September 2016
Volume 57, Issue 11
Open Access
Anatomy and Pathology/Oncology  |   September 2016
Characterization of Antigen-Presenting Macrophages and Dendritic Cells in the Healthy Human Sclera
Author Affiliations & Notes
  • Simona L. Schlereth
    Department of Ophthalmology University of Cologne, Cologne, Germany
  • Stefan Kremers
    Department of Ophthalmology University of Cologne, Cologne, Germany
  • Falk Schrödl
    Department of Ophthalmology, Research Program for Experimental Ophthalmology and Glaucoma Research, Paracelsus Medical University, Salzburg, Austria
    Department of Anatomy, Paracelsus Medical University, Salzburg, Austria
  • Claus Cursiefen
    Department of Ophthalmology University of Cologne, Cologne, Germany
  • Ludwig M. Heindl
    Department of Ophthalmology University of Cologne, Cologne, Germany
  • Correspondence: Simona L. Schlereth, University of Cologne, Department of Ophthalmology, Kerpener Str. 62, 50924 Cologne, Germany; [email protected]
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 4878-4885. doi:https://doi.org/10.1167/iovs.15-18552
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      Simona L. Schlereth, Stefan Kremers, Falk Schrödl, Claus Cursiefen, Ludwig M. Heindl; Characterization of Antigen-Presenting Macrophages and Dendritic Cells in the Healthy Human Sclera. Invest. Ophthalmol. Vis. Sci. 2016;57(11):4878-4885. https://doi.org/10.1167/iovs.15-18552.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: The sclera is mainly made of collagen and fibroblasts. The aim of this study was to analyze whether immune cells are present in the healthy human sclera.

Methods: Ten human anterior episcleral or stromal tissue samples from globe donors were immunohistochemically examined using confocal microscopy. The expression of the macrophage markers CD68, CD163 and CD11b, CD45 (a general leukocyte marker), MHCII (expressed by antigen-presenting cells [APCs]), CD11c (dendritic cell marker), lymphatic endothelium hyaluronan receptor-1 (LYVE1; expressed on lymphatic endothelium and macrophage subsets), chemokine receptor 7 (CCR7, a homing receptor for leukocytes), CXCL12 (expressed by activated leukocytes), CCR2 (a marker for inflammatory monocytes), and glial fibrillary acidic protein (GFAP; expressed by astrocytes) was analyzed and quantified.

Results: In the episclera, a high number of cells (≥40 cells/mm2) were immunoreactive for CD68, CD45, MHCII, CCR7, LYVE1, and CD11b. Lower numbers (<20 cells/mm2) were positive for CXCL12, CCR2, and GFAP. The episclera showed a significantly higher number of cells compared to the stroma (P = 0.008). MHCII+ cells could be double positive for CCR7, CD45, CD11c, or CD11b and seldom CXCL12. Macrophages were most likely from the M1 type (CD68+, CD163−).

Conclusions: The healthy human sclera contains several macrophage populations, which can function as APCs, with the highest density being present in the episclera. Most cells express macrophage markers and may function as APCs. The presence of these cells might indicate that scleral immune cells are important for maintaining physiological functions in the eye and may potentially contribute to blood vessel homeostasis.

