Aim: YCP,
a novel (1,4)-α-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 p
38 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.
Conclusion: We have
demonstrated that YCP-induced NO production occurs through the TLR4 and CR3
membrane receptors in a p38 kinase-dependent manner in macrophages.
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