Amniotic membrane induces apoptosis of interferon-γ activated macrophages in vitro

Wei Li, Hua He, Tetsuya Kawakita, Edgar M. Espana, Scheffer C G Tseng

Research output: Contribution to journalArticle

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Abstract

Amniotic membrane (AM) used as a temporary or permanent graft for ocular surface reconstruction has a potent anti-inflammatory effect. We would like to investigate the mechanism whereby AM induces macrophage apoptosis in vitro. Mouse macrophages, Raw 264.7 cells, were cultured on plastic, type I collagen, corneal stromal slice or AM stromal matrix in serum-free medium with or without interferon-γ (IFN-γ). Cells were stained by LIVE/DEAD assay, Hoechst-33342, and TUNEL assay for cell death and apoptosis. Cell lysates and conditioned media were analysed by Cell Death Detection ELISA assay for quantitation of apoptosis. Conditioned media were also analysed by Griess assay for the nitrite concentration and ELISA assay for tumour necrosis factor alpha (TNF-α) concentration. Lysates of cells were subjected to Western blot analyses of IKK-α, IKK-β, p65 (RelA) subunit of nuclear factor κB (NF-κB), total Akt, phospho-Akt (Ser473), and phospho-FKHR (Thr24)/phosphor-FKHRL1 (Thr32). At 48 hr after cultivation, cells showed a low level of apoptosis when cultured on plastic, type I collagen and corneal stromal slice with or without IFN-γ and on AM without IFN-γ. Nevertheless, cells showed a significant increase of apoptosis when cultured on AM with IFN-γ activation, and this phenomenon became apparent only after 48 hr. IFN-γ-activated macrophages on plastic continuously produced nitric oxide (NO) and TNF-α during 72 hr culturing. In contrast, there was no NO and TNF-α production after 48 hr culture on AM. NO inhibitors, L-NMMA and L-NIL, attenuated NO production of IFN-γ-activated macrophages on AM, while apoptosis was not decreased accordingly. Expression of IKK-α, IKK-β, p65 (RelA) subunit of NF-κB total Akt, phosopho-Akt (Ser473), and phospho-FKHR (Thr24)/FKHRL1 (Thr32) was all down-regulated in IFN-γ-activated macrophages cultured on AM. In conclusion, AM stromal matrix induces apoptosis of IFN-γ activated, but not non-activated macrophages, not through the generation of NO, but instead by down-regulating anti-apoptotic NF-κB and Akt-FKHR signalling pathways.

Original languageEnglish
Pages (from-to)282-292
Number of pages11
JournalExperimental Eye Research
Volume82
Issue number2
DOIs
Publication statusPublished - 2006 Feb
Externally publishedYes

Fingerprint

Amnion
Interferons
Macrophages
Apoptosis
Nitric Oxide
Plastics
Tumor Necrosis Factor-alpha
Conditioned Culture Medium
Collagen Type I
Cell Death
Enzyme-Linked Immunosorbent Assay
In Vitro Techniques
omega-N-Methylarginine
Serum-Free Culture Media
In Situ Nick-End Labeling
Nitrites
Cultured Cells
Anti-Inflammatory Agents
Western Blotting
Transplants

Keywords

  • Amniotic membrane
  • Apoptosis
  • Interferon-γ
  • Macrophages

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems

Cite this

Amniotic membrane induces apoptosis of interferon-γ activated macrophages in vitro. / Li, Wei; He, Hua; Kawakita, Tetsuya; Espana, Edgar M.; Tseng, Scheffer C G.

In: Experimental Eye Research, Vol. 82, No. 2, 02.2006, p. 282-292.

Research output: Contribution to journalArticle

Li, Wei ; He, Hua ; Kawakita, Tetsuya ; Espana, Edgar M. ; Tseng, Scheffer C G. / Amniotic membrane induces apoptosis of interferon-γ activated macrophages in vitro. In: Experimental Eye Research. 2006 ; Vol. 82, No. 2. pp. 282-292.
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N2 - Amniotic membrane (AM) used as a temporary or permanent graft for ocular surface reconstruction has a potent anti-inflammatory effect. We would like to investigate the mechanism whereby AM induces macrophage apoptosis in vitro. Mouse macrophages, Raw 264.7 cells, were cultured on plastic, type I collagen, corneal stromal slice or AM stromal matrix in serum-free medium with or without interferon-γ (IFN-γ). Cells were stained by LIVE/DEAD assay, Hoechst-33342, and TUNEL assay for cell death and apoptosis. Cell lysates and conditioned media were analysed by Cell Death Detection ELISA assay for quantitation of apoptosis. Conditioned media were also analysed by Griess assay for the nitrite concentration and ELISA assay for tumour necrosis factor alpha (TNF-α) concentration. Lysates of cells were subjected to Western blot analyses of IKK-α, IKK-β, p65 (RelA) subunit of nuclear factor κB (NF-κB), total Akt, phospho-Akt (Ser473), and phospho-FKHR (Thr24)/phosphor-FKHRL1 (Thr32). At 48 hr after cultivation, cells showed a low level of apoptosis when cultured on plastic, type I collagen and corneal stromal slice with or without IFN-γ and on AM without IFN-γ. Nevertheless, cells showed a significant increase of apoptosis when cultured on AM with IFN-γ activation, and this phenomenon became apparent only after 48 hr. IFN-γ-activated macrophages on plastic continuously produced nitric oxide (NO) and TNF-α during 72 hr culturing. In contrast, there was no NO and TNF-α production after 48 hr culture on AM. NO inhibitors, L-NMMA and L-NIL, attenuated NO production of IFN-γ-activated macrophages on AM, while apoptosis was not decreased accordingly. Expression of IKK-α, IKK-β, p65 (RelA) subunit of NF-κB total Akt, phosopho-Akt (Ser473), and phospho-FKHR (Thr24)/FKHRL1 (Thr32) was all down-regulated in IFN-γ-activated macrophages cultured on AM. In conclusion, AM stromal matrix induces apoptosis of IFN-γ activated, but not non-activated macrophages, not through the generation of NO, but instead by down-regulating anti-apoptotic NF-κB and Akt-FKHR signalling pathways.

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