Characterisation of a bovine DC-SIGN like molecule

Dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin C-type lectin, DC-SIGN, is nearly exclusively expressed at the cell surface of dendritic cells (DC). In addition to its normal function facilitating contact of DC with T cells (Tc), DCSIGN has been shown to specifically bind, amongst other proteins, the mV-I envelope glycoprotein (gp) 120. In this study, a protein with the same molecular weight as human DC-SIGN was detected by western blot in cell-lysate derived from bovine monocytes-derived DC (MoDC). To functionally characterise this molecule, the uptake of FITC labelled ovalbumin (FITC-OVA) and FITC labelled gp120 (FITC-gpI20) by bovine and human MoDC was assessed. FITC-gpI20 was shown to bind to bovine MoDC in a time- and temperature-dependent manner, similar to that observed in human MoDC. This binding was blocked by a polyclonal anti-DC-SIGN antibody, but not by a control antibody, andú both antibodies did not affect the uptake of FITC-OV A in both cell types. Confocal microscopy analysis. showed that DC-SIGN was expressed in cholera-toxin positive lipid rafts on bovine MoDC. Subsequent pulse-chase studies revealed that FITC-gpI20 was internalised by bovine MoDC after 30 minutes exposure. After 2 hours exposure, FITC-gpI20 co-localised with cellular compartments staining positive for DC-SIGN andMHCII. Having established the binding and uptake of gp120, the ability of bovine MoDC to stimulate a gpl20-specific autologous response in either PBMC or purified CD4+ Tc was assessed. Exposure of gpl20 to bovine MoDC resulted in a weak stimulation of CD4+ Tc, as analysed by 3H-thymidine incorporation. In the last part of this study, the cytokine response of bovine MoDC exposed to gp120 protein was investigated by RT-PCR, Taqman Real-Time PCR and ELISA. Bovine MoDC exposed to gpl20 induced IL-12 but not IL-I0. In summary, there is evidence of a DC-SIGN-like molecule expressed specifically by bovine MoDC, with the ability to bind and internalise mV-I gp120. Furthermore, bovine MoDC stimulated with gpl20 mainly produce IL-12, which are able to present gp120-derived peptides to naIve CD4+ Tc, thus potentially priming a subsequent Thlbiased adaptive immune response.EThOS - Electronic Theses Online ServiceGBUnited Kingdo