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A method to recover, enumerate and identify lymphomyeloid cells present in an inflammatory dermal site: a study in
Abstract:
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 microgram 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/disease 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.
Insights
Researchers developed a novel mouse ear explant culture to study dermal cells. This method identifies immune cells like neutrophils, lymphocytes, and phagocytes, revealing recirculating T CD4 lymphocytes in the dermis.
Area of Science:
- Immunology
- Dermatology
- Cell Biology
Background:
- The dermis harbors diverse immune cells crucial for skin homeostasis and response.
- Studying these dermal cells in situ presents significant methodological challenges.
Purpose of the Study:
- To introduce a new ear explant culture method for isolating and analyzing mouse dermal cells.
- To characterize immune cell populations in the dermis under homeostatic and inflammatory conditions.
Main Methods:
- Mouse ears were processed as explants with dermal sides spread on buffered medium.
- Cells emigrating/sedimenting were identified using immunocytochemistry and flow cytometry.
- The method was validated by intradermal injection of lipopolysaccharide (LPS) or Listeria monocytogenes.
Main Results:
- Identified neutrophils, mononuclear phagocytes, dendritic leukocytes, and T lymphocytes (gamma delta and alpha beta TCRs).
- Observed potential presence of NK cells, mast cells, eosinophils, and progenitors.
- Demonstrated the method's utility in studying cellular responses to LPS and Listeria monocytogenes.
Conclusions:
- The ear explant culture method effectively isolates and identifies major dermal immune cell subsets.
- This technique provides the first evidence of recirculating T CD4 lymphocytes in mouse dermis.
- The method offers advantages over traditional collagenase digestion for studying specific cell populations.