December 2005
Volume 46, Issue 12
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Cornea  |   December 2005
Distinct Populations of Dendritic Cells in the Normal Human Donor Corneal Epithelium
Author Affiliations
  • Satoru Yamagami
    From the Department of Corneal Tissue Regeneration and
  • Seiichi Yokoo
    From the Department of Corneal Tissue Regeneration and
  • Tomohiko Usui
    Department of Ophthalmology, University of Tokyo Graduate School of Medicine, Tokyo, Japan;
  • Hiroko Yamagami
    Department of Ophthalmology, Jichi Medical School Omiya, Saitama, Japan; and
  • Shiro Amano
    Department of Ophthalmology, University of Tokyo Graduate School of Medicine, Tokyo, Japan;
  • Nobuyuki Ebihara
    Department of Ophthalmology, Juntendo University School of Medicine, Tokyo, Japan.
Investigative Ophthalmology & Visual Science December 2005, Vol.46, 4489-4494. doi:10.1167/iovs.05-0054
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      Satoru Yamagami, Seiichi Yokoo, Tomohiko Usui, Hiroko Yamagami, Shiro Amano, Nobuyuki Ebihara; Distinct Populations of Dendritic Cells in the Normal Human Donor Corneal Epithelium. Invest. Ophthalmol. Vis. Sci. 2005;46(12):4489-4494. doi: 10.1167/iovs.05-0054.

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

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Abstract

purpose. To characterize dendritic cells (DC) in normal human corneal epithelium.

methods. Normal human donor corneal epithelium was examined by fluorescence microscopy with single and double staining for multiple markers. Morphologic studies were also performed by confocal microscopy. HLA-DRa, CD1c, and CD16 mRNA expression in the corneal epithelium was examined by RT-PCR. CD45+ cells were separated from the corneal epithelium with magnetic beads and then were stimulated with TNF-α and lipopolysaccharide in vitro.

results. CD45+ cells were mainly located in the basal-cell layer of the corneal epithelium and partly in the wing/surface layers. CD45-positive cell numbers were significantly higher in the peripheral cornea (3–6 mm from the center) than in the central cornea (0–3 mm from the center). All these cells expressed HLA-DR and CD11c but not CD3, CD11b, CD14, CD19, CD56, or CD66, suggesting that these were bone marrow–derived myeloid DC. Some DR+CD11c+ DCs from the periphery expressed CD1c and CD16. HLA-DRa, CD1c, and CD16 mRNAs were detected in normal corneal epithelium. These CD11c+ DCs did not express CD123, CD1a, DC marker (CMRF56), CD40, CD80, or CD86. When CD45+ cells were isolated from the corneal epithelium by magnetic cell sorting, CD40 and CD86 expression were detected after in vitro stimulation with TNF-α and lipopolysaccharide.

conclusions. These findings demonstrate that normal human corneal epithelium contains at least three DC phenotypes, with HLA-DR+ myeloid lineage CD11c+CD16- DCs as the main population plus a small number of CD11c+CD16+ DCs and CD11c+CD1c+ DCs. These cells can be discriminated from bone marrow–derived cells in the human corneal stroma.

