Identification of novel dendritic cell populations in normal mouse retina

Abstract

Purpose: Whether tissue resident or infiltrating antigen-presenting cells (APCs) are involved in modulating immune responses in the retina and initiating inflammation is controversial. In this histologic study, the authors examine the retinas of mice strains with different susceptibility to experimental autoimmune uveoretinitis (EAU) for tissue resident APC.

Methods: Retinal wholemounts from normal and inflamed eyes of B10R III, C57BL/6, BALB/c, and ABH Biozii mice were immunostained for APC markers (33D1, CD11c, CD11b, major histocompatibility complex [MHC] class II, F4/80, CD80, CD86, CD205, mPDCA, B220, and GR1) and analyzed by confocal fluorescence microscopy using emission fingerprinting and three-dimensional reconstruction techniques. Hematoxylin and eosin-stained histologic sections were used to evaluate EAU disease scores and to assess outer blood retina barrier (retinal pigment epithelium [RPE]) structures.

Results: A population of 33D1(+) cells was identified exclusively in the peripheral margins and juxtapapillary areas of the retina in normal, nonimmunized C57BL/6 adult mice. These cells were also MHC class II(high), and their location corresponded to sites of earliest inflammation in EAU. Numbers in the papillary area were very low (less than 10), but this region marked the predominant anatomic site for initiation of inflammation in this moderately susceptible strain. The distribution and phenotype of these cells within the retinas differed between mouse strains exhibiting different disease susceptibility. In EAU-resistant BALB/c mice, many more 33D1(+) dendritic cells were present in the normal retina but were MHC class II(low/-). Conversely, no 33D1(+) or MHC class II (+) dendriform cells could be found in the normal retinas of highly EAU-susceptible B10.RIII mice.

Conclusions: A novel population of 33D1(+) DCs was identified in normal mouse retina. The function of these cells remains to be defined, but increased numbers correlate positively with structural abnormalities in the RPE and increased resistance of the strain to EAU.

Figure 1

Histopathologic features of retinal inflammation in experimental autoimmune uveitis. Photoreceptor rod outer segments and inner and outer nuclear layers are largely unaffected in early inflammation. (A) Inflammation at anterior margin of retina, showing vasculitis around collecting tube venule, vitreous infiltration, and infiltration of choroid and ciliary body. Original magnification, ×125. (B) Inflammation in optic disk, showing moderate leukocyte infiltration in the optic disk and vitreous, with some involvement of adjacent retina. Original magnification, ×62.5. (C-D) Correlation between grade of disease and anatomic location of inflammation. (C) Retinal inflammation with optic disk in the section. (D) Retinal inflammation without optic disk in the section. Sections were scored by two observers, and similar results were obtained. Data presented as percentage of inflamed eyes within each grade spectrum showing the specific inflammatory pattern from one scorer. Ch, choroid; CB, ciliary body; CT, collecting tube; PP, pars plana; JP, juxtapapillary region of retina; ON, optic nerve; OD, optic disk; R, retina; RPE, retinal pigment epithelium.

Figure 2

Leukocyte adhesion and infiltration in retinal wholemounts. Splenocytes from syngeneic mice were in vitro labeled with C-AM and adoptively transferred into day 16 pi EAU-immunized mice (A-B) or OVA-immunized mice (C). Sixteen hours later, mice were injected with 50 μL Evans Blue, and retinal wholemounts were prepared for confocal microscopy. Early leukocyte adhesion and infiltration were observed in the juxtapapillary area (A, arrowhead) and the collecting tube of the peripheral retina (B, open arrow) in day 16 pi EAU mice. No evidence of inflammation was observed in control OVA-immunized mice (C). Bar, 100 μm.

Figure 3

MHC class II⁺ cells in the juxtapapillary and peripheral retina of normal nonimmunized mice. Wholemount retinas from different ages of mice were stained for MHC class II and observed by confocal microscopy. (A, C, E) Juxtapapillary area. (B, D, F) Peripheral retina at margin between retina (R) and ciliary body (CB). Populations of highly dendriform MHC class II⁺ cells were observed in an 8-week-old mouse retina (A, B). These cells were only observed around the optic disk (A) and at the junction of the neural retina and ciliary body (B). No MHC class II⁺ cells could be detected in the retina of a 1-week-old mouse in either location (C, D). Some MHC class II⁺ cells could be detected in the retina from 2 weeks of age (E, F), again located specifically in the juxtapapillary and marginal retina. Scale bar, 50 μm. (G) Retina from an adult mouse 12 weeks pi and reconstitution with GRP⁺ bone marrow cells shows two GFP MHC class II double-positive cells in the retinal margin area. (H) MHC class II⁺ cells in the juxtapapillary and peripheral retina increases significantly with age. Data are expressed as mean ± SD. n = 6 in 1-week, 2-week, and 4-week groups; n = 20 in 8-week group. R, retina; CB, ciliary body; OD, optic disk.

