Guluronate and mannuronate oligomers with various degree of polymerization were prepared from polyguluronate (PG) and polymannuronate (PM) with an alginate lyase from a Pseudoalteromonas sp., and their activities to induce cytokine secretion from mouse macrophage cell line RAW264.7 cells were examined. Enzymatically depolymerized unsaturated alginate oligomers induced tumor necrosis factor (TNF)-a secretion from RAW264.7 cells in a structure-depending manner, while the activities of saturated alginate oligomers prepared by acid hydrolysis were fairly low or only trace levels. These results suggest that unsaturated end-structure of alginate oligomers was important for the TNF-a-inducing activity. Among the unsaturated guluronate (G3-G9) and mannuronate (M3-M9) oligomers, G8 and M7 showed the most potent activity, respectively. BioPlex assay revealed that interleukin (IL)-1a, IL-1b, and IL-6 secretion from RAW264.7 cells were also induced by unsaturated alginate oligomers with similar structure-activity relationship profiles as seen in TNF-a, and the most potent activities were observed with G8 and M7. These results suggest that G8 and M7 may have the most suitable molecular size or entire structural conformation as stimulant for cytokine secretion. Since antibodies to Toll-like receptor (TLR)2 and TLR4 effectively inhibited the G8-and M7-induced production of TNF-a, these alginate oligomers may stimulate innate immunity through the pattern recognition receptors on macrophages similar to microbial products.

Recently we discovered a bacterial strain (MS‐02–063) that produces large amounts of red pigment from coastal area of Nagasaki Prefecture, Japan. Comparative 16S rDNA gene sequencing analysis revealed that strain MS‐02–063 was phylogenetically closely related to γ‐proteobacterium Hahella sp. MBIC 3957 that produces prodigiosin. However, some physiological and biochemical differences between strain MS‐02–063 and Hahella sp. MBIC 3957 were observed. The red pigment (RP‐063) produced by this isolate was highly purified from the culture supernatant. It was speculated that RP‐063 might be prodigiosin‐like pigment in physical properties and biological activities such as antibacterial and cytotoxic activity. Antibacterial activity of RP‐063 was examined by an agar dilution method. The results indicated that RP‐063 showed antibacterial activity for specific for pathogenic gram‐positive bacteria such as Staphylococcus aureus. The potency of antibacterial activity against S. aureus was nearly equal to those of tetracycline. Moreover, RP‐063 showed inhibition of the superoxide generation by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐stimulated mouse macrophage RAW 264.7 cell line. Prodigiosin members have a wide variety of biological properties, including anticancer and antimalarial, etc. Especially, potent immunosuppressive properties have been reported for prodigiosin members with the mechanism of action different from that of the other well known immunosuppressors in atopic dermatitis therapy such as cyclosporin A, FK506 and rapamycin. It is suggested that RP‐063 may be able to arrest the inflammation caused by superantigens secreted from S. aureus, which colonized skin on atopic dermatitis as well as suppression of activated lymphocyte proliferation and superoxide generation from leucocytes.

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We investigated the cytokine-inducing activities of guluronate (G3-G6) and mannuronate (M3-M6) oligomers on RAW264.7 cells with the Bio-Plex assay system. Relatively high levels of tumor necrosis factor-alpha (TNF-alpha), granulocyte colony-stimulating factor (G-CSF), monocyte chemoattractant protein-1 (MCP-1), regulated upon activation normal T cell expressed and secreted (RANTES), granulocyte macrophage (GM)-CSF, and eotaxin were induced by alginate oligomers to different extents depending on the oligomer structures, and low but significant levels of interleukin (IL)-1alpha, IL-1beta, IL-6, IL-9, and IL-13 were also induced. Throughout all cytokines tested, M-oligomers tended to be more potent than G-oligomers in terms of cytokine induction, and this tendency was evident in differences between G3 and M3.

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