Dendritic cells (DC) mediate the transport of antigens from sites of peripheral inflammation to lymphoid organs and then present the antigens to naïve T cells. 1 2 Immature DCs are endocytic and thus can capture antigens. They become mature DCs after stimulation with lipopolysaccharide (LPS), other pathogen-derived substances, and inflammatory cytokines, resulting in the upregulation of CD40 and CD86. 3 4 5 Although macrophages are also professional antigen-presenting cells (APC) in the immune system, DCs are much more potent at initiating and expanding the secondary immune responses than are macrophages. 6 DCs share the features of MHC class II expression and an absence of lineage-specific markers CD3, CD14, CD56, and CD19 (Lin). 7 8 9 Three different pathways are proposed for DC maturation, i.e., a lymphoid-related pathway, myeloid-related DC pathway, and a CD1a+ Langerhans (LC)-type DC pathway that is generally assumed to be related to the myeloid lineage. 1 Various human DC subsets have also been revealed by studies with cell surface markers. 1 3 8 10 11  
For the identification of DC or LC in normal human corneal epithelium, a number of investigations have been performed by using the MHC class II HLA-DR antigen as a marker of DC or LC. 12 13 14 15 16 17 18 19 20 21 22 23 HLA-DR antigen-positive cells are reported to be present in the corneal epithelium, including the central cornea, 12 16 21 22 whereas other reports indicate that HLA-DR antigen-positive cells are only localized in the limbus or the peripheral corneal epithelium. 14 18 19 20 The localization of LC based on CD1a expression is also controversial. 12 24 25 Surprisingly, an MHC class II negative DC population was recently found in the mouse corneal epithelium by the examination of cell surface markers. 26 Characterization of leukocytes with cell surface markers, however, has not yet been done in the normal human corneal epithelium. 
In the present study, the epithelium of normal human donor corneas was examined by immunofluorescence studies using cross-sections and fluorescence microscopy, while the morphology of leukocytes was assessed using flat mounts and confocal microscopy. We tried to characterize the leukocytes based on recent advances in knowledge about cell surface markers for leukocyte subsets. 
Methods
Donor Human Corneas and Preparation for Immunohistochemistry
This study was conducted in accordance with the declaration of Helsinki. Corneas (donors aged 46–68 years) were obtained from the Rocky Mountain Lions’ Eye Bank at 3 to 5 days postmortem and were kept in solution (Optisol GS; Bausch & Lomb, Rochester, NY) in 4°C until use. For immunohistochemistry of cross-sections of whole corneas (n = 7), two pieces of sclera, 1-mm wide, were removed from locations on opposite sides of the cornea (180° apart) to determine the central area of the donor cornea. Then corneal tissue was embedded in optimal cutting temperature (OCT) compound and cut on a cryostat into sections 10-μm thick. Cell numbers in the cross-section were counted on both of the scleral defects. For flat-mount examination of the corneal epithelium by confocal microscopy, the epithelium was carefully removed from the corneal stroma by scraping the outer surface of the cornea (n = 4). The cross-sections of the cornea were air-dried, and immunostaining was done without fixation. Flat-mount examination of the epithelium was performed after fixation in acetone for 10 minutes, because no positive staining was detected after omitting fixation. The donor corneas, with removal of the epithelium, were not used in this study. 
Antibodies
The primary monoclonal antibodies (mAb) used for immunohistochemistry, were obtained from BD Biosciences (San Diego, CA) unless noted. 
Immunohistochemical Studies
Specimens were prepared without fixation for immunohistochemical studies under fluorescence microscopy. Frozen cross-sections (10 μm) were cut on a cryostat, air-dried for 10 minutes, and washed in PBS. The sections were blocked with anti-Fc receptor (FcR) blocker (Miltenyi Biotec, Bergisch Gladbach, Germany) and isotype-matched Ig for each antibody (5 μg/mL mouse IgG1, 5 μg/mL IgG2a, or 5 μg/mL IgG2b; DakoCytomation, Carpinteria, CA) diluted in PBS for 30 minutes to eliminate nonspecific staining. Then fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated mAbs or isotype-matched control antibodies (mouse IgG1, κ-FITC isotype control, MOPC-31C, mouse IgG2a, κ-FITC, G155 to 178, mouse IgG2b, κ-PE, 27 to 35) were applied for 30 minutes. Nonspecific staining of the corneal epithelium with FITC- or PE-conjugated isotype controls (IgG1, IgG2a, and IgG2b) was not detected after pretreatment with the anti-Fc FcR blocker and isotype-matched Ig for each antibody. The specimens were covered with mounting medium (Vector, Laboratories, Burlingame, CA) after four washes in PBS and were examined under a fluorescent microscope (model BH2-RFL-T3 or BX50; Olympus, Tokyo, Japan) and a confocal microscope (Leica TCS 4D; Lasertechnik, Heidelberg, Germany). Sections were stained with FITC-conjugated anti-CD45 mAb (HI30) with an antifading mounting medium containing propidium iodide (PI; Vectashield; Vector Laboratories), and a coverslip was