Dectin 1 is a member of group V CLRs and serves as a PRR to sense the B glucan in fungal cell wall. To determine if Dectin 1 plays a role in PS F2 recognition, RAW264. 7 cells were stimulated with PS F2 or zymosan in the pres ence of anti Dectin 1 new blocking antibodies. As expected, the stimulatory activity of zymosan was blocked by the antibodies. Anti Dectin 1 anti bodies also significantly suppressed PS F2 stimulated TNF production, indicating that PS F2 sti mulates macrophages, in part, via Dectin 1. It is likely that Dectin 1 recognizes B glucan in PS F2. However, since ligands other than B glucan may also bind Dectin 1, it remains possible that Dectin 1 may interact with other unique polysaccharide structures in PS F2.
CR3 is another receptor which recognizes fungal B glucan, and the interaction is via a cation independent lectin site located C terminal to the I domain of CD11b, which can be blocked by anti CD11b mAb M1/70. Indeed, we found that this mAb was able to block zymosan stimulated TNF production in macrophages. To determine if CR3 is involved in the recognition of the G. formosanum polysaccharides, PS F2 stimulation was performed in the presence of M1/70 blocking antibodies for CR3. Results showed that TNF production was blocked by the anti CR3 antibody in a dose dependent way. Dectin 1 and CR3 are, therefore, both involved in recognition of PS F2. Laminarin is the B glucan from brown algae and non stimulatory, but blocks the stimulatory effects of various fungal B glucans.
When stimulation was performed in the presence of laminarin, results showed that laminarin markedly and specifically inhibited PS F2 induced macrophage activation . in contrast, laminarin had no effect on LPS stimulation. The strong inhibition of PS F2 stimulation by laminarin suggests that PS F2 interaction with certain B glucan binding receptor is responsible for macro phage activation. We have tried to determine whether the stimulatory function of PS F2 is provided by B glucan by treating PS F2 with laminarinase. however the laminari nase of commercial source contained certain contaminants which also stimulated the activation of macrophages. Over all, these data suggest that the B glucan receptors Dectin 1 and CR3 both play important roles in AV-951 the recognition of PS F2. Role of TLR4 in PS F2 stimulation Previous studies report that, in addition to Dectin 1 and CR3, TLR4 also recognizes fungal B glucans, in particu lar the polysaccharides extracted from G. lucidum. Although the polysaccharides purified from the sub merged culture of G. formosanum appear distinct in sugar compositions from the polysaccharides extracted from G. lucidum, we also examined whether TLR4 plays a role in PS F2 recognition.