Comparative study on modeccin- and phytohemagglutinin (PHA)-induced secretion of cytokines and nitric oxide (NO) in RAW264.7 cells.

The effects of cytotoxic lectins, modeccin and phytohemagglutinin (PHA) on mouse macrophage cell line RAW264.7 was studied by detecting the induction of inflammatory mediators. Results showed that modeccin induced the release of tumor necrosis factor-α (TNF-α) from RAW264.7 cells with a bell-shape concentration-dependent profile. PHA that showed no significant cytotoxicity on RAW264.7 cells up to 100,000 ng/ml induced much higher level of TNF-α than modeccin. PHA simultaneously induced the secretion of granulocyte colony stimulation factor (G-CSF) from RAW264.7 cells with even much higher level than that of TNF-α, whereas modeccin did not. Furthermore, PHA induced the secretion of nitric oxide (NO) in RAW264.7 cells, while no significant level of NO was detected in the modeccin-treated cells. NH4Cl (a lysomotoropic agent) and cycloheximide (a ribosome inhibitor) strongly inhibited modeccin-induced TNF-α secretion, but no significant inhibitory effects of these reagents on the PHA-induced TNF-α secretion were observed. Contrary to modeccin-induced TNF-α secretion, even slightly increased TNF-α secretion was observed in PHA-treated cells in the presence of 10 mM NH4Cl. In addition, the inhibition profiles of modeccin-induced TNF-α secretion by various kinase inhibitors were different from those of PHA. These results suggested that the action mode of modeccin to stimulate RAW264.7 cells leading to the secretion of inflammatory molecules, including TNF-α, is distinct from that of PHA. On the other hand, significantly increased translocation of activator protein-1 (AP-1), a crucial transcription factor involved in expression of inflammatory molecules, into nucleus was observed in RAW264.7 cells treated with PHA and modeccin.

Mitogenic activity of CEL-I, an N-acetylgalactosamine (GalNAc)-specific C-type lectin, isolated from the marine invertebrate Cucumaria echinata (Holothuroidea).

An N-acetylgalactosamine (GalNAc)-specific Ca(2+)-dependent lectin (C-type lectin), isolated from the marine invertebrate Holothuroidea (Cucumaria echinata), CEL-I, showed potent mitogenic activity toward normal mouse spleen cells. The mitogenic activity of CEL-I, which reached a maximum at 100 microg/ml, was inhibited by GalNAc in a concentration-dependent manner. The mitogenic effect of CEL-I at 10 microg/ml on T cell- enriched splenocytes was at a similar level due to a well-known T cell mitogen, concanavalin A (Con A), at 10 microg/ml. Furthermore, CEL-I evoked a mitogenic response from nude mouse spleen cells, while no significant effects of Con A on this cell population were observed over a wide range of concentrations. These results suggest that CEL-I is a potent mitogenic lectin with the ability to stimulate both T and B cells.

Comparative analysis of the production of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) from macrophages exposed to high virulent and low virulent strains of Edwardsiella tarda.

We previously reported that high virulent strain (NUF251) of Edwardsiella tarda has an ability to prevent the production of reactive oxygen species by macrophages, and is even capable of surviving and multiplying within Japanese flounder (Paralichthys olivaceus) peritoneal macrophages, whereas the low virulent strain (NUF194) has no such ability. In this study, we found that NUF251 and NUF194 induced NO and TNF-alpha production from Japanese flounder peritoneal macrophages, and NUF251 caused faster induction of NO release and much higher level of TNF-alpha production than NUF194. In addition, similar differences between two strains in terms of the induction of NO and TNF-alpha production were also observed in mouse macrophage cell line RAW264.7 cells. Our results suggest that the potent ability to induce the production of NO and TNF-alpha from macrophages may be one of the factors responsible for the virulence of E. tarda.

CEL-I, an N-acetylgalactosamine (GalNAc)-specific C-type lectin, induces nitric oxide production in RAW264.7 mouse macrophage cell line.

We found that CEL-I, a GalNAc-specific C-type lectin isolated from the marine invertebrate Holothuroidea (Cucumaria echinata), induces inducible nitric oxide synthase (iNOS) expression and NO production in RAW264.7 cells. The NO production was inhibited by an iNOS inhibitor, L-NAME, but was not by a lipopolysaccharide (LPS) inhibitor, polymyxin B. In the presence of 0.1-M GalNAc, increased NO production by CEL-I-treated RAW264.7 cells was observed rather than the inhibition. Bovine serum albumin (BSA) significantly inhibited the CEL-I-induced NO production as well as the binding of FITC-labelled CEL-I on RAW264.7 cells. Three MAP kinase inhibitors (specific to extra-cellular regulated kinase, c-jun NH(2)-terminal kinase and p38 MAP kinase) inhibited CEL-I-induced NO production with different extents. Heat-treatment of CEL-I resulted in a decreased activity of CEL-I depending on the temperature. These results suggest that CEL-I induces NO production in RAW264.7 cells through the protein-cell interaction rather than the binding to the specific carbohydrate chains on the cell surface.

CEL-I, an invertebrate N-acetylgalactosamine-specific C-type lectin, induces TNF-alpha and G-CSF production by mouse macrophage cell line RAW264.7 cells.

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-alpha (TNF-alpha) 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-alpha 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 NH(2)-terminal kinase (JNK) and p38 MAP kinase occurred at relatively early time following CEL-I treatment. Furthermore, the secretion of TNF-alpha 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.