Use of murine embryonic fibroblasts to define Toll-like receptor activation and specificity.
Toll-like receptors (TLRs) are critically involved in the innate immune response to bacterial, viral and fungal pathogens. We have studied human peripheral blood mononuclear cells, murine embryonic fibroblasts (MEFs) and a panel of human cell lines, including HEK, HeLa, AGS, ECV304 and U373 cells, for expression of TLR-specific mRNAs and for TLR-ligand dependent cytokine secretion. Peripheral blood cells expressed multiple TLRs; however, many studies have shown that blood contains multiple, heterogeneous cell populations with distinct patterns of TLR expression. Cell lines had variable expression of TLRs, and in most cases lacked TLR2 and TLR8 expression and only weakly expressed mRNAs for TLR5, TLR7 and TLR9. In contrast, MEFs expressed high levels of mRNA for TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8 and TLR9. MEFs were highly responsive to TLR-ligand activation and secreted high levels of both IL-6 and MCP-1 in response to TLR ligands. MEFs from mice with targeted deletions of TLR2, TLR4 and MyD88 demonstrated profound defects in their IL-6 response to their specific ligands, consistent with studies of macrophages and tissues from adult knockout animals. MEF cultures are homogenous and amenable to biochemical analysis and should allow rigorous studies of the contribution of individual TLRs to the innate immune response.
Kurt-Jones
Evelyn A
EA
Department of Medicine, University of Massachusetts Medical Center, 364 Plantation Street, Lazare Research Building Room 226, Worcester, Massachusetts 01605, USA. Evelyn.Kurt-Jones@umassmed.edu
Sandor
Frantisek
F
Ortiz
Yasdel
Y
Bowen
Glennice N
GN
Counter
Stacy L
SL
Wang
Timothy C
TC
Finberg
Robert W
RW
D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000818:Animals; D016207:Cytokines / Q000276:immunology / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004622:Embryo, Mammalian / Q000166:cytology*; D005260:Female; D005347:Fibroblasts / Q000166:cytology* / Q000187:drug effects / Q000276:immunology*; D006367:HeLa Cells; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D008297:Male; D008562:Membrane Glycoproteins / Q000187:drug effects / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D012333:RNA, Messenger / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000235:genetics / Q000276:immunology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051194:Toll-Like Receptor 1; D051195:Toll-Like Receptor 2; D051196:Toll-Like Receptor 3; D051197:Toll-Like Receptor 4; D051198:Toll-Like Receptor 5; D051199:Toll-Like Receptor 7; D051200:Toll-Like Receptor 8; D051217:Toll-Like Receptor 9; D051193:Toll-Like Receptors
D000276:Adjuvants, Immunologic; D016207:Cytokines; D017931:DNA Primers; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; C495342:TLR2 protein, human; C495344:TLR3 protein, human; C495345:TLR4 protein, human; C495346:TLR5 protein, human; C495348:TLR7 protein, human; C495349:TLR8 protein, human; C495371:TLR9 protein, human; D051194:Toll-Like Receptor 1; D051195:Toll-Like Receptor 2; D051196:Toll-Like Receptor 3; D051197:Toll-Like Receptor 4; D051198:Toll-Like Receptor 5; D051199:Toll-Like Receptor 7; D051200:Toll-Like Receptor 8; D051217:Toll-Like Receptor 9; D051193:Toll-Like Receptors