placed. All staining procedures were done at room temperature. The central area of the cornea was defined as the region within 3 mm from the corneal center. The periphery was defined as being within a 3- to 6-mm radius from the center. Each area was determined by marking the surface of the coverslip with a measure and a marker. The central and peripheral areas of each cornea were assessed separately. The percentage of CD45-positive cells in each corneal stromal area was determined by counting CD45-positive cells among the PI-positive cells in four sections. The average CD45-positive cell count was calculated for five different corneas. CD45-positive cell numbers in the center and the periphery were determined as the average number of CD45-positive cells in four areas each. Cell counts in the limbal epithelium were not examined because the exact limbal epithelial area could not be specified on the cross-sections. In the flat-mount confocal microscope study, corneas from different donors were cut into quarters for examination. 
Isolation of CD45-Positive Human Corneal Epithelial Cells
For isolation of corneal epithelial cells, the peripheral cornea (including the limbal region) was dissected away from the stroma to avoid possible contamination by limbal epithelial cells. The dissected epithelium was incubated overnight at 37°C in serum-free basal medium containing trypsin/EDTA (Sigma-Aldrich, St. Louis, MO). After washing 3 times with PBS, single cells were dissociated by trituration with a fire-polished Pasteur pipette. Then CD45-positive cells were positively isolated (Magnetic Activated Cell Sorter; Miltenyi Biotec) according to the manufacturer’s instructions. For separation of CD45-positive and CD45-negative cells, 3 to 5 corneal epithelial specimens were processed together and used for cell culture. To determine leukocyte number per corneal epithelium, corneal epithelial specimen was processed separately (n = 5), and the isolated cells with magnetic beads were applied to poly-l-lysine-coated glass slides and stained with PE-conjugated anti-CD11c mAb (B-ly6). 
RNA Preparation and RT-PCR
Total RNA was isolated from corneal epithelial cells using reagent (Isogen; Nippon Gene, Tokyo, Japan) according to the manufacturer’s instructions (n = 5). Water was used as the negative control. First-strand cDNA was synthesized from the total RNA (Reverse Transcription System; Promega Corp., Tokyo, Japan). The PCR reaction mixture comprised 1% cDNA, 10 mM Tris-Cl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTPs, 20 pmol oligonucleotides, and 2.5 U DNA polymerase (AmpliTaq Gold; Perkin Elmer, Wellesley, MA) in a total volume of 50 μL. After incubation at 95°C for 9 minutes, amplification was performed at 94°C for 30 seconds, and then at 60°C for 30 seconds (iCycler; Bio Rad Laboratories, Hercules, CA). Samples were separated on 2% agarose gel, and the products were detected with ethidium bromide staining. The primer sequences and estimated product sizes are listed in Table 1 . The PCR primer pair was selected to discriminate between cDNA and genomic DNA by using primers specific for different exons. 
In Vitro Stimulation of CD45-Positive and CD45-Negative Cells
The isolated CD45-positive cells were cultured with Dulbecco’s modified Eagle’s medium (DMEM) containing 1% FCS (Basal medium group) or DMEM containing 1% FCS, TNF-α (20 ng/mL), and LPS (100 ng/mL) for 3 days (TNF-α and LPS group). Unstimulated (Unstimulated group) and cultured cells were diluted with PBS to obtain the optimal concentration for cell counting. The diluted cells were then applied to poly-l-lysine-coated glass slides and dried for 10 minutes. The glass slides were incubated in PBS containing collagenase (0.02%) for 3 hours at 37°C. After incubation with anti-FcR blocker and isotype-matched Ig (5 μg/mL mouse IgG1; DakoCytomation) diluted in PBS for 30 minutes to block nonspecific staining, the mAbs were applied for 30 minutes. Nonspecific staining with the FITC-conjugated isotype control (IgG1) was not detected after pretreatment with anti-FcR blocker and the isotype-matched Ig for each antibody. Cells were stained with PE-conjugated anti-CD11c mAb (B-ly6) and FITC-conjugated anti-CD40 mAb (5C3) or FITC-conjugated anti-CD86 mAb (FUN-1), and the double-positive cell rate was calculated. The specimens were covered with mounting medium and were examined under a fluorescent microscope. 
Statistical Analysis
In the CD40- and CD86-positive cell rates, one-way ANOVA and Fisher’s protected least significant difference post hoc test were used to compared band densities. 
Results
CD45-Positive Cells in the Epithelium of Normal Donor Corneas
