Proposal for a new evaluation of phagocytosis using different sizes of fluorescent polystyrene microspheres

Riyo Enomoto, Makoto Imamori, Ayoumi Seon, Kozue Yoshida, Aya Furue, Hirofumi Tsuruda, Eibai Lee-Hiraiwa

Laboratory of Pharmacology, Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Kobe, Japan.
Laboratory of Pharmacology, Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Kobe, Japan 2Cooperative Research Center of Life Sciences, Kobe Gakuin University, Kobe, Japan.
Laboratory of Pharmacology, Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Kobe, Japan 2Cooperative Research Center of Life Sciences, Kobe Gakuin University, Kobe, Japan Cooperative Research Center of Life Sciences, Kobe Gakuin University, Kobe, Japan.
Laboratory of Pharmacology, Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Kobe, Japan Cooperative Research Center of Life Sciences, Kobe Gakuin University, Kobe, Japan 2Cooperative Research Center of Life Sciences, Kobe Gakuin University, Kobe, Japan.

DOI: 10.4236/abc.2013.36064                

Abstract

To investigate phagocytosis, peritoneal-resident and J774.1 macrophages were incubated with fluorescent polystyrene microspheres measuring 1.0 μm in diamter at 200 particles per cell. The amount of phagocytized microspheres increased with incubation time, and both cell types had similar phagocytic activity. Further, we investigated the phagocytosis of different sizes of microspheres by J774.1 macrophages. To adequately evaluate phagocytosis, varying amounts of different sizes of microspheres were added to J774.1 cells, and their phagocytic activities were evaluated. When the microspheres were added at a density of 20 particles per cell, few small microspheres (<1.0 μm in diameter) were phagocytized. This result suggested that their low amount caused difficulty in evaluating phagocytosis. In contrast, when the same variety of microspheres was added at a density of 200 particles per cell, phagocytosis of large microspheres (>3 μm in diameter) could not be evaluated because of cytotoxicity. Thus, the amount of different sizes of microspheres added is important for precisely evaluating phagocytic activity. When the amount of different sizes of microspheres added was standardized to provide a set amount of total surface area, phagocytosis of these microspheres could be adequately evaluated and compared. To determine the effects of phagocytosis on cell viability and proliferation, cells incubated with different sizes of microspheres were assayed using a cell counting kit. We found that phagocytosis had no effect on cell viability or proliferation and was independent of particle size. Furthermore, cells already phagocytized microspheres retained their phagocytic activity.

 

Keywords

Macrophage; Phagocytosis; Polystyrene Microsphere; Total Surface Area

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Savill, J. (1998) Apoptosis. Phagocytic docking without shocking. Nature, 392, 442-443. http://dx.doi.org/10.1038/33025
[2]	Shanbhag, A.S., Jacobs, J.J., Black, J., Galante, J.O. and Glant, T.T. (1994) Macrophage/particle interactions: Effect of size, composition and surface area. Journal of Biomedical Materials Research, 28, 81-90. http://dx.doi.org/10.1002/jbm.820280111
[3]	Smith, A.M., Duan, H., Mohs, A.M. and Nie, S. (2008) Bioconjugated quantum dots for in vivo molecular and cellular imaging. Advanced Drug Delivery Reviews, 60, 1226-1240. http://dx.doi.org/10.1016/j.addr.2008.03.015
[4]	Sulahian, T.H., Imrich, A., Deloid, G., Winkler, A.R. and Kobzik, L. (2008) Signaling pathways required for macrophage scavenger receptor-mediated phagocytosis: Analysis by scanning cytometry. Respiratory Research, 9, 59. http://dx.doi.org/10.1186/1465-9921-9-59
[5]	Wu, Y.C. and Horvitz, H.R. (1998) C. elegans phagocytosis and cell-migration protein CED-5 is similar to human DOCK180. Nature, 392, 501-504. http://dx.doi.org/10.1038/32195
[6]	Olynyk, J.K., et al. (1994) Differential production of TNF by Kupffer cells after phagocytosis of E. coli and C. albicans. American Journal of Physiology, 267, G213-G219.
[7]	Heneweer, C., Gendy, S.E. and Penate-Medina, O. (2012) Liposomes and inorganic nanoparticles for drug delivery and cancer imaging. Therapeutic Delivery, 3, 645-656. http://dx.doi.org/10.4155/tde.12.38
[8]	Kreyling, W.G., Semmler-Behnke, M., Takenaka, S. and Moller, W. (2013) Differences in the Biokinetics of inhaled nanoversus micrometer-sized particles. Accounts of Chemical Research, 46, 714-722. http://dx.doi.org/10.1021/ar300043r
[9]	Costantini, L.M., Gilberti, R.M. and Knecht, D.A. (2011) The phagocytosis and toxicity of amorphous silica. PLoS One, 6, e14647. http://dx.doi.org/10.1371/journal.pone.0014647
[10]	Gilberti, R.M., Joshi, G.N. and Knecht, D.A. (2008) The phagocytosis of crystalline silica particles by macrophages. American Journal of Respiratory Cell and Molecular Biology, 39, 619-627. http://dx.doi.org/10.1165/rcmb.2008-0046OC
[11]	Joshi, G.N. and Knecht, D.A. (2013) Silica phagocytosis causes apoptosis and necrosis by different temporal and molecular pathways in alveolar macrophages. Apoptosis, 18, 271-285. http://dx.doi.org/10.1007/s10495-012-0798-y