Labeling of Peripheral Blood Polymorphonuclear Leukocytes with Indium-111: A New Method for the Quantitation of In-Vivo Accumulation of PMNLs in Rabbit Skin

Abstract

A precise method for quantitation of polymorphonuclear leukocyte (PMNL) accumulation in skin in vivo, has been developed so that the proinflammatory effects of various agents can be compared. This method can also be used to evaluate the effect of therapeutic agents on PMNL accumulation in vivo. Rabbit PMNLs were purified from heparinized blood by dextran sedimentation, hypotonic lysis, and separation on Ficoll-Hypaque. The PMNLs were labeled with 3-5 microCi per 10(6) cells of 111In oxine and reinfused coincidentally with different concentrations of different chemotactic and proinflammatory materials injected intradermally into the back. In some experiments, varying concentrations of acetic acid were applied topically. Four to 18 hours later, the rabbits were sacrificed. Eight-millimeter punch biopsies were obtained from the injection sites and counted in a gamma counter. The number of PMNLs infiltrating the dermis was also quantitated in histologic sections. A significant correlation was found between the percent increase in radioactivity and the percent increase in PMNL accumulation morphologically. Dose-response curves were generated using such proinflammatory materials as formyl-methionyl-leucyl-phenylalanine, lipopolysaccharide, activated serum, trypsin, glycogen, and acetic acid. These curves were highly reproducible from animal to animal. Using this assay, we found that as little as 1 microgram of trypsin induced detectable PMNL accumulation. This is 2-3 logs more sensitive than injecting mice intraperitoneally with trypsin. Diisopropyl fluorophosphate-inactivation of trypsin inhibited PMNL accumulation. This sensitive and quantitative bioassay of PMNL accumulation permits evaluation of multiple agents in the same animal, which decreases animal to animal variation.