YangH, SchwendenerRA, BaikJE, HanSH, AlitaloK, KohGY. Critical role of CD11b+ macrophages and VEGF in inflammatory lymphangiogenesis, antigen clearance, and inflammation resolution. Blood, 2009; 113:5650–5659.
20.
KawaiY, HosakaK, KaidohM, MinamiT, KodamaT, OhhashiT. Heterogeneity in immunohistochemical, genomic, and biological properties of human lymphatic endothelial cells between initial and collecting lymph vessels. Lymphat Res Biol, 2008; 6:15–27.
21.
LivakKJ, SchmittgenTD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 2001; 25:402–408
22.
MaglottD, OstellJ, PruittKD, TatusovaT. Entrez Gene: Gene-centered information at NCBI. Nucleic Acids Res, 2005; 33:D54–D58.
23.
MakinenT, AdamsRH, BaileyJ, LuQ, ZiemieckiA, AlitaloK, KleinR, WilkinsonGA. PDZ interaction site in ephrinB2 is required for the remodeling of lymphatic vasculature. Genes Dev, 2005; 19:397–410.
24.
MaruyamaK, IiM, CursiefenC, JacksonDG, KeinoH, TomitaM, Van RooijenN, TakenakaH, D'AmorePA, Stein-StreileinJ, LosordoDW, StreileinJW. Inflammation-induced lymphangiogenesis in the cornea arises from CD11b-positive macrophages. J Clin Invest, 2005; 115:2363–2372.
25.
MatsuoM, KoizumiK, YamadaS, TomiM, TakahashiR, UedaM, TerasakiT, ObinataM, HosoyaK, OhtaniO, SaikiI. Establishment and characterization of conditionally immortalized endothelial cell lines from the thoracic duct and inferior vena cava of tsA58/EGFP double-transgenic rats. Cell Tissue Res, 2006; 326:749–758.
26.
ModiS, StantonAW, SvenssonWE, PetersAM, MortimerPS, LevickJR. Human lymphatic pumping measured in healthy and lymphoedematous arms by lymphatic congestion lymphoscintigraphy. J Physiol, 2007; 583:271–285.
27.
MuthuchamyM, GashevA, BoswellN, DawsonN, ZawiejaD. Molecular and functional analyses of the contractile apparatus in lymphatic muscle. FASEB J, 2003; 17:920–922.
RocksonSG. Lymphedema. Am J Med, 110:288–295. 2001.
39.
RocksonSG, RiveraKK. Estimating the population burden of lymphedema. Ann N Y Acad Sci, 1131:147–154. 2008.
40.
RutkowskiJM, MoyaM, JohannesJ, GoldmanJ, SwartzMA. Secondary lymphedema in the mouse tail: Lymphatic hyperplasia, VEGF-C upregulation, and the protective role of MMP-9. Microvasc Res, 72:161–171. 2006.
41.
SchenaM, ShalonD, DavisRW, BrownPO. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science, 270:467–470. 1995.
42.
Schmid-SchonbeinGW. Microlymphatics and lymph flow. Physiol Rev, 70:987–1028. 1990.
43.
SchmittgenTD, ZakrajsekBA. Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR. J Biochem Biophys Methods, 46:69–81. 2000.
ShinJW, HuggenbergerR, DetmarM. Transcriptional profiling of VEGF-A and VEGF-C target genes in lymphatic endothelium reveals endothelial-specific molecule-1 as a novel mediator of lymphangiogenesis. Blood, 112:2318–2326. 2008.
46.
SmythGK. Limma: linear models for microarray data. Bioinformatics and Computational Biology Solutions using R and Bioconductor. GentlemanR, CareyV, DudoitS, IrizarryR, HuberW. New York: Springer, 2005; 397–420.
47.
SmythGK. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol, 3,Article32004.
48.
SrinivasanRS, DillardME, LagutinOV, LinFJ, TsaiS, TsaiMJ, SamokhvalovIM, OliverG. Lineage tracing demonstrates the venous origin of the mammalian lymphatic vasculature. Genes Dev, 21:2422–2432. 2007.
49.
SwartzMA. The physiology of the lymphatic system. Adv Drug Deliv Rev, 50:3–20. 2001.
50.
SwartzMA, HubbellJA, ReddyST. Lymphatic drainage function and its immunological implications: from dendritic cell homing to vaccine design. Semin Immunol, 20:147–156. 2008.
51.
SzaboG, MagyarZ. Pressure measurements in various parts of the lymphatic system. Acta Med Acad Sci Hung, 23:237–241. 1967.
TabibiazarR, WagnerRA, LiaoA, QuertermousT. Transcriptional profiling of the heart reveals chamber-specific gene expression patterns. Circ Res, 93:1193–1201. 2003.
55.
TammelaT, SaaristoA, HolopainenT, LyytikkaJ, KotronenA, PitkonenM, Abo-RamadanU, Yla-HerttualaS, PetrovaTV, AlitaloK. Therapeutic differentiation and maturation of lymphatic vessels after lymph node dissection and transplantation. Nat Med, 13:1458–1466. 2007.
56.
TomeiAA, SiegertS, BritschgiMR, LutherSA, SwartzMA. Fluid Flow Regulates Stromal Cell Organization and CCL21 Expression in a Tissue-Engineered Lymph Node Microenvironment. J Immunol, 2009.
57.
WangW, NepiyushchikhZ, ZawiejaDC, ChakrabortyS, ZawiejaSD, GashevAA, DavisMJ, MuthuchamyM. Inhibition of myosin light chain phosphorylation decreases rat mesenteric lymphatic contractile activity. Am J Physiol Heart Circ Physiol, 297:H726–H734. 2009.
58.
YongC, BridenbaughEA, ZawiejaDC, SwartzMA. Microarray analysis of VEGF-C responsive genes in human lymphatic endothelial cells. Lymphat Res Biol, 3:183–207. 2005.
59.
YuanZ, RodelaH, HayJB, OreopoulosD, JohnstonMG. Lymph flow and lymphatic drainage of inflammatory cells from the peritoneal cavity in a casein-peritonitis model in sheep. Lymphology, 27:114–128. 1994.
60.
ZhangB, KirovS, SnoddyJ. WebGestalt: an integrated system for exploring gene sets in various biological contexts. Nucleic Acids Res, 33:W741–W748. 2005.
ZhongH, SimonsJW. Direct comparison of GAPDH, beta-actin, cyclophilin, and 28S rRNA as internal standards for quantifying RNA levels under hypoxia. Biochem Biophys Res Commun, 259:523–526. 1999.
63.
ZweifachBW, PratherJW. Micromanipulation of pressure in terminal lymphatics in the mesentery. Am J Physiol, 228:1326–1335. 1975.
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