Fluorescence microscopy of cross-sections of the corneal epithelium was performed with FITC-conjugated anti-CD45 (panleukocyte) mAb, and nuclear staining was done with PI. A small number of CD45-positive cells were seen in the basal-cell layerof the central cornea (Fig. 1A) . At the periphery, most CD45-positive cells were located in the basal-cell layer (Fig. 1B) , but some were in the wing and the surface cell layer of the epithelium. CD45-positive cells were also present in the limbal epithelium (Fig. 1C) . The mean number of CD45-positive cells in the center (0–3-mm radius from the center) and periphery (3–6 mm radius from the center) was 1.1 ± 0.7 and 8.6 ± 4.2 (n = 5), respectively. Significantly fewer CD45-positive cells were seen in the center than in the peripheral cornea (Wilcoxon test, P < 0.01). No apparent differences of cell population and distribution were detected between young and old donor corneas analyzed in this study. Confocal microscopy of flat-mount normal human corneal epithelium (n = 4) showed that approximately 70% of CD45-positive cells had DC-like dendrites (Fig. 1D) , and the other cells did not have evident dendrites. 
Characterization of CD45+ Cells with Various Markers
To investigate the characteristics of the CD45+ cells, we performed single staining of cross-sections with various markers, as well as double staining using each positive marker and CD45. Single staining of the cornea was positive with the mAbs for HLA-DRa (MHC class II, G46-6) and CD11c (B-ly6). As shown in Figure 2 , all CD45+ cells were stained by CD11c in both the central (Fig. 2A)and peripheral corneal epithelium. PE-conjugated CD11c+ cells were uniformly stained with FITC-conjugated anti-HLA-DR mAb (G46-6; Fig. 2B ) and FITC-conjugated anti-HLA-DR mAb (TAL.1B5; Chemicon International, Temecula, CA [not shown]). HLA-DRa mRNA was also detected in the corneal epithelium (Fig. 2C) . In contrast, no positive staining with the mAbs for CD3 (T cell, UCHT1), CD11b (monocyte/granulocyte, ICRF44), CD19 (B-cell, SJ25C1), CD56 (NK cell, B159), CD66 (granulocyte, B6.2/CD66), CD40 (5C3), CD80 (B7.1, L307.4), and CD86 (B7.2, FUN-1) was seen in the corneal epithelium (not shown). The epithelium was weakly positive for CD14 (monocyte or pre-DC, M5E2), but positive staining that overlapped CD45-positive cells was not detected. These findings meant that the target cells could not be T cells, monocytes, B cells, granulocytes, NK cells, or mature DCs. 
To characterize the CD45+DR+CD11c+ DCs further, we next stained the corneal epithelium with anti-CD123 (9F5), BDCA-3 (Miltenyi Biotec, AD5-14H12), CD16 (3G8), and CD1c mAbs (AD5-8E7), because Lin myeloid DCs can be classified by these cell surface markers. Some of the CD45+ (DR+CD11c+) cells in the peripheral and limbal epithelium expressed CD1c (Fig. 3A)and CD16 (Fig. 3B) , but not CD123, CD1a (HI149), or DC marker (CMRF-56). CD1c- and CD16- mRNAs were also detected in the corneal epithelium (Figs. 3A 3B) . CD1c- and CD16-positive DCs were not detected in the central area. The mean number of CD1c- and CD16-positive cells in the periphery (3–6 mm from the center) was 1.5 ± 1.2 and 1.8 ± 1.0 (n = 5), respectively. BDCA-3-positive staining was not assessed because of strong positive staining of all layers of the corneal epithelium. A Langerhans-type cell marker (CD1a-) and an activated DC marker (CMRF-56) were not identified in the corneal epithelium. 
CD40 and CD86 Expression after In Vitro Stimulation
After CD45 magnetic bead isolation, the leukocyte number per cornea was determined by cell counting by immunocytochemistry with PE conjugated anti-CD11c mAb. No positive stainings were observed with FITC-conjugated nonimmunized mouse IgG1Ab. Averaged CD11c-positive cell number per cornea was (1.8 ± 0.7) × 102 (n = 5). CD45-positive cells (Unstimulated group, n = 5) and cells that had been cultured with DMEM containing 1% FBS (Basal medium group, n = 5) and DMEM containing 1% FBS, TNF-α (20 ng/mL), and LPS (100 ng/mL) (TNF-α and LPS group, n = 5) for 3 days were immunostained using PE-conjugated anti-CD11c and FITC-conjugated anti-CD40 or CD86 mAbs. Anti-CD80 mAb was not used, because the CD80 expression level is extremely low in human DCs. 3 10 27 CD11c-positive cells in the Unstimulated group were not stained by the anti-CD40 and CD86 mAbs. CD40 (Fig. 4A) - and CD86 (Fig. 4B) -positive cell rates in the TNF-α and LPS group were significantly higher than those in Basal medium group (P < 0.001). Representative photographs in the TNF-α and LPS group were shown in the lower panels of Figures 4A(CD40 and CD11c) and 4B (CD86 and CD11c). 
Discussion
We identified HLA-DR+CD45+ CD11c+ cells that were CD3-CD11b-CD14-CD19- CD56-CD66- in normal human corneal epithelium, indicating that these were bone marrow (BM)-derived MHC class II positive myeloid DCs but not lymphoid DCs. The lack of expression of DC marker (CMRF-56), 28 CD40, and CD86 suggested that these cells were immature DCs. In the case of peripheral blood DCs, CD16, 29 CD1c, 10 30 and BDCA-3, 10 have been used as markers to subdivide the CD11c+ DC population. CD123-, CD123low, and CD123hi subpopulations are found in Lin preparations. 3 28 31 Human CD14-negative blood DCs can be separated into CD11c+CD16- DC, CD11c+CD16+ DC, CD123hi DC, CD1c+ DC, and BDCA-3+ DC with these DC-related markers. 30 CD1c and CD16 expression in the peripheral corneal epithelium implies that normal human corneal epithelium contains at least three types of myeloid lineage DCs: CD11c+CD16- DCs are the main population, while there is also a small number of CD11c+CD16+ DCs and CD11c+CD1c+ DCs. Although blood DC phenotypes have not been studied in mice yet, the fact that MHC class II negative DCs exist in the center of mouse corneal epithelium is different from the distribution of DCs in the human corneal epithelium. 26  
