IDENTIFICATION OF RABBIT EOSINOPHILS AND HETEROPHILS IN CUTANEOUS HEALING WOUNDS

The study of wound healing has traditionally used the rabbit as an exp erimental model. We have recently localized the production of the mult ifunctional 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 granulocy tes by virtue of their endogenous peroxidases and differential resista nce to blockade by inhibitors. In sections that have been blocked by h ydrogen peroxide, the peroxidase substrate 3,3'-diaminobenzidine, toge ther with nickel chloride (DAB-Ni), preferentially stained the cytopla sm of rabbit eosinophils while sparing those of heterophils. This sele ctive DAB staining of rabbit eosinophil peroxidase in H2O2-blocked rab bit wounds was verified at the ultrastructural level by electron micro scopy. We applied this technique to quantify eosinophil and heterophil infiltration into the 21-day rabbit cutaneous healing wound model. He terophils were found infiltrated into all three layers of the wound (d ot > 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 heterophi ls, eosinophils in the granulation layer of the wound continued to inc rease up to day 21. The continually increased and sustained presence o f the eosinophils together with their demonstrated production of TGF-a lpha, in the granulation layer of the healing wound suggests that thes e cells play an important role in the organizational aspects of healin g wounds.