Alternatively activated macrophages have been implicated in the therapeutic activity of biodegradable chitosan on wound healing, however, the mechanisms of phenotypic differentiation are still unclear. In vitro, macrophages stimulated with high doses of chitosan (>= 500 mu g/mL) were reported to produce low-level markers associated with alternative activation (arginase-1) as well as classical activation (nitric oxide), and to undergo apoptosis. In this study, we tested the hypothesis that 40 kDa biodegradable chitosan (5-500 mu g/mL) is sufficient to polarize mouse bone marrow-derived macrophages (BMDM) in vitro to an alternatively activated phenotype. Control cultures were stimulated with IL-4 (alternative activation), IFN-gamma/LPS (classical activation), 1 mu m diameter latex beads (phagocytosis), or left untreated. After 48 h of in vitro exposure. BMDM phagocytosed fluorescent chitosan particles or latex beads, and remained viable and metabolically active, although some cells detached with increasing chitosan and latex bead dosage. Arginase-1 was over 100-fold more strongly induced by IL-4 than by chitosan, which induced only sporadic and weak arginase-1 activity over untreated BMDM, and no nitric oxide. IFN-gamma/LPS stimulated nitric oxide production and arginase-1 activity and high concentrations of inflammatory cytokines (IL-6, IL-1 beta, TNF-alpha, MIP-1 alpha/MIP-1 beta), while latex beads stimulated nitric oxide and not arginase-1 activity. Chitosan or latex bead exposure, but not IL-4, tended to promote the release of several chemokines (MIP-1 alpha/beta, GM-CSF, RANTES, IL-1 beta), while all treatments promoted MCP-1 release. These data show that chitosan phagocytosis is not sufficient to polarize BMDM to the alternative or the classical pathway, suggesting that biodegradable chitosan elicits alternatively activated macrophages in vivo through indirect mechanisms. (C) 2011 Elsevier Inc. All rights reserved.