Abstract
BackgroundMacrophages 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.MethodsIn 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.ResultsAcquired 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.ConclusionThe 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.
Highlights
Macrophages are equipped with several receptors for the recognition of foreign particles and pathogens
Influence of thapsigargin concentration and medium composition on phagocytosis The influence of the thapsigargin concentration on the phagocytosis of the fluorescent beads by differentiated Human monocyte like cell line (U937) and Murine alveolar macrophage cell line (MH-S) cells was analyzed in RPMI1640 medium supplemented with 10% Fetal calf serum (FCS)
The effect of 10 nM thapsigargin on the phagocytosis efficiency of differentiated U937 cells was investigated in RPMI1640 medium with 10% FCS (Figure 2A) in RPMI1640 medium without FCS (Figure 2B) and in external Ca2+-free, Mg2+-free and FCS-free Hank’s Balanced Salt Solution (HBSS) buffer
Summary
Macrophages are equipped with several receptors for the recognition of foreign particles and pathogens. 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 by an influx of external calcium upon activation of the calcium channels. Besides phagocytosis of microorganisms and erythrocytes [6], macrophages can uptake percoll and particles, such as polystyrene beads by endocytosis, which enables the investigation of cellular and molecular mechanisms of phagocytosis [7]. This is the most important mechanism for the clearance of particles and fibers in the lung. Biodegradable nanoparticles, functionalized with mannose for targeted and enhanced uptake by macrophages, which bear reactive oxygen species detoxifying catalase, were demonstrated to be scavenged by macrophages [9]
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