Flow cytometric analysis of macrophage response to ceramic and polyethylene particles: effects of size, concentration, and composition.

Abstract

Using the J774 macrophage cell line, we designed an in vitro model to analyze by flow cytometry the effects of size, concentration, and composition of ceramic (Al2O3 and ZrO2) and high density polyethylene (HDP) particles on phagocytosis and cell mortality. Inflammatory mediator (TNF-alpha) also was measured by ELISA. Kinetic studies revealed that phagocytosis of the particles begins very early after cell exposure, increasing with time and particle concentration and reaching a plateau after 15 h. This implies that the optimum period to evaluate cellular response to particulate debris is between 15 and 24 h of incubation. Results also showed that phagocytosis increases with concentration for particles up to 2 microns. For larger particles (up to 4.5 microns), phagocytosis seems to reach a plateau independent of size and concentration, which suggests a saturation of phagocytosis that is most likely dependent on overall particle volume ingested. We did not detect any significant difference in phagocytosis between Al2O3 and ZrO2 at 0.6 microns. Al2O3 seems to be more easily phagocytosed than HDP at the same size (4.5 microns) and concentrations. Cytotoxicity studies revealed that macrophage mortality increases with particle size and concentration for sizes greater than 2 microns. Smaller particles (0.6 microns) cause cell mortality only at higher concentrations (from 1,250 particles per cell), but the mortality is still very low (10%). No significant difference in cell mortality and TNF-alpha release was found between Al2O3 and ZrO2. Effects of Al2O3 and HDP at 4.5 microns were compared by measuring TNF-alpha release. Results showed that TNF-alpha release increases with particle concentrations and is higher with HDP than with Al2O3.