Genetic immunization with LYVE-1 cDNA yields function-blocking antibodies against native protein.
LYVE-1 is a surface bound hyaluronic acid (HA) receptor that is preferentially expressed by lymphatic endothelial cells (LEC). cDNA encoding full-length human LYVE-1 was coated onto gold particles that were then delivered via helium-assisted jet propulsion (gene gun) into the skin of Balb/C mice. LYVE-1 antisera, but not control pre-immune sera, recognized LYVE-1-transfected 293T cells by flow cytometry. While 40-70% of cultured human dermal microvascular endothelial cells (HMEC) were positive for LYVE-1 staining, human lung microvascular endothelial cells (LMEC) were negative. LYVE-1 antisera was used to effectively separate HMEC into LYVE-1 (hi) and LYVE-1(lo) populations that were enriched or depleted, respectively, for podoplanin, another LEC marker. By immunohistochemistry, LYVE-1 antisera detected CD31(lo) podoplanin(hi) lymphatic channels in normal and psoriatic human skin as well as in human tonsil. LYVE-1 antisera also blocked binding of FITC-labeled HA to HMEC (but not LMEC), demonstrating that these antibodies recognized regions of LYVE-1 required for HA binding. In summary, gene gun-assisted delivery of cDNA encoding LYVE-1 into skin resulted in reliable production of antisera that specifically and functionally recognized native LYVE-1 protein.
Cardones
Adela R
AR
Dermatology, National Cancer Institute, National Institutes of Health, 10 Center Drive, Room 12N258, Bethesda, MD 20892, USA.
Leitner
Wolfgang W
WW
Fang
Lei
L
Murakami
Takashi
T
Kapoor
Veena
V
Udey
Mark C
MC
Hwang
Sam T
ST
D000818:Animals; D019138:Antibodies, Blocking / Q000494:pharmacology*; D019470:Biolistics; D015415:Biomarkers; D002460:Cell Line; D002469:Cell Separation; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D004729:Endothelium, Lymphatic / Q000166:cytology / Q000276:immunology; D005260:Female; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes; D006023:Glycoproteins / Q000235:genetics / Q000276:immunology*; D006801:Humans; D006820:Hyaluronic Acid / Q000378:metabolism*; D007114:Immunization; D007155:Immunologic Factors / Q000494:pharmacology*; D026901:Membrane Transport Proteins; D051379:Mice; D008807:Mice, Inbred BALB C; D012333:RNA, Messenger / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D012867:Skin / Q000166:cytology
D019138:Antibodies, Blocking; D015415:Biomarkers; D018076:DNA, Complementary; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006023:Glycoproteins; D006820:Hyaluronic Acid; D007155:Immunologic Factors; D026901:Membrane Transport Proteins; D012333:RNA, Messenger; C492273:LYVE1 protein, mouse