The sclera is the outer covering and protective layer of the eye. It is made of dense connective tissue, mainly of type I collagen providing stability to the eye. The ocular stability and maintenance of intraocular pressure are crucial for proper vision.1 The sclera also offers attachment points for the extraocular muscles.2 The sclera can be separated into three main layers: (1) the superficial episclera facing the orbit with a tight network of blood vessels; (2) the scleral stroma as the middle layer, mainly made of dense collagen; and (3) the profound lamina fusca located toward the uvea.2,3 The lamina fusca is part of ongoing discussions as to whether it should be considered as a scleral layer or as a part of the choroid.3 Until recently, the sclera itself has not received much attention from anatomists and ophthalmologists. A reason for this is the general opinion that besides fibroblasts, no other cells contribute to the scleral matrix and that the sclera is thereby relatively acellular. However, in adult and fetal scleral tissues it has been recently demonstrated that the episcleral blood vessels are surrounded by lymphatic endothelium hyaluronan receptor-1 (LYVE1)+ CD68+ macrophages in adults and by LYVE1+ CD68− cells in fetal samples.4,5 Although many LYVE1+ cells were found, all scleral layers are devoid of LYVE1+ or podoplanin+ lymphatic vessels.47 Thereby, the sclera constitutes the outer border to the lymphatic-free inner eye.6,8,9 The function of the high number of perivascular extraluminal macrophages in the sclera remains unclear so far. They might be involved in the clearance of perivascular fluid or debris. In contrast, in other tissues, such as heart and dermis capillaries, cremaster muscle, glomeruli, and mesenteric vessels, monocytes have been shown to adhere to the luminal side of the endothelium.1014 
Perivascular macrophages have been shown in the brain, where they play distinct roles in normal central nervous system (CNS) functions15,16 and in various inflammatory diseases.1719 In the CNS, the perivascular cells are known as CD45+ antigen-presenting cells (APCs) that can detect neuronal injury and death, and are also able to phagocytose degradation products and small molecules.20 Interestingly, these macrophages have been shown to play a protective role during inflammatory conditions by facilitating the influx of inflammatory leukocytes across the blood–brain barrier.19 Similarly, soon after an infection with the simian immunodeficiency virus, perivascular macrophages accumulated around CNS vessels.21 For the scleral perivascular macrophages, all of this is currently unknown. 
While isolated scleral diseases are rare, still some are able to rapidly destroy the eye, as is the case in necrotizing scleritis. Even in milder cases without necrosis, other complications are frequent and include uveitis, peripheral keratitis, cataract, and glaucoma. Therefore, a better understanding of the sclera and the physiological presence of immune cells is needed that might improve therapies for diseases like scleritis or associated complications such as glaucoma. Other examples are ocular tumors, such as uveal or conjunctival melanoma. Both can infiltrate the sclera, but the involvement of scleral immune cells in tumor defense or in metastasis is not understood.2225 
This study aimed to further characterize the status of human scleral immune cells by using a broad panel of immunohistochemical markers. The macrophage markers CD68, CD163, and CD11b were used in double or triple staining with other markers. 
Major histocompatibility complex class II (MHCII) was used to identify professional APCs. C-C chemokine receptor 7 (CCR7) is expressed on leukocytes and regulates the homing to lymph nodes.26 With regard to macrophages, it has been shown that macrophages that coexpress CCR7 and CD68 can be assigned to the “proinflammatory” M1 subset, whereas CD68+ CD163+ macrophages most likely belong to the more “anti-inflammatory” M2 subset.2729 C-C chemokine receptor 2 (CCR2) is expressed on recruited inflammatory phagocytes. It can also be expressed by macrophages, as has been demonstrated in atherosclerotic plaques,30 and was used here to exclude an atherosclerotic macrophage enhancement around blood vessels. 
The use of CD45 identified leukocytes and a potential bone marrow–derived origin.31,32 C-X-C-motif chemokine ligand 2 (CXCL12) plays an important role in the regulation of cellular functions, such as migration, proliferation, survival, and angiogenesis33 but represents also, among others, a marker for endothelial progenitor cells. Glial fibrillary acidic protein (GFAP) was used to help differentiate macrophages from astrocytes.34 CD11c was used in combination with MHCII to detect dendritic cells.35,36 
With this large marker panel, we aimed to further characterize the episcleral and stromal immune cells to get more information about their activation levels and draw conclusions about their functions. 
Materials and Methods
Human Globe Donors
Human sclera was obtained from the Eye Bank of the Department of Ophthalmology, University of Cologne, Germany, in accordance with the Declaration of Helsinki and with approval of the local Ethics Committee and had approval for scientific examination (n = 10; mean age 63.1 ± 11.3 years, four male and six female, maximum postmortem time 24 hours). Specimens in this study derived from eyes showing no pathologic alterations as revealed by slit-lamp examination and fundoscopy, and further available clinical records did not show any history of eye diseases. 
Confocal Microscopy of Scleral Tissue
From each of the 10 globes, 10 to 15 samples from the episclera and 10 to 15 samples from the stroma were taken to analyze all antibodies mentioned in the Table for the double staining and to perform negative controls. Immunohistochemistry and confocal microscopy were performed as previously described.4,37 Briefly, scleral samples were fixated in 96% ethanol (Merck Chemicals, Darmstadt, Germany) for 15 minutes and transferred into 15% sucrose in phosphate-buffered saline (PBS) for 24 to 36 hours. The globes were then frozen in liquid nitrogen and kept at −20°C until further use. At the anterior scleral margin, defined by the corneoscleral trepanation, 1-cm2-sized tissue samples were cut using scissors. Recent data indicated that the highest amounts of blood vessels and cells are found in this area.4 The samples were laminated by cutting thin horizontal layers using a scalpel.37 Nonspecific binding was blocked by incubation in PBS-containing 5% calf serum (1 hour, room temperature) and the primary antibodies were added (Table). The primary antibodies were incubated overnight at 4°C. Samples were then washed three times for 5 minutes with PBS using a shaking device. Primary antibodies were detected with corresponding fluorescent-labeled secondary antibodies (2 hours at 20°C) (Table), diluted in PBS containing 2% normal goat serum (Dako, Glostrup, Denmark) to avoid nonspecific binding. Due to the autofluorescence of the scleral collagen especially in the green channel, secondary antibodies like fluorescein isothiocyanate (FITC) or 488 were avoided when possible. 
Table
 
