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. 2015 Nov 19;6(11):e1986.
doi: 10.1038/cddis.2015.334.

Membrane-bound and Soluble Fas Ligands Have Opposite Functions in Photoreceptor Cell Death Following Separation From the Retinal Pigment Epithelium

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Free PMC article

Membrane-bound and Soluble Fas Ligands Have Opposite Functions in Photoreceptor Cell Death Following Separation From the Retinal Pigment Epithelium

H Matsumoto et al. Cell Death Dis. .
Free PMC article

Abstract

Fas ligand (FasL) triggers apoptosis of Fas-positive cells, and previous reports described FasL-induced cell death of Fas-positive photoreceptors following a retinal detachment. However, as FasL exists in membrane-bound (mFasL) and soluble (sFasL) forms, and is expressed on resident microglia and infiltrating monocyte/macrophages, the current study examined the relative contribution of mFasL and sFasL to photoreceptor cell death after induction of experimental retinal detachment in wild-type, knockout (FasL-/-), and mFasL-only knock-in (ΔCS) mice. Retinal detachment in FasL-/- mice resulted in a significant reduction of photoreceptor cell death. In contrast, ΔCS mice displayed significantly more apoptotic photoreceptor cell death. Photoreceptor loss in ΔCS mice was inhibited by a subretinal injection of recombinant sFasL. Thus, Fas/FasL-triggered cell death accounts for a significant amount of photoreceptor cell loss following the retinal detachment. The function of FasL was dependent upon the form of FasL expressed: mFasL triggered photoreceptor cell death, whereas sFasL protected the retina, indicating that enzyme-mediated cleavage of FasL determines, in part, the extent of vision loss following the retinal detachment. Moreover, it also indicates that treatment with sFasL could significantly reduce photoreceptor cell loss in patients with retinal detachment.

Figures

Figure 1
Figure 1
FasL mediates photoreceptor cell death induced by retinal detachment. (a) Time course of TUNEL-positive cell density in outer nuclear layer (ONL) in BALB/c WT and BALB/c FasL−/− mice (n=6 each group and time point). The peak of TUNEL-positive cell density was day 1 in both groups. BALB/c FasL−/− mice showed significantly less photoreceptor cell death than BALB/c WT mice 1 day after retinal detachments (**P<0.01). (b) TUNEL (green) and TO-PRO-3 (blue) staining at 24 h after retinal detachment. Only ONL showed TUNEL-positive cells in both BALB/c WT and BALB/c FasL−/− mice. (c) ONL/INL (inner nuclear layer) ratio 7 days after retinal detachment (n=6 each). ONL/INL ratio was significantly higher in BALB/c FasL−/− than in BALB/c WT mice (*P<0.05). (d) Time course of TUNEL-positive cell density in ONL in B6129 WT and B6129 ΔCS mice (n=6 each group and time point). The peak of TUNEL-positive cell density was on day 1 in both groups. B6129 ΔCS mice showed significantly more photoreceptor cell death than B6129 WT mice at 24 h (*P<0.05), 120 h (*P<0.05), and 168 h (*P<0.001) after retinal detachment. (e) TUNEL (green) and TO-PRO-3 (blue) staining at 24 h after retinal detachment. Only ONL showed TUNEL-positive cells in both B6129 WT and B6129 ΔCS mice. (f) ONL/INL ratio 7 days after retinal detachment (n=6 each). ONL/INL ratio was significantly lower in B6129 ΔCS than in B6129 WT mice (*P<0.05). Scale bar, 50 μm. The graphs show mean±S.E.M. GCL, ganglion cell layer; UT, untreated
Figure 2
Figure 2
FasL mediates both photoreceptor apoptosis and necrosis after retinal detachment. (a) BALB/c FasL−/− mice showed significantly less apoptotic and necrotic photoreceptor cell deaths than BALB/c WT mice at 24 h after retinal detachment (P<0.05 each). (b) B6129 ΔCS mice exhibited significantly more apoptotic photoreceptor cell death than B6129 WT mice at 24 h after retinal detachment (P<0.05 each), whereas there was no significant difference in necrotic photoreceptor cell death between them. A, apoptosis; N, necrosis
Figure 3
Figure 3
Retinal detachment-induced inflammatory cytokine expression is reduced in FasL−/− mice but increased in ΔCS mice. (a and b) ELISA to detect MCP-1 (a) and IL-6 (b) in the BALB/c WT and BALB/c FasL−/− retinas at 24 h after retinal detachment (n=8 each). MCP-1 generation was significantly suppressed in BALB/c FasL−/− mice (*P<0.05). IL-6 expression was reduced (but not significant) in BALB/c FasL−/− than in BALB/c WT mice. (c and d) ELISA to detect MCP-1 (c) and IL-6 (d) in the B6129 WT and B6129 ΔCS retinas at 24 h after retinal detachment (n=8 each). MCP-1 and IL-6 generation was significantly increased in B6129 ΔCS than in B6129 WT mice (*P<0.05)
Figure 4
Figure 4
Classically and alternatively activated subretinal macrophages after induction of retinal detachment. (ac) Ccr2, Ly6c, and Cx3cr1 mRNAs expression in the subretinal space was evaluated by LCM followed by qPCR using BALB/c WT and BALB/c FasL−/− mice at 1 and 7 days after retinal detachment (n=6 each group and time point). Ccr2 mRNA was significantly higher in day 1 than in day 7 in each group (WT: **P<0.01, FasL−/−: *P<0.05) (a). Ly6c mRNA was significantly higher in day 1 as compared with day 7 (WT: *P<0.05, FasL−/−: **P<0.01) (b). On the other hand, Cx3cr1 mRNA was significantly higher in day 7 than in day 1 (WT: ***P<0.001, FasL−/−: ***P<0.001) (c). There were no significant differences in the three mRNAs expression between BALB/c WT and BALB/c FasL−/− mice. (df) Ccr2, Ly6c, and Cx3cr1 mRNAs expression in the subretinal space in B6129 WT and B6129 ΔCS mice at 1 and 7 days after retinal detachment (n=6 each group and time point). Ccr2 mRNA tended to be higher in day 1 than in day 7 in each group; however, the differences did not achieve statistical significance (d). Ly6c mRNA was significantly higher in day 1 as compared with day 7 in B6129 ΔCS mice (**P<0.01). B6129 WT mice showed higher Ly6c mRNA in day 1 than in day 7; however, the difference did not reach statistical significance (e). Cx3cr1 mRNA was significantly higher in day 7 than in day 1 (WT: *P<0.05, ΔCS: ***P<0.001) (f). There were no significant differences in the three mRNAs expression between B6129 WT and B6129 ΔCS mice. The graphs show mean±S.E.M.
Figure 5
Figure 5
Subretinal injection of soluble Fas ligand attenuates photoreceptor cell death after retinal detachment. (a) TUNEL-positive cell density in the outer nuclear layer (ONL) in B6129 ΔCS mice at 24 h after retinal detachment. Recombinant murine soluble Fas ligand (sFasL) or control phosphate-buffered saline (CTR) was injected into the subretinal space when creating retinal detachment (n=12 each group). The eyes with sFasL showed significantly reduced photoreceptor cell death as compared with control eyes (*P<0.05). (b) TUNEL (green) and TO-PRO-3 (blue) staining at 24 h after retinal detachment. The graphs show mean±S.E.M. Scale bar, 50 μm

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