title: Isolation, characterization, and functional analysis of ferret lymphatic endothelial cells.
abstract: The lymphatic endothelium (LE) serves as a conduit for transport of immune cells and soluble antigens from peripheral tissues to draining lymph nodes (LNs), contributing to development of host immune responses and possibly dissemination of microbes. Lymphatic endothelial cells (LECs) are major constituents of the lymphatic endothelium. These specialized cells could play important roles in initiation of host innate immune responses through sensing of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs), including toll-like receptors (TLRs). LECs secrete pro-inflammatory cytokines and chemokines to create local inflammatory conditions for recruitment of naïve antigen presenting cells (APCs) such as dendritic cells (DCs) to sites of infection and/or vaccine administration. In this study, we examined the innate immune potential of primary LEC populations derived from multiple tissues of an animal model for human infectious diseases - the ferret. We generated a total of six primary LEC populations from lung, tracheal, and mesenteric LN tissues from three different ferrets. Standard RT-PCR characterization of these primary LECs showed that they varied in their expression of LEC markers. The ferret LECs were examined for their ability to respond to poly I:C (TLR3 and RIG-I ligand) and other known TLR ligands as measured by production of proinflammatory cytokine (IFNα, IL6, IL10, Mx1, and TNFα) and chemokine (CCL5, CCL20, and CXCL10) mRNAs using real time RT-PCR. Poly I:C exposure induced robust proinflammatory responses by all of the primary ferret LECs. Chemotaxis was performed to determine the functional activity of CCL20 produced by the primary lung LECs and showed that the LEC-derived CCL20 was abundant and functional. Taken together, our results continue to reveal the innate immune potential of primary LECs during pathogen-host interactions and expand our understanding of the roles LECs might play in health and disease in animal models.
authors.affiliation: Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 15261, USA.
authors.forename: Stella J
authors.initials: SJ
authors.lastname: Berendam
authors.affiliation: Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 15261, USA.
authors.forename: Beth A
authors.initials: BA
authors.lastname: Fallert Junecko
authors.affiliation: Department of Microbiology and Molecular Genetics, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261, USA.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Murphey-Corb
authors.affiliation: Department of Microbiology, School of Medicine, University of Washington, Seattle, WA 98195, USA.
authors.forename: Deborah H
authors.initials: DH
authors.lastname: Fuller
authors.affiliation: Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 15261, USA. Electronic address: reinhar@pitt.edu.
authors.forename: Todd A
authors.initials: TA
authors.lastname: Reinhart
mesh_terms: D000818:Animals; D015415:Biomarkers; D018929:Cell Culture Techniques; D054418:Chemokine CCL20 / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology* / Q000502:physiology; D005260:Female; D005289:Ferrets / Q000502:physiology*; D005786:Gene Expression Regulation / Q000276:immunology / Q000502:physiology; D008168:Lung; D010802:Phylogeny; D051193:Toll-Like Receptors / Q000378:metabolism
chemical_list: D015415:Biomarkers; D054418:Chemokine CCL20; D018076:DNA, Complementary; D051193:Toll-Like Receptors
keywords: Chemokine; Ferret; Lymphatic endothelial cell; Lymphatics; Toll-like receptor