Antibodies Used in This Study
Table
 
Antibodies Used in This Study
Samples were rinsed again three times for 5 minutes on the shaker, then incubated with 4′,6-diamidino-2-phenylindole (DAPI) diluted 1:2000 in PBS (Carl Roth, Karlsruhe, Germany; 10 minutes, 20°C), washed again, embedded in fluorescent mounting medium (Dako), and stored at 4°C. Slides were examined using a confocal microscope (LSM Meta 510 BX53; Carl Zeiss AG, Jena, Germany) with ×10, ×20, and ×40 objective lenses. Negative controls were included in the analysis by omission of the primary antibodies and resulted in the absence of immunoreactivity. 
Immune cells were counted in 0.2025-mm2-sized confocal images of anterior episclera or stroma (this corresponds to a picture of a ×20 objective lens). The selected location was in the area with the highest cell density and within the episclera around blood vessels. Blood vessels were detectable in the DAPI staining by enhancement of multiple nuclei in a vessel shape. Three groups were defined, depending on the amount of counted cells: (1) high group (mean ≥ 40 cells/mm2), (2) medium group (mean ≥ 20 to < 40 cells/mm2), and (3) low group (mean < 20 cells/mm2). 
Statistical Analyses
Statistical analysis was performed using Prism 6, V.6.02 (GraphPad Software, San Diego, CA, USA). For the differences between stromal and episcleral cell amounts, a 2-way ANOVA was used. P values < 0.05 were considered statistically significant. 
Results
Healthy Human Episclera Contains CD68+, CD45+, MHCII+, and CCR7+ Cells
Episcleral samples were immunohistochemically analyzed for a panel of various immune cell markers (Fig. 1). In the high group (mean ≥ 40 cells/mm2), cells displayed immunoreactivity for CD68, CD45, MHCII, CCR7, LYVE1, and CD11b. In the low group (mean < 20 cells/mm2) CXCL12+, CCR2+, and GFAP+ cells were detected. Medium cell numbers (mean > 20 cells < 40/mm2) were not detected here. 
Figure 1
 
Examples of images of episcleral samples analyzed for a panel of markers are shown: The healthy episclera contains high numbers of CD68+ (A), CD45+ (B), CCR7+ (C), and MHCII+ (D) cells. Medium numbers were found for LYVE1+ (E) and CD11b+ (F) cells. Only single CXCL12+ (G), CCR2+ (H), or GFAP+ (I) cells were detectable. Nucleus staining was performed with DAPI and is shown in blue. Scale bars: 50 μm.
Figure 1
 
