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Apoptotic signaling in endothelial cells with neutrophil activation

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Abstract

As is the case for tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), degranulated substances (DS) released from polymorphonuclear leukocytes (PMN) and H2O2 cause endothelial cell apoptosis through the phosphorylation of members of the mitogen-activated protein kinase (MAPK) family. Stimulation of human umbilical vein endothelial cells (HUVEC) with IL-1β or TNF-α/cycloheximide (CHX) was found to enhance the phosphorylation of p38 and Jun-N-terminal kinase (JNK) in a time-dependent fashion, but did not affect the time-dependent phosphorylation of extracellular signal-regulated kinase. In addition, IL-1β and TNF-α/CHX induced the phosphorylation of activating transcription factor-2 (ATF-2), but not c-Jun. Moreover, the p38 in HUVEC was phosphorylated by DS released from PMN and also by H2O2, but not by •O2 − induced by myeloperoxidase (MPO) of PMN. On the other hand, caspase 8 in HUVEC was activated by DS, but not by H2O2 and/or •O2 −. In addition, caspases 3 and 7 were cleaved by the treatment of DS and turned into active forms. DS was concentrated, analyzed by electrophoresis, and revealed to contain precursor and subunits of MPO (90, 60, and 14 kDa) and another peptide with a molecular weight of about 28 kDa. Because SB203580 that was an inhibitor of p38 MAPK did not repress phosphorylation of ATF-2 in HUVEC, it was suggested that JNK was more important than p38 in a series of signaling courses. These results suggest the possibility that not only TNF-α/CHX and IL-1β but also DS released from PMN and the cell-permeable reactive oxygen species H2O2 induce blood vessel injury through endothelial apoptosis.

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Abbreviations

TNF-α:

Tumor necrosis factor-α

IL-1β:

Interleukin-1β

DS:

Degranulated substances

PMN:

Polymorphonuclear leukocytes

MAPK:

Mitogen-activated protein kinase

HUVEC:

Human umbilical vein endothelial cells

CHX:

Cycloheximide

JNK:

Jun-N-terminal kinase

ERK:

Extracellular signal-regulated kinase

ATF-2:

Activating transcription factor-2

MPO:

Myeloperoxidase

ROS:

Reactive oxygen species

TNFR:

TNF-α receptor

FMLP:

FormylMet-Leu-Phe

CB:

Cytochalasin B

EGTA:

Ethyleneglycol-bis(2-aminoethylether)tetraacetic acid

PMSF:

Phenylmethylsulfonyl fluoride

PBS:

Phosphate-buffered saline without calcium and magnesium

HBSS:

Hanks’ balanced salt solution

SDS:

Sodium dodecyl-sulfate

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Acknowledgements

We express our sincere thanks to Dr. Yasuo Akanuma (Institute for Diabetes Care and Research, Asahi Life Foundation, Tokyo) and Dr. Akiko Ishida-Okawara (National Institute of Infectious Diseases, Tokyo) for their helpful support and discussions. This study was supported by grants from the Human Science Foundation (Tokyo, Japan).

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Author notes

  1. Tomokazu Nagao & Kazuo Suzuki

    Present address: Department of Infection and Host Defense, Chiba University, 1-8-15 Inohana, Chuo-ku, Chiba, 260-8670, Japan

Authors and Affiliations

  1. Department of Bioactive Molecules, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo, 162-8640, Japan

    Osamu Koshio, Tomokazu Nagao, Ayako Mabuchi & Kazuo Suzuki

  2. Research Division, Institute for Diabetes Care and Research, Asahi Life Foundation, 1-6-1 Marunouchi, Chiyoda-ku, Tokyo, 100-0005, Japan

    Osamu Koshio

  3. Department of Microbiology and Immunology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo, 113-8602, Japan

    Ayako Mabuchi

  4. Department of Physiology, Otago University, Dunedin, 9054, New Zealand

    Ayako Mabuchi

  5. Department of Microbiology and Immunology, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo, 173-8605, Japan

    Osamu Koshio & Yasuo Ono

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  1. Osamu Koshio
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Correspondence to Osamu Koshio.

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Koshio, O., Nagao, T., Mabuchi, A. et al. Apoptotic signaling in endothelial cells with neutrophil activation. Mol Cell Biochem 363, 269–280 (2012). https://doi.org/10.1007/s11010-011-1179-5

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