Keratinocytes in culture accumulate phagocytosed melanosomes in the perinuclear area.
There are many techniques for evaluating melanosome transfer to keratinocytes but the spectrophotometric quantification of melanosomes incorporated by keratinocyte phagocytosis has not been previously reported. Here we describe a new method that allows the spectrophotometric visualization of melanosome uptake by normal human keratinocytes in culture. Fontana-Masson staining of keratinocytes incubated with isolated melanosomes showed the accumulation of incorporated melanosomes in the perinuclear areas of keratinocytes within 48 h. Electron microscopic observations of melanosomes ingested by keratinocytes revealed that many phagosomes containing clusters of melanosomes or their fragments were localized in the perinuclear area. A known inhibitor of keratinocyte phagocytosis which inhibits protease-activated receptor-2, i.e., soybean trypsin inhibitor, decreased melanosome uptake by keratinocytes in a dose-dependent manner. These data suggest that our method is a useful model to quantitate keratinocyte phagocytosis of melanosomes visually in vitro.
Ando
Hideya
H
Skin Aging and Photo-aging Research Center, Doshisha University, Kizugawa, Kyoto, Japan. hando@mail.doshisha.ac.jp
Niki
Yoko
Y
Yoshida
Masaki
M
Ito
Masaaki
M
Akiyama
Kaoru
K
Kim
Jin-Hwa
JH
Yoon
Tae-Jin
TJ
Lee
Jeung-Hoon
JH
Matsui
Mary S
MS
Ichihashi
Masamitsu
M
D018929:Cell Culture Techniques; D002467:Cell Nucleus / Q000378:metabolism / Q000648:ultrastructure*; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism / Q000648:ultrastructure*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism / Q000648:ultrastructure*; D008543:Melanins / Q000378:metabolism; D020460:Melanosomes / Q000378:metabolism / Q000648:ultrastructure*; D046529:Microscopy, Electron, Transmission; D008954:Models, Biological; D010587:Phagocytosis / Q000502:physiology*; D010588:Phagosomes / Q000378:metabolism / Q000648:ultrastructure*; D044464:Receptor, PAR-2 / Q000037:antagonists & inhibitors / Q000378:metabolism; D013053:Spectrophotometry / Q000379:methods; D013194:Staining and Labeling
D004791:Enzyme Inhibitors; D008543:Melanins; D044464:Receptor, PAR-2