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.
4–7 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.
10–14
Perivascular macrophages have been shown in the brain, where they play distinct roles in normal central nervous system (CNS) functions
15,16 and in various inflammatory diseases.
17–19 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.
22–25
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.
27–29 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 angiogenesis
33 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.
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.
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-cm
2-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).
Episcleral samples were immunohistochemically analyzed for a panel of various immune cell markers (
Fig. 1). In the high group (mean ≥ 40 cells/mm
2), cells displayed immunoreactivity for CD68, CD45, MHCII, CCR7, LYVE1, and CD11b. In the low group (mean < 20 cells/mm
2) CXCL12+, CCR2+, and GFAP+ cells were detected. Medium cell numbers (mean > 20 cells < 40/mm
2) were not detected here.
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).
In the Episclera, the Relative Amount of Immune Cells Is Higher Compared to the Scleral Stroma
Healthy Human Scleral Macrophages Are Positive for CCR7, MHCII, and CD45; Single MHCII Cells Were Double Positive for CXCL12
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 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