A method to recover, enumerate and identify lymphomyeloid cells present in an inflammatory dermal site: A study in laboratory mice

We describe a new method to recover and study cells present in the dermis of mouse ear at homeostasis or after intradermal injection of disturbing agents (lipopolysaccharide or Listeria monocytogenes). The ears either left untreated or inoculated were handled and processed as culture explants of the dorsal and ventral leaflets, their dermal sides being spread on a buffered medium. Within this medium emigrate/sediment, with different kinetics: neutrophils, mononuclear phagocytes, dendritic leucocytes, T lymphocytes expressing either gamma delta or alpha beta TCRs, and other minor subsets, the identification of which deserves more relevant reagents: they are likely to be NK, mast cells, eosinophils and their local progenitors. All the major subsets were identified through a combination of immunocytochemical and flow cytometry labeling. Two examples illustrating the advantages and limitations of this new method are given: either 1 mu g of LPS or 10(4) Listeria monocytogenes were injected within the ear 48, 24, 12, 6, 3 h before ear explant culture. This ear explant culture has been further compared to the ear sheet treatment with collagenase/dispase for three cell populations, the epidermal dendritic leucocytes, the gamma delta epidermal T cells as well as the alpha beta T cells recirculating within the steady state dermis. This method provides the first evidence of the existence of recirculating T CD4 lymphocytes in the mouse dermis.