Collectin-11 Is an Important Modulator of Retinal Pigment Epithelial Cell Phagocytosis and Cytokine Production

Abstract

In this paper, we report previously unknown roles for collectin-11 (CL-11, a soluble C-type lectin) in modulating the retinal pigment epithelial (RPE) cell functions of phagocytosis and cytokine production. We found that CL-11 and its carbohydrate ligand are expressed in both the murine and human neural retina; these resemble each other in terms of RPE and photoreceptor cells. Functional analysis of murine RPE cells showed that CL-11 facilitates the opsonophagocytosis of photoreceptor outer segments and apoptotic cells, and also upregulates IL-10 production. Mechanistic analysis revealed that calreticulin on the RPE cells is required for CL-11-mediated opsonophagocytosis whereas signal-regulatory protein α and mannosyl residues on the cells are involved in the CL-11-mediated upregulation of IL-10 production. This study is the first to demonstrate the role of CL-11 and the molecular mechanisms involved in modulating RPE cell phagocytosis and cytokine production. It provides a new insight into retinal health and disease and has implications for other phagocytic cells.

Keywords: CL-K1; Collectin 11; Cytokine production; Opsonophagocytosis; Retinal pigment epithelial cells.

Fig. 1

CL-11 is produced in the neural retina and RPE. a RT-PCR on dissected neural retinas (NR) and RPE-choroid complex (RCC) and kidney tissues (used as a positive control) from WT B6 mice. b RT-PCR on mouse primary RPE cells that had been stimulated with (stimulated) or without (unstimulated) TNF-α/IL-1β for 24 h. c Fluorescence microscopy images of immunohistochemical staining of CL-11 (red) in eye tissue of WT and CL-11^−/− mice; nuclear marker DAPI (blue) is also shown. Bottom and right: higher-magnification images that correspond with boxed areas. CR, choroid; RPE, retinal pigment epithelium; POS, photoreceptor outer segments; PIS, photoreceptor inner segments; ONL, outer nuclear layer. Scale bar, 20 μm. d Fluorescence microscopy images of immunochemical staining for CL-11 (red) and ZO-1 (a tight junction protein) (green) in flat-mounted RPE-choroid complex of WT and CL-11^−/− mice. Scale bar, 10 μm. e Western blot for CL-11 on rCL-11 (used as a positive control) and supernatants of mouse primary RPE cells stimulated with TNF-α/IL-1β for 24 h or were left unstimulated. a–e A representative of 3 experiments is shown.

Fig. 2

Detection of carbohydrate residue expression in PRC and PRE. a Fluorescence microscopy images of staining with GNL and LCA (green), and DAPI (blue) in the mouse eye. Bottom: higher-magnification images that correspond with the boxed areas. Scale bar, 50 μm. b Fluorescence microscopy images of immunochemical staining for CL-11 (red) and GNL (green) in the mouse eye. Scale bar, 10 μm. A representative of 3 experiments is shown.

Fig. 3

CL-11 facilitates opsonophagocytosis of POS/apoptotic cells by RPE cells. a Flow cytometry analysis of CL-11 binding to POS incubated with FITC-labeled rCL-11 (600 ng/mL) for 1 h. b Fluorescence microscopy images of primarily cultured mouse RPE incubated with nonopsonized POS (POS) or CL-11 (600 ng/mL) opsonized POS (CL-11-POS) for 1 h, followed by thorough washing and fluorescence quenching; POS were prelabeled with FITC (green), RPE cells were stained with phalloidin (red), nuclear staining was with DAPI (blue). Scale bar, 50 μm. c Quantification of POS associated with RPE cells corresponds to b. Data are from 6 individual images (from 2 coverslips) per group and representative of 3 independent experiments. ** p < 0.01, unpaired two-tailed Student t test. d Confocal images corresponding to the CL-11-POS image in b. The z-stack image shows POS (green) phagocytosed by RPE cells (stained with phalloidin [red]). Scale bar, 25 μm. e Flow cytometry analysis of uptake of POS in B6-RPE07 cells incubated with nonopsonized POS (POS) or CL-11 (600 ng/mL) opsonized POS (CL-11-POS for 2 h, followed by thorough washing and fluorescence quenching. f Quantification of POS associated with RPE cells (histogram e), shown as geometric mean of fluorescence intensity (GMFI). Data are from 4 cell samples per group and were analyzed by unpaired two-tailed Student t test. ** p < 0.01. g Fluorescence microscopy images of B6-RPE07 cells incubated with nonopsonized apoptotic cells (AP) or rCL-11 (600 ng/mL) opsonized apoptotic cells (CL-11-AP) for 1 h, followed by thorough washing and fluorescence quenching; apoptotic cells were prelabeled with TAMRA (red), RPE was stained with phalloidin (green), nuclear staining was with DAPI (blue). Scale bar, 50 μm. h Quantification of apoptotic cells associated with RPE cells corresponds to e. Data shown are from 6 individual images (from 3 coverslips) per group and were analyzed by unpaired two-tailed Student t test. *** p < 0.0001. i Flow cytometry analysis of uptake of AP in B6-RPE07 cells incubated with AP or CL-11-AP (preincubated with indicated concentrations of rCL-11). Apoptotic cells were prelabeled with TAMRA. j Quantification of apoptotic cells associated with RPE cells (histogram i), shown as geometric mean of fluorescence intensity (GMFI). Data are from 4 cell samples per group and were analyzed with one-way ANOVA, comparing the nonopsonized AP with the CL-11-opsonized AP. e–j A representative of 3 independent experiments is shown.