Our recent study demonstrated that the normal human corneal stroma contains HLA-DR+CD45+ CD11b+CD11c+CD14+ cells that are CD3- CD19- CD56- CD66- cells. 32 The cells in the corneal stroma and the epithelial cells targeted in this study are both of the BM-derived myeloid lineage but not the lymphoid lineage. CD11b- and CD14-negative DCs in the corneal epithelium are, however, different from CD11b- and CD14-positive cells. In the skin, CD1a+CD1c+CD11c+ DCs and CD1a-CD1c+CD11c+ DCs are distinct populations that are distributed in the epidermis and the dermis, respectively. 33 These DCs express a monocyte marker, CD11b, 27 indicating that LCs in the epidermis are phenotypically different from DCs in the corneal epithelium. In the upper airway mucosa (nasal mucosa), CD1a+LC-type DCs are localized in the mucosal epithelium, while CD11b+CD11c+ CD14+ monocyte lineage cells and a few CD1c+CD11c+ DCs are intermingled in the mucosal epithelium and the lamina propria. 27 In the cornea, the LC marker CD1a is totally negative, and CD11c+ myeloid DCs (including a small number of CD1c+ and CD16+ cells) in the epithelium and CD11b+CD14+ cells suggesting monocyte/macrophage lineage in the stroma 32 are separately distributed above and below Bowman’s membrane, respectively. These findings suggest that the microenvironment and the extent of exposure to pathogens may determine further diversification of the APC lineage and subsets of DCs in each tissue. Although the exact mechanisms that control the tissue-specific distribution of DC/monocyte are unknown, specific BM-derived cells may be distributed accurately to each tissue by following local chemokines, or, more likely, prototype cells may be differentiated into tissue-specific cells by the local effects of cytokines and chemical mediators. 
CD1 molecules bind lipids and facilitate T-cell receptor recognition of fatty acids, glycolipids, and lipopeptide antigens of foreign or self-origin. Four human CD1 isoforms (CD1a, -b, -c, and -d) are known to be antigen-presenting molecules with the unique ability to present lipid antigens to T cells. 34 Mature DCs are able to induce the maximum activation of peptide Ag-specific T cells, but DC maturation is an independent process that is not required for lipid Ag presentation by CD1, 35 suggesting a crucial role in the rapid response of host defenses. CD16, FcγRIII, was originally identified as a human NK-cell-associated antigen and is also expressed by DCs, monocytes/macrophages, and granulocytes. 30 CD16-positive DCs show higher phagocytic and oxidative activity than CD16-negative DCs, produce significant amounts of cytokines, 36 and have a marked ability to generate in vitro activation of naïve T cells. 30 Moreover, CD1c+ and CD16+ DCs are stronger stimulators of a response to allo-antigens in the mixed lymphocyte reaction compared with other BDCA-3 and CD123-positive DCs. 3 These findings suggest that CD1c- or CD16-positive DCs in the peripheral cornea may have a role in inflammation of the corneal epithelium as a potent initiator of the innate and adaptive protective immune systems. Conversely, stronger DCs may contribute to rapid allograft rejection and may explain the severity of the immune reaction in the prevascularized corneal bed. 
The existence of myeloid DC, including a central area of corneal epithelium, has important implications for corneal immunity. For example, it has been considered that normal cornea is devoid of a MHC-class-II–positive DC population and priming of T-cells after corneal transplantation depends on host derived APC. 37 38 Myeloid DCs in central cornea have potential to lead donor APC-dependent direct immune response, as described in a mouse corneal transplantation model. 39 40 Moreover, DCs recognize molecular signatures of potential pathogens via Toll-like receptors (TLR). TLRs have been identified as being part of a large family of pathogen-recognition receptors that play a decisive role in the induction of both innate and adaptive immunity. 41 DCs throughout corneal epithelium may represent an efficient system to prevent or to treat certain inflammatory disorders of microbial origin in the ocular surface. Furthermore, the increment of CD40 and CD86 expression after in vitro stimulation suggests that immature DCs in the corneal epithelium can become a mature form in response to inflammation such as bacterial or viral infection in vivo. 
In summary, we characterized MHC-class II–positive BM-derived myeloid lineage cells in the epithelium of normal donor corneas. These were divided into CD11c+CD16− DCs as the main population, as well as CD11c+CD16+ DCs and CD11c+CD1c+ DCs. When isolated DCs were stimulated in vitro, the cells expressed the surface markers of activated DCs. These cells can be discriminated from CD11b-positive bone marrow–derived cells in the normal human corneal stroma and may have a role as a first-line part of the innate and adaptive host immune responses in the human cornea. Further study is required for identification of critical factors to mediate recruitment and mobilization of DCs in the corneal epithelium as revealed in mouse cornea. 42  
 
