The Effect of Enzymatically Polymerised Polyphenols on CD4 Binding and Cytokine Production in Murine Splenocytes

High-molecular weight polymerised polyphenols have been shown to exhibit anti-influenza virus, anti-HIV, and anti-cancer activities. The purpose of this study was to evaluate the immunomodulating activities of enzymatically polymerised polyphenols, and to clarify the underlying mechanisms of their effects. The cytokine-inducing activity of the enzymatically polymerised polyphenols derived from caffeic acid (CA), ferulic acid (FA), and p-coumaric acid (CoA) was investigated using murine splenocytes. Polymerised polyphenols, but not non-polymerised polyphenols, induced cytokine synthesis in murine splenocytes. Polymerised polyphenols induced several cytokines in murine splenocytes, with interferon-γ (IFN-γ) and granulocyte-macrophage colony-stimulating factor (GM-CSF) being the most prominent. The underlying mechanisms of the effects of the polymerised polyphenols were then studied using neutralising antibodies and fluorescent-activated cell sorting (FACS) analysis. Our results show that polymerised polyphenols increased IFN-γ and GM-CSF production in splenocytes. In addition, the anti-CD4 neutralised monoclonal antibody (mAb) inhibited polymerised polyphenol-induced IFN-γ and GM-CSF secretion. Moreover, polymerised polyphenols bound directly to a recombinant CD4 protein, and FACS analysis confirmed that interaction occurs between polymerised polyphenols and CD4 molecules expressed on the cell surface. In this study, we clearly demonstrated that enzymatic polymerisation confers immunoactivating potential to phenylpropanoic acids, and CD4 plays a key role in their cytokine-inducing activity

Slit2–Robo4 signalling promotes vascular stability by blocking Arf6 activity

Slit–Roundabout (Robo) signalling has a well-understood role in axon guidance1–5. Unlike in the nervous system, however, Slitdependent activation of an endothelial-specific Robo, Robo4, does not initiate a guidance program. Instead, Robo4 maintains the barrier function of the mature vascular network by inhibiting neovascular tuft formation and endothelial hyperpermeability induced by pro-angiogenic factors 6. In this study, we used cell biological and biochemical techniques to elucidate the molecular mechanism underlying the maintenance of vascular stability by Robo4. Here, we demonstrate that Robo4 mediates Slit2-dependent suppression of cellular protrusive activity through direct interaction with the intracellular adaptor protein paxillin and its paralogue, Hic-5. Formation of a Robo4–paxillin complex at the cell surface blocks activation of the small GTPase Arf6 and, consequently, Rac by recruitment of Arf-GAPs (ADP-ribosylation factor- directed GTPase-activating proteins) such as GIT1. Consistent with these in vitro studies, inhibition of Arf6 activity in vivo phenocopies Robo4 activation by reducing pathologic angiogenesis in choroidal and retinal vascular disease and VEGF-165 (vascular endothelial growth factor-165)-induced retinal hyperpermeability. These data reveal that a Slit2–Robo4–paxillin–GIT1 network inhibits the cellular protrusive activity underlying neovascularization and vascular leak, and identify a new therapeutic target for ameliorating diseases involving the vascular system