Fig. 4

CL-11 regulates inflammatory cytokine production by RPE cells. B6-RPE07 cells were cultured with or without rCL-11, in the absence or presence of LPS (10 ng/mL) or IFN-γ (500 U/mL) for 24 h. Supernatants were collected and used for ELISA. a IL-10. b IL-6. Data are from 4 individual cell culture plate wells per group and were analyzed with one-way ANOVA, comparing the untreated and CL-11-treated samples. A representative of 3 independent experiments is shown.

Fig. 5

Identification of the receptor responsible for CL-11-mediated opsonophagocytosis by RPE cells. a Detection of calreticulin in B6-RPE07 cells by RT-PCR. b Fluorescence microscopy images of B6-RPE07 cells (from 2 independent experiments) stained for calreticulin (green) and the nuclear marker DAPI (blue). Negative control: the staining was performed using 2nd antibody alone. Scale bar, 10 μm. c Confocal images of B6-RPE07 cells (at low and high magnifications) incubated with rCL-11-opsonized POS for 30 min and subjected to staining for CL-11 (red) and calreticulin (green) and the nuclear marker DAPI (blue). Scale bar, 10 μm. A representative of 2 independent experiments is shown. d Flow cytometry analysis of phagocytosis of POS (prelabeled with FITC) by B6-RPE07 cells pretreated with anti-calreticulin antibody or control antibody (Ctrl Ab), and then incubated with nonopsonized POS (POS) or CL-11-opsonized (CL-11-POS) for 2 h. Data are from 4 cell samples per group and were analyzed with one-way ANOVA with the Tukey post hoc test. ** p < 0.01; ns, no significant difference. A representative of 3 independent experiments is shown.

Fig. 6

Identification of the receptor/molecules involved in CL-11-mediated modulation of cytokine production by RPE cells. a Detection of SIRPα in B6-RPE07 cells by RT-PCR. b Fluorescence microscopy images of B6-RPE07 cells (of 2 independent experiments) stained for SIRPα (green) and nuclear marker DAPI (blue). Negative control: the staining was performed using 2nd antibody alone. Scale bar, 10 μm. c Confocal images of B6-RPE07 cells (at low and high magnifications) incubated with rCL-11 (600 ng/mL) for 1 h and subjected to staining for CL-11 (red) and SIRPα (green) and nuclear marker DAPI (blue). Scale bar, 10 μm. A representative of 2 independent experiments is shown. d B6-RPE07 cells were pretreated with anti-SIPRα antibody (1:100 diluted) or control antibody (Ctrl Ab) and then incubated with or without rCL-11 (600 ng/mL) for 24 h. Supernatants were collected and used for measuring IL-10 by ELISA. Data are from 4 cell samples per group and were analyzed with one-way ANOVA with the Tukey post hoc test. *** p < 0.001; ns, no significance. A representative of 3 independent experiments is shown. e Fluorescence microscopy images of B6-RPE07 cells that had been stained with fluorescence-conjugated GNL, LCA, or LTL (green). Scale bar, 10 μm. f Flow cytometry for the detection of mannosyl residue (MR) expression on B6-RPE07 cells that had been treated with buffer alone or containing α-mannosidase for 2 h, and then stained with fluorescence-conjugated LCA. g Flow cytometry analysis of CL-11 binding to B6-RPE07 cells treated with buffer or containing α-mannosidase for 2 h, incubated with rCL-11 (600 ng/mL) for 1 h, and then subjected to staining for CL-11. h, i B6-RPE07 cells were pretreated with buffer alone or containing α-mannosidase at the indicated concentrations for 2 h and then incubated with rCL-11 (600 ng/mL) for 24 h. Supernatants were collected and used for measuring IL-10 and IL-6 by ELISA. The dashed line indicates the level of IL-10 or IL-6 in RPE cells without CL-11 stimulation. f–i Data are from 4 cell samples per group and were analyzed with one-way ANOVA, comparing the control (buffer alone) and α-mannosidase-treated cells. A representative of 3 independent experiments is shown.

Fig. 7

A schematic diagram illustrates the proposed mechanisms by which CL-11 modulates RPE cell phagocytosis and cytokine production. Locally produced CL-11 (by RPE, photoreceptors, and possibly other cells): (i) binds to shed POS to mediate or upregulate the phagocytosis of POS through the interaction of the CL-11 collagen-like region (CLR) with calreticulin/CD91 on RPE cells, (ii) binds to RPE cells to mediate the upregulation of IL-10 production through the interaction of the CL-11 carbohydrate domain (CRD) with a specific glycoprotein (SIRPα) and/or mannose-containing structures on RPE cells.