Table 1.
 
Oligonucleotide Primers for RT-PCR
Table 1.
 
Oligonucleotide Primers for RT-PCR
Gene PCR Primers Product Size (bp)
GAPDH
5′-GGTGAAGGTCGGTGTGAACGGA-3′
5′-TGTTAGTGGGGTCTCGCTCCTG-3′ 223
HLA.DRa
5′-TCGAAATGGAAAACCTGTCACC-3′
5′-CCCAATAATGATGCCCACCA-3′ 267
CD1c
5′-TGGTGACAATGCAGACGCA-3′
5′-GGAGATGGCACCCATGTTGTA-3′ 414
CD16
5′-TTCATTGACGCTGCCACAGT-3′
5′-CCGCTATCTTTGAGTGTGGCTT-3′ 281
Figure 1.
 
Immunohistochemical staining of CD45+ cells in the epithelium of normal human donor corneas. Transverse cross-sections stained with anti-CD45 mAb were coverslipped using antifading mounting medium containing propidium iodide (PI; red) for nuclear staining. (A) Representative staining of CD45-positive cells (green) in the basal-cell layer of central corneal epithelium. CD45-positive cells are also present in the stroma. (B) In the peripheral epithelium, CD45-positive cells are located in the basal-cell layer, and some cells are in the wing cell layer of the epithelium (not shown). (C) In the limbal epithelium, CD45-positive cells are present in the basal-cell layer and wing-cell layer. (D) Confocal microscopy of a flat mount of the corneal epithelium shows that the CD45-positive cells are dendritic cells. Original magnification: (AC) ×100; (D) ×400.
Figure 1.
 
Immunohistochemical staining of CD45+ cells in the epithelium of normal human donor corneas. Transverse cross-sections stained with anti-CD45 mAb were coverslipped using antifading mounting medium containing propidium iodide (PI; red) for nuclear staining. (A) Representative staining of CD45-positive cells (green) in the basal-cell layer of central corneal epithelium. CD45-positive cells are also present in the stroma. (B) In the peripheral epithelium, CD45-positive cells are located in the basal-cell layer, and some cells are in the wing cell layer of the epithelium (not shown). (C) In the limbal epithelium, CD45-positive cells are present in the basal-cell layer and wing-cell layer. (D) Confocal microscopy of a flat mount of the corneal epithelium shows that the CD45-positive cells are dendritic cells. Original magnification: (AC) ×100; (D) ×400.
Figure 2.
 
Representative photomicrographs of CD45-positive cells stained with various markers. (A) In corneal cross-sections, all the CD45-positive cells (green) in the epithelium are stained with CD11c (red). Double-stained cells are observed as yellow cells (A, yellow). CD11b is universally detected in the corneal stroma but is totally negative here (not shown). The CD11c+ cells in the corneal epithelium are all HLA-DR+ (clone G46-6, green) (B, HLA-DR+: upper, merged: lower). Similar findings were observed with staining for HLA-DR+ (clone TAL.1B5) (not shown). Original magnification: (A, B) ×100. (C) GAPDH (endogenous control, 27 PCR cycles) and HLA-DRa (30 PCR cycles) mRNA was detected in the corneal epithelium. No PCR products are detected in the negative control sample.
Figure 2.
 
