Macrophages and neutrophils are professional phagocytosis cells of the innate immune system. The uptake of large particles (>0.5 μm) occurs in an actin-dependent manner. It has been demonstrated that many cells, such as bladder and thyroid epithelial cells, also accomplish phagocytosis  but professional phagocytes, like macrophages, possess phagocytic receptors that increase the target particle range and the phagocytosis rate . The mechanisms underlying phagocytosis by macrophages are complex and provide important tools for the essential role of the cells in the uptake and degradation of infectious agents, senescent cells, particles, tissue remodeling, immune response and inflammation .
Macrophages are equipped with receptors for Fc-mediated, complement-mediated and pathogenic conserved motifs-mediated phagocytosis . Although ligand binding to all of these receptors promotes phagocytosis, the effects of receptor signaling differ. Whereas FcR-mediated phagocytosis results in the production and secretion of proinflammatory mediators like arachidionic acid and reactive oxygen species, complement receptor (CR)-mediated phagocytosis does not cause the release of these mediators [22, 23]. Interaction of ligands with phagocytic receptors for immunoglobulins (FcγR) and for complement proteins induces the activation of phospholipase C and phospholipase D, which in turn leads to the formation of Ca2+ mobilizing second messengers. This, on the other hand, is involved in the activation of store-operated calcium entry (SOCE) channels in the plasma membrane, resulting in a Ca2+ flux from the extracellular medium. It is generally accepted that an increase in cytosolic Ca2+ is an early signal that accompanies phagocytic ingestion, but the elevation of cytososlic Ca2+ is required for the promotion of an efficient ingestion of foreign particles by some, but not all, receptors. In 1985 Lew et al. showed that antibody/FcR generated phagocytosis relies on an increase of cytosolic Ca2+ concentration upon receptor activation, whereas activation of the complement receptor C3b/bi generates a Ca2+-independent phagocytosis mechanism in human neutrophils. They determined a decrease in Fc-receptor-mediated phagocytosis after increasing the intracellular Ca2+-buffering capacity of quin2, which was more pronounced in the absence of extracellular calcium. In contrast, a decrease in complement-mediated phagocytosis of yeast cells was not observed despite increasing the intracellular Ca2+ buffering by quin2 . Nevertheless, the question of the necessity of Ca2+ for the phagocytosis of foreign particles is controversial . Some studies in murine macrophages verified a decrease of phagocytic ingestion of IgG opsonized or non-opsonized latex beads due to Ca2+ chelation [26–28], whereas others reported effective phagocytosis of IgG opsonized red blood cells at low cytosolic Ca2+ levels [29–31].
In this study, the influence of external calcium levels and thapsigargin concentrations were investigated with regard to the phagocytosis efficiency for non-IgG functionalized, fluorescence-labeled polystyrene beads. The phagocytosis assays were applied on two different reference cell lines: the murine alveolar macrophage derived MH-S  and human lymphoma monocyte like cell line U937 , which were differentiated to adherent, phagocytosis-exhibiting macrophage-like cells by PMA, previous to performing phagocytosis assays.
The data acquired from thapsigargin containing assay set-ups, external Ca2+ free conditions, and a stepwise increase in external Ca2+ concentrations revealed that external calcium concentrations of 1 mM and 2 mM did not influence the uptake of non-functionalized beads by differentiated U937 cells and MH-S cells in HBSS buffer. Furthermore, neither the presence nor the absence of thapsigargin also affected the phagocytosis efficiency of these cells at physiological calcium levels. The phagocytosis efficiency of differentiated U937 cells was not even affected in their phagocytosis efficiency at an external calcium level of 4 mM. This situation changed if 10 nM thapsigargin was present in HBSS buffer. Whereas external free calcium concentrations of 1 mM and 2 mM were still ineffective in increasing the phagocytosis efficiency of differentiated U937 cells, the presence of thapsigargin enhanced phagocytosis efficiency from 9.6% to 17.7% at an external calcium concentration of 4 mM. The cytotoxicity assay showed that the increased calcium levels in HBSS buffer caused a high level of cell death of up to 46%, which was probably caused either by high osmotic pressure or by an increase in intracellular calcium levels, known to cause the death of cells . To exclude decreased phagocytosis efficiency due to the lack of FCS or Mg2+, phagocytosis assays with differentiated U937 cells were performed in 1 m EGTA containing RPMI1640 + 10% FCS medium. This concentration of EGTA was shown to be effective in chelating the complete free calcium of the FCS containing medium (personal communication with Dr. Eva Schwarz, Saarland University). The phagocytosis efficiency of differentiated U937 cells in this medium did not show a significant difference to the phagocytosis efficiency in EGTA-free RPMI1640 + 10% FCS medium. Further increase of external calcium only showed significant enhancement in phagocytosis efficiency at a calcium concentration of 5 mM, which according to the maxchelator software, corresponds to 4 mM free calcium. But, the amount of free calcium in FCS is not known, and probably the calcium level is much higher than 4 mM. This data also provided a hint to the insignificant role of physiological calcium levels on phagocytosis.
