A synthetic analog of alpha-galactosylceramide induces macrophage activation via the TLR4-signaling pathways.
Alpha-galactosylceramide (alpha-GalCer), a bioactive glycolipid isolated from the marine sponge Agelas mauritianus, is a potent immunomodulator with therapeutic potential for the treatment of autoimmune diseases and cancer. The Toll-like receptor 4 (TLR4), one of the promising molecular targets for immune-modulating drugs, is commonly expressed in innate immune cells especially macrophages and dendritic cells. Currently, whether alpha-GalCer can activate TLR4 signaling pathways remains unreported. In this study, we examined the effects of alpha-GalCer and its various structural analogs, CCL-1 approximately 47, on TLR4 activation. We found that one alpha-GalCer analog (CCL-34), but not alpha-GalCer itself, strongly stimulated NF-kappaB activity in RAW 264.7 cells. CCL-34 activated NF-kappaB in a TLR4-dependent manner and stimulated TNF-alpha production in bone marrow cells of TLR4-functional C3H/HeN mice but not in those of TLR4-defective C3H/HeJ mice. Furthermore, CCL-34 treatment stimulated NF-kappaB activation and IL-8 production in a 293 cell line constitutively expressing human TLR4, MD-2 and CD14. Treatment of RAW 264.7 cells with CCL-34 also activated TLR4-downstream mitogen-activated protein kinases (ERK, JNK and p38), induced expression of TLR4-downstream genes (TNF-alpha, IL-6, IL-1beta and iNOS) and promoted production of cytokines characteristic of activated macrophages. CCL-34-treated RAW 264.7 cells acquired a distinct morphology similar to that of LPS-activated macrophages and exhibited higher phagocytotic activity. Moreover, treatment with a TLR4-neutalizing antibody inhibited the CCL-34-induced morphological alteration. In summary, we identify a novel synthetic compound CCL-34 that can activate macrophages via TLR4-dependent signaling pathways. Our results suggest that CCL-34 is an immune modulator and may serve as a potential drug lead for immunotherapy.
Hung
Ling-Chien
LC
Institute of Traditional Medicine, National Yang-Ming University, Taipei 11221, Taiwan.
Lin
Chun-Cheng
CC
Hung
Shih-Kai
SK
Wu
Bing-Ching
BC
Jan
Mi-Dan
MD
Liou
Sheng-Hung
SH
Fu
Shu-Ling
SL
D000818:Animals; D002460:Cell Line; D005699:Galactosylceramides / Q000138:chemical synthesis / Q000737:chemistry; D017930:Genes, Reporter; D008156:Luciferases / Q000378:metabolism; D008262:Macrophage Activation / Q000187:drug effects*; D017737:Macrophages, Peritoneal / Q000187:drug effects*; D051379:Mice; D015394:Molecular Structure; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects*; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*
D005699:Galactosylceramides; D051197:Toll-Like Receptor 4; C493154:alpha-galactosylceramide; D008156:Luciferases