Macrophage receptors of polysaccharide isolated from a marine filamentous fungus Phoma herbarum YS4108.
OBJECTIVE
YCP, a novel (1,4)-alpha-D-glucan, was isolated from the mycelium of the marine filamentous fungus Phoma herbarum YS4108. In this work, we investigated a YCP-binding cellular receptor expressed by macrophages and the intracellular signal transduction pathways involved in YCP-induced macrophage activation.


METHODS
Fluorescence-labeled YCP (fl-YCP) was prepared using the CDAP-activation method. Fluorescence confocal laser microscopy and fluorescence-activated cell sorting (FACS) were used to analyze the effect of fl-YCP on macrophages. To characterize the properties of the YCP receptor, carbohydrates and antibodies were used to inhibit the binding of fl-YCP to macrophages. Moreover, we investigated the role of membrane receptors Toll-like receptor 2 (TLR2), Toll-like receptor 4 (TLR4), Toll-like receptor 6 (TLR6) and complement receptor 3 (CR3). We also examined the role of the p38 kinase pathway in mediating nitric oxide (NO) production.


RESULTS
YCP had an in vitro stimulatory effect on the release of NO in macrophage, and fl-YCP can bind directly to receptors on the surface of macrophages in a time- and dose-dependent manner. Competition studies show that LPS, laminarin, anti-TLR4 antibody and anti-CD11b (CR3) antibody could inhibit fl-YCP binding to macrophages. Conversely, mannose, anti-TLR2 and anti-TLR6 antibody could not. Treatment of RAW264.7 cells with YCP resulted in significant activation of p38 in a time-dependent manner. The specific p38 inhibitor SB203580 abrogated YCP-induced NO generation. Treatment of RAW264.7 cells with anti-TLR4 antibody and anti-CR3 antibody significantly reduced YCP-induced NO production and p38 activation.


CONCLUSIONS
We have demonstrated that YCP-induced NO production occurs through the TLR4 and CR3 membrane receptors in a p38 kinase-dependent manner in macrophages.Acta Pharmacologica Sinica (2009) 30: 1008-1014; doi: 10.1038/aps.2009.93.
Chen
Song
S
School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China.
Yin
Deng-Ke
DK
Yao
Wen-Bing
WB
Wang
Yi-Dan
YD
Zhang
Yi-Ran
YR
Gao
Xiang-Dong
XD
D000818:Animals; D001203:Ascomycota / Q000737:chemistry*; D039481:CD11b Antigen / Q000378:metabolism; D002460:Cell Line; D004789:Enzyme Activation; D005936:Glucans; D006801:Humans; D008070:Lipopolysaccharides / Q000378:metabolism; D008262:Macrophage Activation; D008264:Macrophages / Q000378:metabolism*; D008358:Mannose / Q000378:metabolism; D051379:Mice; D009573:Nitrites / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D012623:Seawater / Q000382:microbiology; D015398:Signal Transduction / Q000502:physiology; D051195:Toll-Like Receptor 2 / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000378:metabolism; D051216:Toll-Like Receptor 6 / Q000378:metabolism; D047071:beta-Glucans / Q000737:chemistry / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D039481:CD11b Antigen; D005936:Glucans; D008070:Lipopolysaccharides; D009573:Nitrites; D011134:Polysaccharides; D011956:Receptors, Cell Surface; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051216:Toll-Like Receptor 6; D047071:beta-Glucans; C008247:laminaran; D048051:p38 Mitogen-Activated Protein Kinases; D008358:Mannose