Expression of toll-like receptors 2 and 4 and CD14 during differentiation of HL-60 cells induced by phorbol 12-myristate 13-acetate and 1 alpha, 25-dihydroxy-vitamin D(3).
Macrophages form a crucial bridge between the innate and adaptive immune response. One of their most important functions is to recognize infectious microorganisms. Toll-like receptors (TLRs) are key elements in pathogen recognition, and among them, TLR2 and TLR4 are most discussed. However, expression patterns of TLRs during myeloid cell differentiation to macrophage are unknown. In this study, we examined differentiation in the model human myeloid cell line, HL-60, treated with phorbol 12-myristate 13-acetate (PMA) or VitD(3). Expression of TLR2, TLR4, and CD14 were measured by reverse transcription-PCR, RNase protection assay, and fluorescence-activated cell sorter assays. After treatment by PMA (1, 10, and 100 nM) for 12, 24, and 48 h, expression of TLR2 and CD14 mRNA was increased in a time- and dose-dependent manner. However, VitD(3) only induced expression of CD14 but not TLR2 in HL-60 cells. TLR4 was expressed constitutively before differentiation and increased slightly after that. Thus, PMA-mediated differentiation of HL-60 cells to macrophages is associated largely with TLR2 expression and, to a much lesser extent, with TLR4. Furthermore, up-regulation of TLR2 and CD14 mRNA expression by PMA was abrogated by a protein kinase C inhibitor, Calphostine C, suggesting the up-regulation of TLR2 and CD14 mRNA is dependent on the activation of protein kinase C. Coexpression of CD14/TLR2 and/or CD14/TLR4 may be essential but not sufficient for the production of tumor necrosis factor-alpha in response to lipopolysaccharide in our system.
Li
Changlin
C
Laboratory of Immune Cells Signaling, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
Wang
Yibing
Y
Gao
Li
L
Zhang
Jingsong
J
Shao
Jie
J
Wang
Shengnian
S
Feng
Weiguo
W
Wang
Xingyu
X
Li
Minglie
M
Chang
Zongliang
Z
D002117:Calcitriol / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D029721:Drosophila Proteins; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D018922:HL-60 Cells; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000096:biosynthesis* / Q000235:genetics; D008264:Macrophages / Q000502:physiology; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics; D009281:Naphthalenes / Q000494:pharmacology; D010587:Phagocytosis / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011956:Receptors, Cell Surface / Q000096:biosynthesis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D015533:Transcriptional Activation; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D029721:Drosophila Proteins; D004791:Enzyme Inhibitors; D018950:Lipopolysaccharide Receptors; D008562:Membrane Glycoproteins; D009281:Naphthalenes; D011956:Receptors, Cell Surface; C495342:TLR2 protein, human; C495345:TLR4 protein, human; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha; D011493:Protein Kinase C; D002117:Calcitriol; C058819:calphostin C; D013755:Tetradecanoylphorbol Acetate