Identification of rabbit eosinophils and heterophils in cutaneous healing wounds.

The study of wound healing has traditionally used the rabbit as an experimental model. We have recently localized the production of the multifunctional cytokine, TGF-alpha, to eosinophils in rabbit skin wounds. It was evident that during the process of TGF-alpha localization, the distinction between the two granulocytic cell types, eosinophils and heterophils, was impossible by conventional histochemical techniques. This paper describes a rapid method to distinguish these two granulocytes by virtue of their endogenous peroxidases and differential resistance to blockade by inhibitors. In sections that have been blocked by hydrogen peroxide, the peroxidase substrate 3,3'-diaminobenzidine, together with nickel chloride (DAB-Ni), preferentially stained the cytoplasm of rabbit eosinophils while sparing those of heterophils. This selective DAB staining of rabbit eosinophil peroxidase in H2O2-blocked rabbit wounds was verified at the ultrastructural level by electron microscopy. We applied this technique to quantify eosinophil and heterophil infiltration into the 21-day rabbit cutaneous healing wound model. Heterophils were found infiltrated into all three layers of the wound (clot > granulation > base), but eventually all disappeared by day 21. As with the heterophils, eosinophils which had infiltrated into the clot and base of the wound had disappeared by day 21. Unlike the heterophils, eosinophils in the granulation layer of the wound continued to increase up to day 21. The continually increased and sustained presence of the eosinophils together with their demonstrated production of TGF-alpha, in the granulation layer of the healing would suggests that these cells play an important role in the organizational aspects of healing wounds.