Abstract We have previously reported that Gr-1+CD11b+F4/80+ cells demonstrated in tissues surrounding the burn site (TSBS) are inhibitory on the production of antimicrobial peptides by epidermal keratinocytes (EK). In this study, the mechanism by which Gr-1+CD11b+F4/80+ cells inhibit the production of β-defensin 3 (MBD-3) by EK was investigated. Burn mice were BALB/c mice exposed to a gas flame (3rd degree). Twelve hrs after burn injury, Gr-1+CD11b+F4/80+ cells were isolated from their TSBS using a magnetic bead technique. To determine the effect of Gr-1+CD11b+F4/80+ cells on MBD-3 production, LPS-stimulated EK prepared from skin tissues of normal mice (1 x 106 cells/ml, lower chamber) were cultured with Gr-1+CD11b+F4/80+ cells (upper chamber) in dual-chamber transwells. Culture fluids harvested were assayed for MBD-3 by ELISA. After stimulation with LPS (0.1 μg/ml), EK produced 25 μg/ml of MBD-3, and only 0.4 μg/ml of MBD-3 was produced by EK transwell-cultured with Gr-1+CD11b+F4/80+ cells. When the same transwell cultures were performed with media supplemented with a mixture of mAbs for CCL2 and IL-10 (2 μg/ml each), 36 μg/ml of MBD-3 was detected in the culture fluids. In contrast, MBD-3 production by EK stimulated with LPS was inhibited by a mixture of recombinant CCL2 (50 ng/ml) and IL-10 (10 ng/ml). CCL2 and IL-10 were detected in the culture fluids of Gr-1+CD11b+F4/80+ cells. These results indicate that, through the production of CCL2 and IL-10, Gr-1+CD11b+F4/80+ cells appearing in association with burn injuries inhibit the production of MBD-3 from EK. This study was supported by SHC NA #8610.