Examples of images of episcleral samples analyzed for a panel of markers are shown: The healthy episclera contains high numbers of CD68+ (A), CD45+ (B), CCR7+ (C), and MHCII+ (D) cells. Medium numbers were found for LYVE1+ (E) and CD11b+ (F) cells. Only single CXCL12+ (G), CCR2+ (H), or GFAP+ (I) cells were detectable. Nucleus staining was performed with DAPI and is shown in blue. Scale bars: 50 μm.
In general, the highest expression of all tested markers was found for CD68. All analyzed episcleral tissue samples contained high numbers of CD68+ macrophages (mean 74 ± 18.9 cells/mm2). Similarly high cell numbers were detected when examining CD45 (mean 58 ± 35.6 cells/mm2), MHCII (mean 69 ± 35.4 cells/mm2), and CCR7 (mean 59 ± 50.7 cells/mm2). CCR7 was present at strong levels in 6 out of 10 samples, while it was almost absent in 4 out of 10 samples. Lower cell numbers were seen for LYVE1 (mean 47 ± 28.9 cells/mm2) and CD11b (mean 45 ± 49.7 cells/mm2). 
Only single cells were positive for CXCL12 (mean 13 ± 12.5 cells/mm2), CCR2 (mean 11 ± 11.0 cells/mm2), and GFAP (mean 8 ± 8.9 cells/mm2). 
Healthy Human Scleral Stroma Contains MHCII+ Cells and CD68+ Macrophages
We next analyzed scleral stroma samples for expression of the above-mentioned marker panel (Fig. 2). Most cells here were CD68 positive (mean 40 ± 16.8 cells/mm2), CD45 positive (mean 48 ± 35.8 cells/mm2) (both high amount group), and MHCII positive (mean 31 ± 18.2 cells/mm2) (medium amount group [mean ≥ 20 to < 40 cells/mm2]). These cell markers were detected in all analyzed samples. MHCII+ cells were elongated and in some areas were attached to each other, forming lines (Fig. 2B). 
Figure 2
 
The healthy scleral stroma contains medium numbers of MHCII+ cells, CD45+ cells, and CD68+ macrophages. Representative pictures of CD45 (A), MHCII (B), and CD68 (C) immunoreactive cells are shown. Note the linear arrangement of several MHCII+ cells (B). Nucleus staining was performed with DAPI and is shown in blue. Scale bars: 25 μm.
Figure 2
 
The healthy scleral stroma contains medium numbers of MHCII+ cells, CD45+ cells, and CD68+ macrophages. Representative pictures of CD45 (A), MHCII (B), and CD68 (C) immunoreactive cells are shown. Note the linear arrangement of several MHCII+ cells (B). Nucleus staining was performed with DAPI and is shown in blue. Scale bars: 25 μm.
In the low number group the following cells were detected: Only single cells in some samples were positive for LYVE1 (mean 17 ± 17.0 cells/mm2), CCR7 (mean 12 ± 12.3 cells/mm2), CD11b (mean 7 ± 12.5 cells/mm2), CXCL12 (mean 11 ± 11.0 cells/mm2), CCR2 (mean 13 ± 14.8 cells/mm2), and GFAP (mean 2 ± 3.3 cells/mm2). 
In the Episclera, the Relative Amount of Immune Cells Is Higher Compared to the Scleral Stroma
Comparing the number of immune cells in the scleral stroma and episclera, significantly fewer cells were found in the stroma (P = 0.008) (Fig. 3). The only cell type that was slightly more present in the stroma as compared to the episclera was CCR2+ cells, whereby the amount was very low in both tissue areas and the difference was not significant. 
Figure 3
 
Episcleral and stromal immune cells. Quantification of (A) episcleral and (B) stromal cells revealed that in general the amount of cells was significantly higher in the episclera compared to the stroma (P = 0.008). In both groups CD68, CD45, and MHCII were the most frequent cells. The following means were detectable in the episclera: CD68+ (74.1 cells/mm2), MHCII+ (68.7 cells/mm2), CD45+ (57.8 cells/mm2), CCR7+ (58.8 cells/mm2), LYVE1+ (46.9 cells/mm2), CD11b+ (45.5 cells/mm2), CXCL12+ (12.8 cells/mm2), CCR2+ (10.9 cells/mm2), and GFAP+ (7.9 cells/mm2). In the stroma the number of cells was lower, with the following mean values: CD68+  (39.5 cells/mm2, equivalent to a decrease of 47% compared to the episclera), MHCII+ (30.6 cells/mm2, decrease of 55%), CD45+ (48.4 cells/mm2; decrease of 24%), CCR7+ (12.4 cells/mm2, decrease of 79%), LYVE1+ (17.3 cells/mm2, decrease of 64%), CD11b+ (7.4 cells/mm2, decrease of 84%), CXCL12+ (11.4 cells/mm2, decrease of 11%), CCR2+ (13.3 cells/mm2, increase of 12% compared to the episclera), and GFAP+ (2.5 cells/mm2, decrease of 68%). A total of 10 scleral samples were analyzed for the marker panel in episclera and stroma, and a 2-way ANOVA was performed to analyze for statistical differences. A P value < 0.05 was considered statistically significant.
Figure 3
 