Representative photomicrographs of CD45-positive cells stained with various markers. (A) In corneal cross-sections, all the CD45-positive cells (green) in the epithelium are stained with CD11c (red). Double-stained cells are observed as yellow cells (A, yellow). CD11b is universally detected in the corneal stroma but is totally negative here (not shown). The CD11c+ cells in the corneal epithelium are all HLA-DR+ (clone G46-6, green) (B, HLA-DR+: upper, merged: lower). Similar findings were observed with staining for HLA-DR+ (clone TAL.1B5) (not shown). Original magnification: (A, B) ×100. (C) GAPDH (endogenous control, 27 PCR cycles) and HLA-DRa (30 PCR cycles) mRNA was detected in the corneal epithelium. No PCR products are detected in the negative control sample.
Figure 3.
 
CD1c- and CD16 expression in the corneal epithelium. (A) Among CD45-positive cells from the peripheral corneal epithelium, CD1c (red) cells are detected in the basal layer and wing/surface cell layers. Representative photomicrograph shows that CD1c-positive cells (red) are detected among CD45-positive cells (green) in the peripheral cornea. These CD1c and CD45-positive cells are yellow (double labeled). The CD1c gene was detected in the corneal epithelium by RT-PCR (35 cycles). (B) Among CD45-positive cells (green), some are stained with CD16- mAb (red-yellow). The CD16 gene was detected in the corneal epithelium by RT-PCR (35 cycles), but not in the negative control sample. Original magnification: (A, B) ×100.
Figure 3.
 
CD1c- and CD16 expression in the corneal epithelium. (A) Among CD45-positive cells from the peripheral corneal epithelium, CD1c (red) cells are detected in the basal layer and wing/surface cell layers. Representative photomicrograph shows that CD1c-positive cells (red) are detected among CD45-positive cells (green) in the peripheral cornea. These CD1c and CD45-positive cells are yellow (double labeled). The CD1c gene was detected in the corneal epithelium by RT-PCR (35 cycles). (B) Among CD45-positive cells (green), some are stained with CD16- mAb (red-yellow). The CD16 gene was detected in the corneal epithelium by RT-PCR (35 cycles), but not in the negative control sample. Original magnification: (A, B) ×100.
Figure 4.
 
CD40 and CD86 expression after in vitro stimulation. Magnetic bead-isolated CD45-positive cells (Unstimulated group), and cells stimulated for 3 days with DMEM containing 1% FBS (Basal medium group) and DMEM containing 1% FBS, TNF-α and LPS (TNF-α and LPS group) were immunostained using PE-conjugated anti-CD11c (red) and FITC-conjugated anti-CD40 or CD86 (green) mAbs. Unstimulated cells are not stained by anti-CD40 or CD86 mAbs. CD40 (A)- and CD86 (B)-positive cell rates in CD11c-positive cells in the TNF-α and LPS group are significantly high compared with those in the Basal medium group. Lower panels of A (CD40+CD11c+, yellow) and B (CD86+CD11c+, yellow) are representative photographs. Red cells are CD11c-single positive. Original magnification, ×200. *P < 0.001.
Figure 4.
 
CD40 and CD86 expression after in vitro stimulation. Magnetic bead-isolated CD45-positive cells (Unstimulated group), and cells stimulated for 3 days with DMEM containing 1% FBS (Basal medium group) and DMEM containing 1% FBS, TNF-α and LPS (TNF-α and LPS group) were immunostained using PE-conjugated anti-CD11c (red) and FITC-conjugated anti-CD40 or CD86 (green) mAbs. Unstimulated cells are not stained by anti-CD40 or CD86 mAbs. CD40 (A)- and CD86 (B)-positive cell rates in CD11c-positive cells in the TNF-α and LPS group are significantly high compared with those in the Basal medium group. Lower panels of A (CD40+CD11c+, yellow) and B (CD86+CD11c+, yellow) are representative photographs. Red cells are CD11c-single positive. Original magnification, ×200. *P < 0.001.
The authors thank Toshiya Osawa and Kayo Aoyama for excellent technical support. 
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Figure 1.
 
