Journal of Biochemistry Advance Access originally published online on September 10, 2007
Journal of Biochemistry 2007 142(5):587-595; doi:10.1093/jb/mvm164
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© 2007 The Japanese Biochemical Society
CEL-I, an Invertebrate N-Acetylgalactosamine-specific C-Type Lectin, Induces TNF-
and G-CSF Production by Mouse Macrophage Cell Line RAW264.7 Cells
1Division of Biochemistry, Faculty of Fisheries and 2Department of Applied Chemistry, Faculty of Engineering, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
*To whom correspondence should be addressed. Tel: +81-95-819-2831, Fax: +81-95-819-2799, E-mail: t-oda{at}nagasaki-u.ac.jp
Received July 8, 2007; Accepted August 9, 2007
 |
Abstract |
|---|
Our previous studies demonstrated that CEL-I, an N-acetylgalactosamine (GalNAc)-specific C-type lectin purified from the marine invertebrate Cucumaria echinata (Holothuroidea) showed potent cytotoxicity to several cell lines such as HeLa, MDCK and XC cells. In this study, we found that CEL-I induced increased secretion of tumour necrosis factor-
(TNF-) and granulocyte colony stimulation factor (G-CSF) by mouse macrophage cell line RAW264.7 cells in a dose-dependent manner, whereas this cell line was highly resistant to CEL-I cytotoxicity. The cytokine-inducing activity of CEL-I was stronger than that of phytohaemagglutinin (PHA-L). A binding study using FITC-labelled CEL-I (F-CEL-I) indicated that the amount of bound F-CEL-I on RAW264.7 cells was greater than that of F-PHA-L, suggesting that the greater activity of CEL-I to induce cytokine secretion by RAW264.7 cells is partly due to the higher binding ability. Since the cell binding and cytokine-inducing activity of CEL-I were partly but significantly inhibited by the specific sugar (GalNAc), it is considered that the binding of CEL-I to cell-surface-specific saccharide moieties, which may be recognized by CEL-I with higher affinity than GalNAc, is essential for the induction of cytokine secretion. The secretion of TNF- and G-CSF from CEL-I-treated RAW264.7 cells were almost completely prevented by brefeldin A (BFA), whereas increase in mRNA levels of these cytokines were not affected by BFA. Bio-Plex beads assay suggested that temporal increase in phosphorylation of extracellular-regulated kinase (ERK), c-jun NH2-terminal kinase (JNK) and p38 MAP kinase occurred at relatively early time following CEL-I treatment. Furthermore, the secretion of TNF- and G-CSF were inhibited by specific inhibitors for these MAP kinases. These results suggest that the intracellular signal transduction through the activation of MAP kinase system is involved in CEL-I-induced cytokine secretion.
Key Words:
C-type lectin, Cucumaria echinata, cytokines, granulocyte colony stimulating factor, macrophage cell line, tumour necrosis factor-
Abbreviations: BFA, brefeldin A; C-type lectin, Ca2+-dependent lectin; DMEM, Dulbecco's modified Eagle's minimal essential medium; ERK, extracellular-regulated kinase; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; F-CEL-I, FITC-labelled CEL-I; FITC, fluorescein isothiocyanate; GalNAc, N-acetylgalactosamine; G-CSF, granulocyte colony stimulating factor; GlcNAc, N-acetylglucosamine; JNK, c-jun NH2-terminal kinase; LPS, lipopolysaccharide; MAP kinase, mitogen-activated protein kinase; PBS, phosphate-buffered saline; PHA-L, phytohaemagglutinin-L (Phaseolus vulgaris agglutinin); TNF, tumour necrosis factor; WGA, wheat germ agglutinin