Episcleral and stromal immune cells. Quantification of (A) episcleral and (B) stromal cells revealed that in general the amount of cells was significantly higher in the episclera compared to the stroma (P = 0.008). In both groups CD68, CD45, and MHCII were the most frequent cells. The following means were detectable in the episclera: CD68+ (74.1 cells/mm2), MHCII+ (68.7 cells/mm2), CD45+ (57.8 cells/mm2), CCR7+ (58.8 cells/mm2), LYVE1+ (46.9 cells/mm2), CD11b+ (45.5 cells/mm2), CXCL12+ (12.8 cells/mm2), CCR2+ (10.9 cells/mm2), and GFAP+ (7.9 cells/mm2). In the stroma the number of cells was lower, with the following mean values: CD68+  (39.5 cells/mm2, equivalent to a decrease of 47% compared to the episclera), MHCII+ (30.6 cells/mm2, decrease of 55%), CD45+ (48.4 cells/mm2; decrease of 24%), CCR7+ (12.4 cells/mm2, decrease of 79%), LYVE1+ (17.3 cells/mm2, decrease of 64%), CD11b+ (7.4 cells/mm2, decrease of 84%), CXCL12+ (11.4 cells/mm2, decrease of 11%), CCR2+ (13.3 cells/mm2, increase of 12% compared to the episclera), and GFAP+ (2.5 cells/mm2, decrease of 68%). A total of 10 scleral samples were analyzed for the marker panel in episclera and stroma, and a 2-way ANOVA was performed to analyze for statistical differences. A P value < 0.05 was considered statistically significant.
Healthy Human Scleral Macrophages Are Positive for CCR7, MHCII, and CD45; Single MHCII Cells Were Double Positive for CXCL12
Next, we analyzed whether these immune cells showed simultaneous expression of markers. For that, the macrophage markers CD11b or CD68 were analyzed in combination with other markers. In the episclera, almost all MHCII-positive cells were also CD45 positive (Figs. 4A–C). Several MHCII+ cells also displayed CCR7 expression (Figs. 4D–F). The CD11b-positive macrophages expressed MHCII (Figs. 4G–I). While the majority of MHCII+ cells were lacking CXCL12-immunoreactivity, singular MHCII+ cells displayed CXCL12 (Figs. 4J–L). 
Figure 4
 
Almost all (AC) MHCII+ cells were positive for CD45. MHCII-positive cells colocalized for CCR7 (DF) and CD11b (GI). White arrowheads exemplify double-positive cells, and yellow arrows exemplify single-positive cells. Single cells were immunoreactive for CXCL12 and MHCII (JL). The majority of MHCII+ cells were negative for CXCL12. Scale bars: 50 μm.
Figure 4
 
Almost all (AC) MHCII+ cells were positive for CD45. MHCII-positive cells colocalized for CCR7 (DF) and CD11b (GI). White arrowheads exemplify double-positive cells, and yellow arrows exemplify single-positive cells. Single cells were immunoreactive for CXCL12 and MHCII (JL). The majority of MHCII+ cells were negative for CXCL12. Scale bars: 50 μm.
Immune-Activating Potential of the Sclera: The Human Sclera Contains Several CD11c+ MHCII+ Dendritic Cells, and the Macrophages Are Most Likely Derived From the M1 Type
The samples were analyzed for the coexpression of dendritic cell markers CD11c and MHCII and to further characterize M1 and M2 macrophage markers for the coexpression of CD163 and CD68 (Fig. 5). Several CD11c+ MHCII+ dendritic cells were detectable in the episclera (Fig. 5A) and very low amounts in the stroma (Fig. 5B). When analyzing simultaneous expression of CD68 and CD163, CD163 was mainly negative in the episclera (Fig. 5C) and stroma (Fig. 5D), indicating CD68+ CD163−, most likely M1 macrophages in the sclera. 
Figure 5
 
