Influence of external calcium and thapsigargin on the uptake of polystyrene beads by the macrophage-like cell lines U937 and MH-S

Abstract

Background: Macrophages are equipped with several receptors for the recognition of foreign particles and pathogens. Upon binding to these receptors, particles become internalized. An interaction of particles with macrophage surface receptors is accompanied by an increase in cytosolic calcium concentration. This calcium is provided by intracellular stores and also by an influx of external calcium upon activation of the calcium channels. Nevertheless, the role of calcium in phagocytosis remains controversial. Some researchers postulate the necessity of calcium in Fc-receptor-mediated phagocytosis and a calcium-independent phagocytosis of complement opsonized particles. Others refute the need for calcium in Fc-receptor-mediated phagocytosis by macrophages. Methods: In this study, the influence of external calcium concentrations and thapsigargin on the phagocytosis of polystyrene latex beads by the macrophage-like cell lines MH-S (murine) and differentiated U937 (human) was analyzed. The phagocytosis efficiency was determined by flow cytometry and was evaluated statistically by ANOVA test and Dunett's significance test, or ANOVA and Bonferroni's Multiple Comparison. Results: Acquired data revealed an external calcium-independent way of internalization of non-functionalized polystyrene latex beads at free calcium concentrations ranging from 0 mM to 3 mM. The phagocytosis efficiency of the cells is not significantly decreased by a complete lack of external calcium. Furthermore, the presence of thapsigargin, known to lead to an increase of cytosolic calcium levels, did not have a significant enhancing influence on bead uptake by MH-S cells and only an enhancing effect on bead uptake by macrophage-like U937 cells at an external calcium concentration of 4 mM. Conclusion: The calcium-independent phagocytosis process and the decrease of phagocytosis efficiency in the presence of complement receptor inhibitor staurosporine lead to the assumption that besides other calcium independent receptors, complement receptors are also involved in the uptake of polystyrene beads. The comparison of the phagocytosis efficiencies of both cell types in bivalent cation-free HBSS buffer and in cell medium, leads to the conclusion that it is more likely that other media ingredients such as magnesium are of greater importance for phagocytosis of non-functionalized polystyrene beads than calcium.