Monocyte chemoattractant protein 1 mediates retinal detachment-induced photoreceptor apoptosis

Abstract

Photoreceptor apoptosis is a major cause of visual loss in retinal detachment (RD) and several other visual disorders, but the underlying mechanisms remain elusive. Recently, increased expression of monocyte chemoattractant protein 1 (MCP-1) was reported in vitreous humor samples of patients with RD and diabetic retinopathy as well as in the brain tissues of patients with neurodegenerative diseases, including Alzheimer's disease and multiple sclerosis. Here we report that MCP-1 plays a critical role in mediating photoreceptor apoptosis in an experimental model of RD. RD led to increased MCP-1 expression in the Müller glia and increased CD11b+ macrophage/microglia in the detached retina. An MCP-1 blocking antibody greatly reduced macrophage/microglia infiltration and RD-induced photoreceptor apoptosis. Confirming these results, MCP-1 gene-deficient mice showed significantly reduced macrophage/microglia infiltration after RD and very little photoreceptor apoptosis. In primary retinal mixed cultures, MCP-1 was cytotoxic for recoverin+ photoreceptors, and this toxicity was eliminated through immunodepleting macrophage/microglia from the culture. In vivo, deletion of the gene encoding CD11b/CD18 nearly eliminated macrophage/microglia infiltration to the retina after RD and the loss of photoreceptors. Thus, MCP-1 expression and subsequent macrophage/microglia infiltration and activation are critical for RD-induced photoreceptor apoptosis. This pathway may be an important therapeutic target for preventing photoreceptor apoptosis in RD and other CNS diseases that share a common etiology.

Fig. 1.

MCP-1 expression is up-regulated after retinal detachment (RD) in mice. (A–D) Up-regulation of MCP-1 after RD. (A) Quantitative real-time PCR data for MCP-1 mRNA 72 h after RD (n = 6). (B) ELISA to detect MCP-1 protein 72 h after RD (n = 6). ∗∗, P < 0.01. (C–E) Immunoreactivity of MCP-1 in control retina (C) or after RD in WT mice (D) or in MCP-1^−/− mice (E). (F–H) MCP-1 localization in Müller glia. Double IHC was carried out with antibodies against MCP-1 (F) and glutamine synthetase, a marker for Müller cells (G). Arrows indicate colocalization. (H) Overlay. (Scale bar: 50 μm.)

Fig. 2.

An MCP-1 blocking antibody prevents RD-induced photoreceptor loss. (A and B) TUNEL in retinal sections with subretinal injection of control antibody (A) or MCP-1 blocking antibody (B). (Scale bar: 100 μm.) (C) Quantification of TUNEL⁺ photoreceptors 72 h after RD (n = 8 each). ∗∗, P < 0.01.

Fig. 3.

Cytotoxic effect of MCP-1 on RD-induced photoreceptor apoptosis. (A and B) TUNEL 72 h after RD in WT mice (A) and MCP-1^−/− mice (B). (Scale bar: 50 μm.) (C and D) TEM photomicrographs through the ONL 72 h after RD in WT mice (C) and MCP-1^−/− mice (D). Note the increased presence of apoptotic photoreceptors in WT mice compared with MCP-1^−/− mice (arrows). (Scale bar: 10 μm.) (E) Quantification of TUNEL⁺ cells (n = 8 each). ∗, P < 0.05.

Fig. 4.

Markedly reduced number of CD11b⁺ cells in MCP-1^−/− mice. (A and B) IHC with an antibody against CD11b 72 h after RD in WT mice (A) or MCP-1^−/− mice (B). Macrophage/microglia was recruited in the IPL (short arrows) and subretinal space (arrows). (Scale bar: 50 μm.) (C and D) Quantification of CD11b⁺ cells in the OPL (C) and in the subretinal space (D) (n = 8 each). ∗, P < 0.05. (E and F) TEM photomicrographs in the ONL 72 h after RD in WT mice (E) and MCP-1^−/− mice (F). Apoptotic photoreceptors (short white arrow) and invading cells (long white arrows) are more prevalent in WT mice than in MCP-1^−/− mice. The disruption of synaptic structures such as cone pedicles and rod spherules was more severe in WT mice than in MCP-1^−/− mice (black arrows). (Scale bar: 10 μm.)

Fig. 5.

CD11b⁺ cells mediate the cytotoxic effect of MCP-1 on cultured photoreceptors. (A and B) Recoverin⁺ photoreceptors in retinal primary culture with (B) or without (A) MCP-1. (C and D) CD11b⁺ cells before (C) or after (D) depletion by immunopanning. Arrows indicate CD11b⁺ cells. (E and F) Recoverin⁺ photoreceptors with (F) or without (E) MCP-1 (1 ng/ml) after depletion of CD11b⁺ cells. (G and H) Recoverin⁺ photoreceptors (red) and peripheral macrophage (green) in culture in the presence (H) or absence (G) of MCP-1 (1 ng/ml) after depletion of CD11b⁺ cells. (Scale bar: 100 μm.) (I) Dose–response curve of MCP-1 cytotoxicity on the cultured photoreceptors in the presence or absence of resident CD11b⁺ cells. ∗ (P < 0.05) and ∗∗ (P < 0.01) represent the significance when compared with controls without MCP-1. Catalase (2 μg/ml) suppresses MCP-1-induced photoreceptor loss. (J) Dose–response curve of MCP-1 cytotoxicity on cultured photoreceptors after added peripheral macrophage (PM).

Fig. 6.

Deletion of the Mac-1 gene prevents RD-induced photoreceptor apoptosis. (A and B) TUNEL in retinal sections of WT mice (A) or Mac-1^−/− mice (B). (Scale bar: 100 μm.) (C and D) TEM photomicrographs in the ONL 72 h after RD in WT mice (C) and Mac-1^−/− mice (D). Apoptotic photoreceptors (arrow) are more prevalent in WT mice than in Mac-1^−/− mice. (Scale bar: 10 μm.) (E) Quantification of TUNEL⁺ photoreceptors 72 h after RD (n = 8 each). (F) Quantification of TUNEL⁺ photoreceptors with or without PBN treatment. ∗∗, P< 0.01.