Detection of CD11c+ MHCII+ dendritic cells in the sclera: Several CD11c+ MHCII+ cells (gray arrowheads) were detectable in the episclera (A), and very few CD11c+ MHCII+ cells (white arrows) in (B) were seen in the scleral stroma. To differentiate M1 and M2 macrophages, simultaneous detection of CD68 and CD163 was performed. CD163 were mainly negative in the episclera (C) and stroma (D), indicating most likely M1 macrophages (CD68+ CD163−, gray arrows in [D]) in the human sclera.
Figure 5
 
Detection of CD11c+ MHCII+ dendritic cells in the sclera: Several CD11c+ MHCII+ cells (gray arrowheads) were detectable in the episclera (A), and very few CD11c+ MHCII+ cells (white arrows) in (B) were seen in the scleral stroma. To differentiate M1 and M2 macrophages, simultaneous detection of CD68 and CD163 was performed. CD163 were mainly negative in the episclera (C) and stroma (D), indicating most likely M1 macrophages (CD68+ CD163−, gray arrows in [D]) in the human sclera.
Discussion
The sclera is the main covering and stabilizing layer of the eye. This study aimed to characterize the immunophenotype of scleral cells and therefore identify potential immunologic functions. Our results indicate that the episclera and to a much lesser extent also the scleral stroma contain numerous APCs such as CD11c+/MHCII+ dendritic cells and CD11b+/ MHCII+ macrophages, which are yolk sac or bone marrow derived (CD45+).32 Furthermore, CCR7+ MHCII+ macrophages were detected in healthy episclera, although not in all individuals. 
Only very few studies have investigated human scleral immune cells so far: One group suggests that scleral fibroblasts usually do not express MHC II, but are able to express it when cocultured with recombinant human gamma-interferon in vitro.38 Our results support these findings, as MHCII was mainly expressed by CD11b+ macrophages. In mice, macrophages and dendritic cells have been detected in the sclera.39 
Flugel et al.40 showed that MHCII+ CD45+ cells can be found in the human trabecular meshwork, ciliary meshwork, Schlemm's channel, and iris and in the vicinity of scleral vessels. When comparing our results with studies of other ocular tissues, single CCR7+ cells were obtained in human iris.41 In the cornea, it has been shown that CCR7+ cells are found in inflamed corneal stroma in mice.42 In our study, most of the CCR7+ cells were macrophages. The CCR7+ cells could be proinflammatory M1 macrophages. The reason for this activation is not clear. It might be due to systemic influences that secondarily influence the perivascular episcleral cells or due to prolonged postmortem times that led to hypoxia and cell stress. 
In the sclera, MHC II+ CD45+ macrophages accumulate around blood vessels. This is different compared to, for example, the dermis, where T cells or dendritic cells, but not macrophages, surround blood vessels.43 Similar perivascular macrophages have been found in the CNS. Here, perivascular macrophages are well known, and they have important functions in normal CNS function44 as well as in inflammatory diseases.4547 Although scleritis and encephalitis are not connected, there are some similarities between scleral and cerebral perivascular macrophages: They are both CD45+, they have antigen-presenting functions, and are mainly located around blood vessels. In the CNS, these perivascular cells are known to connect the CNS with the peripheral immune system.20 In cases of inflammation, they migrate from the periphery; they respond to neuronal cell damage but also to cytokines from the peripheral blood.20,48 This would also be a possible option in the sclera, which provides the outer border of the immune-privileged inner eye. As the sclera has perforating vessels, these cells might be involved in buoying the intraocular immune privilege. 
In pathologic situations, episcleritis or scleritis is often associated with blood vessel involvement, leading to vasculitis and/or thrombosis.49 The role of the macrophages and dendritic cells during the disease is not well understood, but due to their large numbers as detected via immunohistochemistry50 in necrotizing scleritis, one group suggests that CD68+ macrophages might play an important role in this disease. Bernauer et al.51,52 predominantly found T- and B-cells in cases of scleritis. B-cells were mainly located around blood vessels. Fong et al.49 found vascular immunodeposits in 93% of scleral biopsies from patients with necrotizing scleritis.49 It therefore seems likely that the physiologically existing macrophages are deregulated in scleritis, attracting lymphocytes, accumulating around blood vessels, and contributing to autoimmunity, similar to the above-mentioned CNS macrophages. Again, further studies are needed to better understand the role of macrophages in this process, but are currently not feasible to carry out due to the lack of availability of human tissues with active scleritis. 
In summary, this study shows that the sclera is not an acellular tissue containing only fibroblasts, but is full of APCs including dendritic cells and macrophages. These cells are potentially involved in physiological processes in the sclera, such as immune defense or maintaining blood vessel homeostasis. This work is the basis for further studies analyzing the human sclera in pathologic situations, where the macrophage quantity or activation status might be different. 
Acknowledgments
The authors thank Günther Simons and Sabine Hackbart from the Eye Bank of the University of Cologne and Jennifer Austin (University of Cologne, Department of Ophthalmology) for their support and Thomas Langmann and Maria Notara (University of Cologne, Department of Ophthalmology) for helpful scientific discussions. We thank the members of the imaging facility at the Cologne Cluster of Excellence in Cellular Stress Responses in Aging-associated Dieseases (CECAD) for great technical support. 
Supported by German Research Foundation (FOR 2240 (Lymph)Angiogenesis and Cellular Immunity in Inflammatory Diseases of the Eye to CC and LMH); HE 6743/2-1 and HE 6743/3-1 to LMH; CU 47/6-1, Cu 47/9-1, Cu 47/12-1 to CC; German Cancer Aid (to LMH and CC); GEROK Program University of Cologne (to SLS and LMH); EU COST BM1302 Joining Forces in Corneal Regeneration (to CC); Research Fund of the Paracelsus Medical University (PMU-FFF R15_05_067-KAS to FS). 
Disclosure: S.L. Schlereth, None; S. Kremers, None; F. Schrödl, None; C. Cursiefen, None; L.M. Heindl, None 
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Figure 1
 
