Fluorescent Probe-Labeled Lipid Microsphere Uptake by Human Endothelial Cells: A Flow Cytometric Study

Suzuki, Kunihiro; Takahashi, Kimiko; Matsuki, Yasunori; Kawakami, Makoto; Kawaguchi, Yasushi; Hidaka, Toshihiko; Sekiyama, Yasushi; Mizukami, Yuichi; Kawagoe, Mikio; Nakamura, Haruo

doi:10.1254/jjp.60.349

Cited by 16 publications

(16 citation statements)

References 23 publications

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“…2). During the uptake experiments, there was very little, if any, liberation of Dil from labelled LM [6].…”

Section: Discussionmentioning

confidence: 95%

“…Lipid emulsions (Dil-labelied LM; Dil-LM and LM) were prepared as previously described [6]. With Aex fixed at 488 nm.…”

Section: Reagentsmentioning

confidence: 99%

“…The fluorescence of Dil-LM at the Aex of 488 nm and Aem of 570 nm was augmented linearly in accordance with the concentration of Dil. The fluorescence of Dil-LM obtained under the same condition was little affected at any pH of the lipid emulsion between 3-3 and 8-9 [6]. The concentration of the labelled lipid emulsion was expressed as weight of Dil/volume of Iipid emulsion.…”

Section: Reagentsmentioning

confidence: 99%

“…Dil accumulated in the cells is visible with a fluorescent microscope andean be easily quiintified by flow cytometry [7][8][9][10]. Utilizing Dil-labelled LM (Dil-LM), in vitro human endothelial cells were shown to preferentially take up LM [6].…”

Section: Introductionmentioning

confidence: 99%

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Activated CD4+ T cells preferentially take up lipid microspheres, but resting cells do not

Suzuki

1995

Clinical and Experimental Immunology

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SUMMARY Lipid microspheres (LM) used as drug carriers increase the effectiveness and reduce the toxicity of incorporated drugs. The present study is designed to determine whether or not activated T lymphocytes, which were the cells chosen first from the ‘inflammatory cells’, can take up LM in vitro. LM were labelled with a fluorescent probe, DiI (Dil‐LM), to examine the kinetics. Flow cytometric analysis demonstrated that in freshly isolated peripheral blood mononuclear cells (PBMC), monocytes principally took up Dil‐LM, while lymphocytes and granulocytes did not. When PBMC were stimulated with immobilized anti‐CD3 MoAb and IL‐2, cells expressing CD3, CD4, CD8 and CD 16 incorporated Dil‐LM. Purified CD4+ T cells, obtained by positive panning selection, were stimulated with this system. They were CD25, CD71, LFA‐1‐positive, and also showed an ability to take up Dil‐LM, which resting cells did not. The findings were confirmed by flow cytometry and quantitative analysis of Dil. Confocal micrographs showed fluorescent granules from the probe in the cytoplasm of stimulated CD4+ T cells after incubation with DiI‐LM. These results suggest that immunomodulatory agents incorporated into LM might selectively regulate the function of CD4+ or CD8+ T cells when these are activated.

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“…2). During the uptake experiments, there was very little, if any, liberation of Dil from labelled LM [6].…”

Section: Discussionmentioning

confidence: 95%

“…Lipid emulsions (Dil-labelied LM; Dil-LM and LM) were prepared as previously described [6]. With Aex fixed at 488 nm.…”

Section: Reagentsmentioning

confidence: 99%

Section: Reagentsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Activated CD4+ T cells preferentially take up lipid microspheres, but resting cells do not

Suzuki

1995

Clinical and Experimental Immunology

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“…Selective uptake of LM by endothelial cells has been demonstrated by Suzuki et al [10]. Since endothelial cells transport serum pro teins and other high molecular weight sub stances by transcytosis [11,12], the cells might possibly use the same mechanism to move LM from the luminal cavity to the space surrounding the vessels.…”

Section: Introductionmentioning

confidence: 91%

Transcytosis of Lipid Microspheres by Human Endothelial Cells

Takahashi¹,

Suzuki²,

Ichiki

et al. 1996

Pharmacology

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Human endothelial cells were cultivated on microporous membranes mimicking the luminal and basal spaces of blood vessels. When fluorescence-labeled lipid microspheres (LM) were added to the upper chambers of the model cultures, confluent monolayers of endothelial cells transported considerable levels of fluorescence to lower chambers. The transport was time dependent and was diminished by the addition of cytochalasin B. The uptake of LM into the endothelial cytoplasm was confirmed by electron microscopy and laser scanning confocal imaging. The amounts of fluorescence in the lower chamber were reduced when the endothelial cell layer was fixed with formaldehyde. These observations suggest that endothelial cells can transport LM by transcytosis. Endothelial cells seem to carry the LM without processing, since only minimal amounts of free fluorescence were detected even after longer cultivation periods. The fluorescence in the lower chambers of cell cultures treated with interleukin 1β was 3.7-fold higher than that of untreated cells; interleukin 2 and tumor necrosis factor alpha treatments had no discernible effect on LM transport. The interleukin 1β induced increase of transcytosis in endothelial cells would explain why LM preferentially accumulate in inflammatory tissues.

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Characterization of tissue factor expression on the human endothelial cell line ECV304

et al. 1997

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The endothelial cell line ECV304 is a spontaneously transformed cell line established from human umbilical vein. The characterization of tissue factor (TF) expression by ECV304 cells has been accomplished in this study. ECV304 cells expressed both TF mRNA and antigen (TF ag) constitutively. In ECV304 cell lysates, the levels of TF ag (1.4 ± 0.3 ng of TF ag /10 6 cells) were considerably higher than in THP-1 monocytoid cells (0.07 ± 0.03 ng of TF ag /10 6 cells). TF ag was also detected on the ECV304 cell surface by flow cytometric studies. In binding analyses, 3.5 ± 0.7 × 10 4 molecules of TF per cell were estimated, similar to the amounts found in ECV304 cell lysates (2.9 ± 0.6 × 10 4 molecules/ cell), suggesting that all TF ag was translocated to the cell surface. Phorbol myristate acetate (PMA) stimulation of ECV304 cells resulted in an increase of TF mRNA levels, which was abrogated when gene transcription was impaired, suggesting a transcriptional regulation of the TF gene by PMA. In contrast, TF ag was not elevated by PMA-stimulation, indicating the existence of additional posttranscriptional mechanisms. Thus, ECV304 cells constitute a singular endothelial cell model for exploring the regulation of TF expression. Am. J. Hematol. 56:71-78, 1997.

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Fluorescent Probe-Labeled Lipid Microsphere Uptake by Human Endothelial Cells: A Flow Cytometric Study

Cited by 16 publications

References 23 publications

Activated CD4+ T cells preferentially take up lipid microspheres, but resting cells do not

Activated CD4+ T cells preferentially take up lipid microspheres, but resting cells do not

Transcytosis of Lipid Microspheres by Human Endothelial Cells

Characterization of tissue factor expression on the human endothelial cell line ECV304

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