A heterogeneous Ly-6B2+ leukocyte population consists of yet undescribed iNOS-expressing cell types in murine skin wounds

The gaseous mediator nitric oxide (NO) is a central regulatory molecule during the inflammatory phase of cutaneous tissue repair. The inducible NO-synthase (iNOS) represents the main isoform of the three NO producing enzymes at the wound site. In particular, keratinocytes and macrophages are described as main sources of iNOS-derived NO in skin wounds. Here we provide experimental evidence that Ly-6B2+ leukocytes are an additional cellular source of iNOS-derived NO in wounds. As wound iNOS protein expression temporally coincides with both macrophage and neutrophil infiltration, we used immunohistochemistry (IHC) and fluorescence-activated cell sorting (FACS) to address iNOS expression in both macrophages and neutrophil subsets. IHC analyses excluded F4/80+ macrophages as iNOS producers, but indicated Ly-6G/C (Gr-1)+ neutrophils to express iNOS in wound granulation tissue. A subsequent FACS-based analysis from cellular wound tissue preparations revealed an iNOS-expressing fraction of Ly-6B2-determined leukocytes that consisted of Ly-6G+ and Ly-6G− cells, meaning that mainly mature neutrophils (Ly-6B2+/Ly-6G+) as well as inflammatory monocytes (Ly-6B2+/Ly-6G−) are dominant iNOS-expressing cell types in the developing granulation tissue of acute wounds.