Figure 4

Precise anatomic location of MHC class II⁺ cells in normal retina. Wholemount retinas (n = 6) were double stained with anti-mouse CD31 (green) and MHC class II⁺ (red) and were analyzed by confocal microscopy. Venules were identified by their anatomic location within the retinal vascular tree, comparative vessel diameter, and predominant expression of CD31. Z-stacks (65-100 μm) of the whole thickness of the retina were taken and reconstructed to the front view (A, C) or side view (B, D) for image analysis. (A-B) MHC class II⁺ cells were observed in the layer between inner and outer retinal vasculatures and surrounding the collecting tube in the peripheral retina. (C-D) MHC class II⁺ cells were observed surrounding the optic nerve and extending throughout the entire retinal thickness in the juxtapapillary area.

Figure 5

Phenotype of MHC-class II⁺ cells. (A-B) Wholemount retinas (n > 4) from normal nonimmunized C57BL/6 mice were double stained with anti-mouse CD11b FITC (A, green) or mouse DC maker 33D1 FITC (B, green) and MHC class II PE (red). (C) Spleen sections from a normal mouse were stained for 33D1-FITC and F4/80 PE using a similar protocol for retinal wholemounts. (D-H) Wholemount retinas (n > 6) from day 16 to 28 pi EAU mice were double stained for CD31 FITC (D, E, H) or mouse DC marker 33D1 FITC (F) or CD3 FITC (G) and MHC class II PE and were analyzed by confocal microscopy. Increased numbers of MHC class II⁺ perivascular cells compared with normal controls were observed in the venules of the peripheral margin (D) and the juxtapapillary area (E). (F) All MHC class II⁺ cells stained positively for mouse DC marker 33D1. (G) CD3 T cells were observed clustering with MHC class II⁺ cells (open arrows). (F) Vascular endothelial cells stained negatively for MHC class II molecules. a, arteriole; v, venule; CB, ciliary body.

Figure 6

Mouse retinal DCs in other strains of mice. B10.RIII retina (A), BALB/c retina (B), and ABH Biozzi retina (C) were double stained for 33D1 (green) and MHC class II (red). Each panel shows single-channel collection and a merged image to show colocalization of signal. (A) No 33D1⁺ or MHC class II⁺ cells were observed in any B10.RIII retinas. (B) A large number of 33D1⁺ cells were detected in the retina of BALB/c, but they were MHC class II negative. (C) 33D1⁺ MHC II⁺ cells were observed in the ciliary body but not in the retina of an ABH Biozzi mouse. (D, E) Retinas from day 12 pi IRBP-immunized B10R III mice (D) or BALB/c mice with acute ocular herpes simplex infection were double stained for 33D1 FITC and MHC class II PE and observed by confocal microscopy. (D) All MHC class II⁺ cells stained positively for 33D1 in inflamed B10.RIII mouse retina. (E) Increased numbers of 33D1⁺ cells over control noninfected eyes (B) were observed and displayed altered morphology compared with control and remained MHC class II negative. OD, optic disk. All images represent retinal wholemounts from 3 to 6 mice in each group.

Figure 7

Outer blood retina barrier of normal retinal juxtapapillary areas. Eyes from normal nonimmunized mice were processed for hematoxylin and eosin (H-E) staining. (A-B) Eyes are from C57BL/6 mice and (C-D) from BALB/c mice. Juxtapapillary retinas with optic nerve are shown (A-C). Note (A) the RPE layer ends at the optic nerve sheet, whereas (B) there is a gap between the RPE and optic nerve sheet (open arrow). (C) Anatomic abnormality is more pronounced, showing large gaps in RPE (arrow) with extensive overlap between neural retina and optic nerve sheath. In the peripheral retina, RPE cells often appear highly attenuated or even absent, allowing close apposition between vascular choroid and neural retina (D, arrow). Histogram (E) showing the percentage of mouse eyes (n > 40; n = 12 for Biozzi mice) from each strain that show structural abnormality at the RPE/optic nerve sheath. Ch, choroid; ON, optic nerve; RPE, retinal pigment epithelium. Original magnification, ×62.5.