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

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 (IFN-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 IFN-α during 72 hr culturing. In contrast, there was no NO and IFN-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.

Immobilized Heavy Chain-Hyaluronic Acid Polarizes Lipopolysaccharide-activated Macrophages toward M2 Phenotype

“…2). Collectively, these data corroborate the previous studies based on AM (33,34,40) and AME (41) while beginning to explain the mechanism of their anti-inflammatory effects via promotion of apoptosis of activated neutrophils (33,34,37), macrophages (35,37,39), and probably lymphocytes (36).…”

“…Application of cryopreserved human AM reduces infiltration of neutrophils in the excimer-ablated rabbit corneal stroma while promoting apoptosis of neutrophils (33,34); traps and causes apoptosis of monocytes and macrophages during the treatment of human corneal epithelial defects (35); induces rapid regression of corneal stromal edema and inflammation by reducing infiltration of lymphocytes (36), neutrophils (37), and macrophages (37,38); and promotes M2a phenotype in the corneal stroma of murine HSV-1 necrotizing keratitis (39). These in vivo data are supported by in vitro findings that human AM induces apoptosis of IFN-␥ activated monocyte/macrophage RAW264.7 cells (40).…”

On the influence of neutrophils in corneas with necrotizing HSV-1 keratitis following amniotic membrane transplantation

“…Macrophages with ingested apoptotic cells markedly decrease expression of CD40, a co-stimulatory factor for T-lymphocytes (Barker et al, 1999) of proinflammatory cytokines and increased production of prostaglandin E2 and transforming growth factor-b1 (Fadok et al, 1998), both factors involved as regulators of physiological T cell homeostasis, activation, differentiation, and effector function. Recently, it has been shown that cells of a macrophage cell-line cocultured with AM only underwent apoptosis when activated with IFN-γ, while apoptosis was not found when IFN-γ was absent (Li et al, 2006).…”

On the origin of amniotic stem cells: of mice and men

“…Suppression of proteinase and MMP activation by amniotic membrane leads to decreased infiltration of inflammatory cells (Kim et al, 2000). Moreover, pro-apoptotic activity of the amniotic membrane was also reported -amnion can promote the apoptosis of leucocytes (Li et al, 2006;Zhou et al, 2003). Amniotic epithelial cells express the apoptosis-inducing genes Fas L, TNF, and TRAIL (Li et al, 2005).…”