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Uptake and distribution of fullerenes in human mast cells.

ABSTRACT: Fullerenes are carbon cages of variable size that can be derivatized with various side chain moieties resulting in compounds that are being developed into nanomedicines. Although fullerene use in several preclinical in vitro and in vivo models of disease has demonstrated their potential as diagnostic and therapeutic agents, little is known about how they enter cells, what organelles they target, and the time course for their cellular deposition. Fullerenes (C(70)) that have already been shown to be potent inhibitors of mast cell (MC)-mediated allergic inflammation were conjugated with Texas red (TR) and used in conjunction with confocal microscopy to determine mechanisms of uptake, the organelle localization, and the duration they can be detected in situ. We show that C(70)-TR are nonspecifically endocytosed into MCs, where they are shuttled throughout the cytoplasm, lysosomes, mitochondria, and into endoplasmic reticulum at different times. No nuclear or secretory granule localization was observed. The C(70)-TR remained detectable within cells at 1 week. These studies show that MCs endocytose fullerenes, where they are shuttled to organelles involved with calcium and reactive oxygen species production, which may explain their efficacy as cellular inhibitors. From the clinical editor: Fullerenes are carbon cages of variable size that have already been shown to be potent inhibitors of mast cell (MC)-mediated allergic inflammation. These were conjugated with Texas red (TR) and used in conjunction with confocal microscopy to determine mechanisms of uptake, the organelle localization, and duration, demonstrating that MCs endocytose fullerenes, which are shuttled to organelles involved with calcium and reactive oxygen species production. This intracellular trafficking may explain the efficacy of fullerenes as cellular inhibitors.

SUBMITTER: Dellinger A

PROVIDER: S-EPMC2888797 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Uptake and distribution of fullerenes in human mast cells.

Dellinger Anthony A Zhou Zhiguo Z Norton Sarah K SK Lenk Robert R Conrad Daniel D Kepley Christopher L CL

Nanomedicine : nanotechnology, biology, and medicine 20100204 4

Fullerenes are carbon cages of variable size that can be derivatized with various side chain moieties resulting in compounds that are being developed into nanomedicines. Although fullerene use in several preclinical in vitro and in vivo models of disease has demonstrated their potential as diagnostic and therapeutic agents, little is known about how they enter cells, what organelles they target, and the time course for their cellular deposition. Fullerenes (C(70)) that have already been shown to ...[more]

PMID: 20138243

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Project description:Studies of mast cell biology are dependent on relevant and validated in vitro models. Here, we present detailed information concerning the phenotype of both freshly isolated human skin mast cells (MCs) and of in vitro cultures of these cells that were obtained by analyzing their total transcriptome. Transcript levels of MC-related granule proteins and transcription factors were found to be remarkably stable over a 3-week culture period. Relatively modest changes were also seen for important cell surface receptors including the high-affinity receptor for IgE, FCER1A, the low-affinity receptor for IgG, FCGR2A, and the receptor for stem cell factor, KIT. FCGR2A was the only Fc receptor for IgG expressed by these cells. The IgE receptor increased by 2-5-fold and an approximately 10-fold reduction in the expression of FCGR2A was observed most likely due to the cytokines, SCF and IL-4, used for expanding the cells. Comparisons of the present transcriptome against previously reported transcriptomes of mouse peritoneal MCs and mouse bone marrow-derived MCs (BMMCs) revealed both similarities and major differences. Strikingly, cathepsin G was the most highly expressed granule protease in human skin MCs, in contrast to the almost total absence of this protease in both mouse MCs. Transcript levels for the majority of cell surface receptors were also very low compared to the granule proteases in both mouse and human MCs, with a difference of almost two orders of magnitude. An almost total absence of T-cell granzymes was observed in human skin MCs, indicating that granzymes have no or only a minor role in human MC biology. Ex vivo skin MCs expressed high levels of selective immediate early genes and transcripts of heat shock proteins. In validation experiments, we determined that this expression was an inherent property of the cells and not the result of the isolation process. Three to four weeks in culture results in an induction of cell growth-related genes accompanying their expansion by 6-10-fold, which increases the number of cells for in vitro experiments. Collectively, we show that cultured human skin MCs resemble their ex vivo equivalents in many respects and are a more relevant in vitro model compared to mouse BMMCs for studies of MC biology, in particular human MC biology.

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Uptake and distribution of fullerenes in human mast cells.

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Uptake and distribution of fullerenes in human mast cells.

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