Uptake of Neutrophil-Derived Ym1 Protein Distinguishes Wound Macrophages in the Absence of Interleukin-4 Signaling in Murine Wound Healing

The determination of regenerative wound-healing macrophages as alternatively activated macrophages is currently questioned by the absence of IL-4 in wound tissue. Yet, murine wound tissue expressed high levels of Ym1 (chitinase 3-like 3), an established marker of the IL-4-induced alternatively activated macrophage phenotype. Ym1 was expressed in wound neutrophils but not in macrophages. Initially, Ym1-free wound-healing macrophages, invading from the wound margins, became gradually positive for the protein in the absence of IL-4 signaling and Stat6 activation, as they entered the neutrophil-populated wound regions. IL-4 failed to induce Ym1 protein in ex vivo-cultured wound tissue explants containing wound-healing macrophages. Recombinant Ym1 protein was selectively taken up by macrophages but not by keratinocytes and endothelial cells. Cultured macrophages lost the ability to take up the recombinant protein when four highly conserved residues and the 70-amino acid small alpha+beta domain essential for Ym1 function were removed. The data suggest that the IL-4/Stat6-independent presence of Ym1 protein in wound-healing macrophages is of exogenous origin, with Ym1 taken up from wound neutrophils as the cellular source. The data suggest that in situ determination of wound-healing macrophages, often defined by Ym1, might not essentially describe an IL-4-dependent macrophage phenotype. Consequently, wound-healing macrophages should not be classified by the established categories of the well-accepted but simplified paradigm of M1/M2 macrophage activation.