Objectives: Our objective was to determine if HBD3 binding to rHagB alters the binding of rHagB to the surface of human myeloid dendritic cells.
Methods: To test this, human myeloid dendritic cells and mouse JAWS II cells were incubated with 0.1 mM rHagB, 1.0 mM HBD3+0.1 mM rHagB (10:1 molar ratio), 1.0 mM HBD3, or 0.1 M PBS, pH 7.2. After 5 minutes, cells were rapidly fixed and processed for confocal microscopy. Surface bound rHagB and HBD3 were detected with monoclonal MoAb 1858 to rHagB, polyclonal rabbit to rHagB, and polyclonal 500-P241 rabbit antibody to HBD3 and visualized with either secondary fluorescent labeled anti-mouse or anti-rabbit antibodies by confocal microscopy.
Results: For confocal microscopy, 6.3 x 104 dendritic cells in chamber slides were incubated with rHagB and had more fluorescent label on the cell surface than cells incubated with HBD3+rHagB, HBD3, or 0.1 M PBS, pH 7.2. Interestingly, there was noticeable fluorescence detected by confocal microscopy in dendritic cells treated with HBD3, rabbit anti-HBD3 antibody, and fluorescent labeled anti-rabbit antibody.
Conclusions: Overall, these results strongly suggest that HBD3 binding to rHagB alters the binding of rHagB to the surface of human myeloid dendritic cells, likely as a mechanism in which HBD3 attenuates a pro-inflammatory response of rHagB in dendritic cells. This work was supported by NIH, NIDCR grant R01 DE014390.