Examples of images of episcleral samples analyzed for a panel of markers are shown: The healthy episclera contains high numbers of CD68+ (A), CD45+ (B), CCR7+ (C), and MHCII+ (D) cells. Medium numbers were found for LYVE1+ (E) and CD11b+ (F) cells. Only single CXCL12+ (G), CCR2+ (H), or GFAP+ (I) cells were detectable. Nucleus staining was performed with DAPI and is shown in blue. Scale bars: 50 μm.
Figure 1
 
Examples of images of episcleral samples analyzed for a panel of markers are shown: The healthy episclera contains high numbers of CD68+ (A), CD45+ (B), CCR7+ (C), and MHCII+ (D) cells. Medium numbers were found for LYVE1+ (E) and CD11b+ (F) cells. Only single CXCL12+ (G), CCR2+ (H), or GFAP+ (I) cells were detectable. Nucleus staining was performed with DAPI and is shown in blue. Scale bars: 50 μm.
Figure 2
 
The healthy scleral stroma contains medium numbers of MHCII+ cells, CD45+ cells, and CD68+ macrophages. Representative pictures of CD45 (A), MHCII (B), and CD68 (C) immunoreactive cells are shown. Note the linear arrangement of several MHCII+ cells (B). Nucleus staining was performed with DAPI and is shown in blue. Scale bars: 25 μm.
Figure 2
 
The healthy scleral stroma contains medium numbers of MHCII+ cells, CD45+ cells, and CD68+ macrophages. Representative pictures of CD45 (A), MHCII (B), and CD68 (C) immunoreactive cells are shown. Note the linear arrangement of several MHCII+ cells (B). Nucleus staining was performed with DAPI and is shown in blue. Scale bars: 25 μm.
Figure 3
 
Episcleral and stromal immune cells. Quantification of (A) episcleral and (B) stromal cells revealed that in general the amount of cells was significantly higher in the episclera compared to the stroma (P = 0.008). In both groups CD68, CD45, and MHCII were the most frequent cells. The following means were detectable in the episclera: CD68+ (74.1 cells/mm2), MHCII+ (68.7 cells/mm2), CD45+ (57.8 cells/mm2), CCR7+ (58.8 cells/mm2), LYVE1+ (46.9 cells/mm2), CD11b+ (45.5 cells/mm2), CXCL12+ (12.8 cells/mm2), CCR2+ (10.9 cells/mm2), and GFAP+ (7.9 cells/mm2). In the stroma the number of cells was lower, with the following mean values: CD68+  (39.5 cells/mm2, equivalent to a decrease of 47% compared to the episclera), MHCII+ (30.6 cells/mm2, decrease of 55%), CD45+ (48.4 cells/mm2; decrease of 24%), CCR7+ (12.4 cells/mm2, decrease of 79%), LYVE1+ (17.3 cells/mm2, decrease of 64%), CD11b+ (7.4 cells/mm2, decrease of 84%), CXCL12+ (11.4 cells/mm2, decrease of 11%), CCR2+ (13.3 cells/mm2, increase of 12% compared to the episclera), and GFAP+ (2.5 cells/mm2, decrease of 68%). A total of 10 scleral samples were analyzed for the marker panel in episclera and stroma, and a 2-way ANOVA was performed to analyze for statistical differences. A P value < 0.05 was considered statistically significant.
Figure 3
 
