Expression of CCL2 and its receptor in activation and migration of microglia and monocytes induced by photoreceptor apoptosis

Abstract

Purpose: To explore the effect of the CCL2 and CCR2 system on the activation and migration of microglia and monocytes in light-induced photoreceptor apoptosis.

Methods: At 1 day, 3 days, 7 days, and 14 days after light exposure, OX42 and ED1 immunostaining were used to label the activation and migration of microglia and monocytes. Double immunostaining of CCL2 with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), OX42, or glial fibrillary acidic protein (GFAP) was applied to explore the relationships among CCL2, apoptotic photoreceptors, activated microglia and monocytes, and macroglial cells (Müller cells and astrocytes). Real-time PCR was used to evaluate the mRNA levels of retinal CCL2 and CCR2 and the proinflammatory factors interleukin (IL)-1 beta and tumor necrosis factor (TNF)-alpha.

Results: Real-time PCR analyses showed that CCL2 and CCR2 expression gradually increased after light exposure and peaked at 3 days, coinciding with the infiltration of OX42-positive cells and the expression of IL-1 beta and TNF-alpha in the outer retina. Double immunostaining of CCL2 with TUNEL revealed that CCL2 was expressed robustly in about 30% of the apoptotic photoreceptors at the early stage. As degeneration progressed, immunostaining of CCL2 with OX42 showed that activated and migrated microglia and monocytes expressed CCL2. At the late stage, Müller cells became the main source of CCL2, which was illustrated by CCL2 immunostaining with GFAP.

Conclusions: Light exposure led to apoptosis of photoreceptors, which expressed CCL2, accelerating an inflammation-mediated cascade by activating and attracting microglia and monocytes and promoting their secretion of CCL2 in the injured position.

Figure 1

Microglia/monocyte activation and infiltration in light-induced photoreceptor degeneration. A, E: In healthy retinas, OX42-positive microglia were seen only in the retinal ganglion cells and the inner plexiform layer with long, slim processes, while no ED1-positive monocytes were seen in the retina. B, F: One day after exposure to light, a small number of monocytes/microglia began to infiltrate into the outer nuclear layer (ONL) and subretinal space. C: The number of OX42-positive cells peaked at 3 days, with activated amoeboid morphology. D, H: At 7 days, many OX42-positive cells remained in the thinned ONL and subretinal space, while the number of ED1-positive cells in the outer retina peaked at that time.

Figure 2

Expression of CCL2 and CCR2 in the retina after extensive light exposure. A: CCL2 was scarcely expressed in the retinal ganglion cells (RGCs) of a healthy retina. B, C: At 1 day after light exposure, there was a small amount of CCL2 expression in the outer nuclear layer (ONL), which increased in the RGCs and the inner nuclear layer (INL) at 3 days. D: At 7 days, CCL2 was observed in cells with radially oriented processes (yellow arrows) throughout the RGCs. E: CCR2 was scarcely expressed in a healthy retina. F, G: At 1 day, CCR2 was observed in the RGCs of the retina, and it was expressed robustly in the INL and the ONL at 3 days after light exposure. H: CCR2 was richly expressed in the ONL at 7 days.

Figure 3

Localization of CCL2 in a healthy retina and apoptotic photoreceptors by double immunofluorescence staining. A, D: In a healthy retinal slice and flat mount, CCL2 was mainly expressed in the retinal ganglion cell layer. B, E: Glial fibrillary acidic protein (GFAP) staining was localized in the astrocytes. C, F: The merged images revealed that CCL2 was expressed by astrocytes in a healthy retina. G: At 1 day after light exposure, CCL2 was expressed robustly in the outer nuclear layer (ONL). H: Large numbers of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)–positive cells were observed in the ONL. I: The merged image shows that some of the CCL2 expression was colocalized in TUNEL-positive cells. J, K, L: Amplified images of G, H, and I, respectively.

Figure 4

Localization of CCL2 in activated microglia and monocytes by double immunofluorescence staining. A, B: At 3 days after light exposure, in the retinal slice, CCL2 expression was observed in the outer nuclear layer (ONL), and many OX42-positive cells were activated and migrated into the ONL and subretinal space. C: The merged image shows that CCL2 was colocalized in those activated and migrated microglia and monocytes. D, E, F: Amplified images of A, B, and C, respectively. G, H: In the retinal flat mount, CCL2 was expressed richly in the outer retina, where OX42-positive cells were found. I: The merged image shows that CCL2 was colocalized in those microglia and monocytes. J, K, L: Amplified images of G, H, and I, respectively.

Figure 5

Localization of CCL2 in the late stage of photic injury by double immunofluorescence staining. A, B, C: In the flat mount, CCL2 was colocalized in OX42-positive cells with amoeboid morphology at 3 days after light exposure. D, E, F: At 7 days, no expression of CCL2 was observed in OX42-positive cells. G: However, expression of CCL2 was observed in cells with radially oriented processes throughout the inner nuclear layer. H: Strong immunoreactivity to glial fibrillary acidic protein (GFAP) appeared from the internal limiting membrane to the inner plexiform layer at 7 days. I: The merged image shows that CCL2 was colocalized in GFAP-positive Müller glia. J, K, L: Amplified images of G, H, and I, respectively. M, N, O: Immunofluorescent staining in the retinal flat mount confirmed the observation in the retinal slice.

Figure 6

Real-time PCR of CCL2 and CCR2 and IL-1 beta and TNF-alpha expression after extensive light exposure. A, B: The mRNA of CCL2 and CCR2 began to increase as early as 1 day, peaked at 3 days, and slowly decreased. C, D: The expression pattern of interleukin (IL)-1 beta and tumor necrosis factor (TNF)-alpha was consistent with the patterns of CCL2 and CCR2. GAPDH expression served as the loading control. The mean ± standard deviation of data from three independent experiments is shown, **, p<0.01.

Figure 7

Western blot analysis of CCL2 and CCR2 expression after extensive light exposure. A, B: CCL2 protein expression began to increase at 1 day and peaked at 3 days. A, C: CCR2 protein expression began to increase at 3 days and peaked at 7 days after light exposure. Actin protein expression served as the loading control. The mean ± standard deviation of data from three independent experiments is shown, *,p<0.05; **,p<0.01.

Figure 8

Comparative changes in microglia/monocyte infiltration, as well as CCL2 and CCR2 and IL-1 beta and TNF-alpha expression. A: The number of OX42-positive cells increased rapidly at as early as 1 day after light exposure and peaked at 3 days. B: The increasing number of ED1-positive cells in the outer retina occurred just following the infiltration of OX42-positive cells and peaked at 7 days. A, B: The expression patterns of CCL2 and CCR2 and interleukin (IL)-1 beta and tumor necrosis factor (TNF)-alpha were consistent with the infiltration of OX42-positive cells, with the peak at 3 days.