MH-S cells showed some differences in phagocytosis efficiency at an external calcium concentration of 4 mM. In the absence of thapsigargin, the presence of 4 mM external free calcium in HBSS buffer increased the phagocytosis efficiency of MH-S cells significantly from 35% to 44%, whereas in the presence of 10 nM thapsigargin, no enhancement of the phagocytosis efficiency of MH-S cells was determined. Nevertheless, neither cell line showed any differences in their phagocytosis efficiency in the range of free external calcium at the range of 0 mM to 2 mM in HBSS buffer. We can conclude that, at physiological free calcium levels, which, according to free calcium measurements of the sera of healthy individuals, is at the range of 1.175 mM – 1.375 mM , the efficiency of phagocytosis of beads by MH-S cells and differentiated U937 cells is not affected by calcium.
It was previously demonstrated that the tumor promoter thapsigargin increases intracellular free Ca2+-levels by inhibiting the Ca2+ reuptake from the cytosol into the ER-lumen by sarco-endoplasmic reticulum ATPases [13, 14]. In this study, experimental set-ups with increasing thapsigargin concentrations in RPMI1640 + 10% FCS revealed that the phagocytosis behavior of differentiated U937 cells and MH-S cells is not affected by the presence of thapsigargin, even at concentrations of 1 μM. As mentioned, a concentration of 10 nM thapsigargin was only effective in enhancing the phagocytosis efficiency of U937 cells in HBSS buffer with 4 mM calcium, but not at lower extracellular calcium levels. According to the cytotoxicity assay, differentiated U937 cells seem to be resistant to the presence of thapsigargin even at high concentrations. MH-S cells, on the other hand, were highly sensitive to the presence of thapsigargin, leading to a cell lysis of up to 25.5%.
The uptake of beads by differentiated U937 cells in Ca2+-free, Mg2+-free HBSS buffer was low, compared to the phagocytosis efficiency in FCS-free RPMI 1640 medium, which, according to the manufacturer, contains 1 mM free Ca2+. However, after verifying that this external Ca2+ concentration does not have an increasing effect on phagocytosis efficiency, it can be concluded that other supplements such as magnesium, which is also not present in HBSS, could have a more pronounced influence on the phagocytosis efficiency of these cells. In fact, the suppression of phagocytosis caused by lowered extracellular levels of magnesium was previously demonstrated in rat alveolar macrophages [34, 35].
All of the data acquired in this study indicate that the uptake of non-functionalized polystyrene beads with a size of 1 μm by MH-S cells and by differentiated U937 cells does not depend on external calcium at physiological levels or on the addition of thapsigargin. The beads used in this study, were not opsonized with antibodies or with complement proteins but the latter type of opsonization cannot be excluded in phagocytosis assays performed in FCS-containing medium. As mentioned above, complement-mediated phagocytosis was demonstrated to be Ca2+ independent in human neutrophils . Therefore, a complement-mediated, Ca2+- independent phagocytosis of polystyrene beads is a possible way of bead uptake by the reference macrophage-like cell lines used. This conclusion is further supported by the results obtained from phagocytosis studies performed in the presence of staurosporine. The uptake of beads by both cell types was significantly reduced when the complement receptor inhibitor staurosporine was added to the medium. Therefore, the calcium- independent phagocytosis process and the influence of staurosporine on bead uptake reveal a possible complement receptor-dependent way of polystyrene bead entry. However, taking into account that staurosporine did not completely inhibit the bead uptake, interaction of beads with other receptors could also cause the uptake of beads in an Ca2+-independent manner.
In conclusion, a comparison of the particle uptake by differentiated U937 cells and MH-S cells in Ca2+-containing HBSS with that in FCS-free RPMI 1640 medium indicated that other PRMI1640 ingredients, for example magnesium ions, could be more necessary for an efficient phagocytosis than free external calcium ions. Experiments to reveal the influence of external magnesium are in preparation and should shed some light onto the mechanisms involved in the uptake of polystyrene beads by macrophage-like U937 cells and MH-S cells.