Episcleral and stromal immune cells. Quantification of (A) episcleral and (B) stromal cells revealed that in general the amount of cells was significantly higher in the episclera compared to the stroma (P = 0.008). In both groups CD68, CD45, and MHCII were the most frequent cells. The following means were detectable in the episclera: CD68+ (74.1 cells/mm2), MHCII+ (68.7 cells/mm2), CD45+ (57.8 cells/mm2), CCR7+ (58.8 cells/mm2), LYVE1+ (46.9 cells/mm2), CD11b+ (45.5 cells/mm2), CXCL12+ (12.8 cells/mm2), CCR2+ (10.9 cells/mm2), and GFAP+ (7.9 cells/mm2). In the stroma the number of cells was lower, with the following mean values: CD68+  (39.5 cells/mm2, equivalent to a decrease of 47% compared to the episclera), MHCII+ (30.6 cells/mm2, decrease of 55%), CD45+ (48.4 cells/mm2; decrease of 24%), CCR7+ (12.4 cells/mm2, decrease of 79%), LYVE1+ (17.3 cells/mm2, decrease of 64%), CD11b+ (7.4 cells/mm2, decrease of 84%), CXCL12+ (11.4 cells/mm2, decrease of 11%), CCR2+ (13.3 cells/mm2, increase of 12% compared to the episclera), and GFAP+ (2.5 cells/mm2, decrease of 68%). A total of 10 scleral samples were analyzed for the marker panel in episclera and stroma, and a 2-way ANOVA was performed to analyze for statistical differences. A P value < 0.05 was considered statistically significant.
Figure 4
 
Almost all (AC) MHCII+ cells were positive for CD45. MHCII-positive cells colocalized for CCR7 (DF) and CD11b (GI). White arrowheads exemplify double-positive cells, and yellow arrows exemplify single-positive cells. Single cells were immunoreactive for CXCL12 and MHCII (JL). The majority of MHCII+ cells were negative for CXCL12. Scale bars: 50 μm.
Figure 4
 
Almost all (AC) MHCII+ cells were positive for CD45. MHCII-positive cells colocalized for CCR7 (DF) and CD11b (GI). White arrowheads exemplify double-positive cells, and yellow arrows exemplify single-positive cells. Single cells were immunoreactive for CXCL12 and MHCII (JL). The majority of MHCII+ cells were negative for CXCL12. Scale bars: 50 μm.
Figure 5
 
Detection of CD11c+ MHCII+ dendritic cells in the sclera: Several CD11c+ MHCII+ cells (gray arrowheads) were detectable in the episclera (A), and very few CD11c+ MHCII+ cells (white arrows) in (B) were seen in the scleral stroma. To differentiate M1 and M2 macrophages, simultaneous detection of CD68 and CD163 was performed. CD163 were mainly negative in the episclera (C) and stroma (D), indicating most likely M1 macrophages (CD68+ CD163−, gray arrows in [D]) in the human sclera.
Figure 5
 
Detection of CD11c+ MHCII+ dendritic cells in the sclera: Several CD11c+ MHCII+ cells (gray arrowheads) were detectable in the episclera (A), and very few CD11c+ MHCII+ cells (white arrows) in (B) were seen in the scleral stroma. To differentiate M1 and M2 macrophages, simultaneous detection of CD68 and CD163 was performed. CD163 were mainly negative in the episclera (C) and stroma (D), indicating most likely M1 macrophages (CD68+ CD163−, gray arrows in [D]) in the human sclera.
Table
 
Antibodies Used in This Study
Table
 
Antibodies Used in This Study
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