Immunohistochemical staining of CD45+ cells in the epithelium of normal human donor corneas. Transverse cross-sections stained with anti-CD45 mAb were coverslipped using antifading mounting medium containing propidium iodide (PI; red) for nuclear staining. (A) Representative staining of CD45-positive cells (green) in the basal-cell layer of central corneal epithelium. CD45-positive cells are also present in the stroma. (B) In the peripheral epithelium, CD45-positive cells are located in the basal-cell layer, and some cells are in the wing cell layer of the epithelium (not shown). (C) In the limbal epithelium, CD45-positive cells are present in the basal-cell layer and wing-cell layer. (D) Confocal microscopy of a flat mount of the corneal epithelium shows that the CD45-positive cells are dendritic cells. Original magnification: (AC) ×100; (D) ×400.
Figure 1.
 
Immunohistochemical staining of CD45+ cells in the epithelium of normal human donor corneas. Transverse cross-sections stained with anti-CD45 mAb were coverslipped using antifading mounting medium containing propidium iodide (PI; red) for nuclear staining. (A) Representative staining of CD45-positive cells (green) in the basal-cell layer of central corneal epithelium. CD45-positive cells are also present in the stroma. (B) In the peripheral epithelium, CD45-positive cells are located in the basal-cell layer, and some cells are in the wing cell layer of the epithelium (not shown). (C) In the limbal epithelium, CD45-positive cells are present in the basal-cell layer and wing-cell layer. (D) Confocal microscopy of a flat mount of the corneal epithelium shows that the CD45-positive cells are dendritic cells. Original magnification: (AC) ×100; (D) ×400.
Figure 2.
 
Representative photomicrographs of CD45-positive cells stained with various markers. (A) In corneal cross-sections, all the CD45-positive cells (green) in the epithelium are stained with CD11c (red). Double-stained cells are observed as yellow cells (A, yellow). CD11b is universally detected in the corneal stroma but is totally negative here (not shown). The CD11c+ cells in the corneal epithelium are all HLA-DR+ (clone G46-6, green) (B, HLA-DR+: upper, merged: lower). Similar findings were observed with staining for HLA-DR+ (clone TAL.1B5) (not shown). Original magnification: (A, B) ×100. (C) GAPDH (endogenous control, 27 PCR cycles) and HLA-DRa (30 PCR cycles) mRNA was detected in the corneal epithelium. No PCR products are detected in the negative control sample.
Figure 2.
 
Representative photomicrographs of CD45-positive cells stained with various markers. (A) In corneal cross-sections, all the CD45-positive cells (green) in the epithelium are stained with CD11c (red). Double-stained cells are observed as yellow cells (A, yellow). CD11b is universally detected in the corneal stroma but is totally negative here (not shown). The CD11c+ cells in the corneal epithelium are all HLA-DR+ (clone G46-6, green) (B, HLA-DR+: upper, merged: lower). Similar findings were observed with staining for HLA-DR+ (clone TAL.1B5) (not shown). Original magnification: (A, B) ×100. (C) GAPDH (endogenous control, 27 PCR cycles) and HLA-DRa (30 PCR cycles) mRNA was detected in the corneal epithelium. No PCR products are detected in the negative control sample.
Figure 3.
 
CD1c- and CD16 expression in the corneal epithelium. (A) Among CD45-positive cells from the peripheral corneal epithelium, CD1c (red) cells are detected in the basal layer and wing/surface cell layers. Representative photomicrograph shows that CD1c-positive cells (red) are detected among CD45-positive cells (green) in the peripheral cornea. These CD1c and CD45-positive cells are yellow (double labeled). The CD1c gene was detected in the corneal epithelium by RT-PCR (35 cycles). (B) Among CD45-positive cells (green), some are stained with CD16- mAb (red-yellow). The CD16 gene was detected in the corneal epithelium by RT-PCR (35 cycles), but not in the negative control sample. Original magnification: (A, B) ×100.
Figure 3.
 
CD1c- and CD16 expression in the corneal epithelium. (A) Among CD45-positive cells from the peripheral corneal epithelium, CD1c (red) cells are detected in the basal layer and wing/surface cell layers. Representative photomicrograph shows that CD1c-positive cells (red) are detected among CD45-positive cells (green) in the peripheral cornea. These CD1c and CD45-positive cells are yellow (double labeled). The CD1c gene was detected in the corneal epithelium by RT-PCR (35 cycles). (B) Among CD45-positive cells (green), some are stained with CD16- mAb (red-yellow). The CD16 gene was detected in the corneal epithelium by RT-PCR (35 cycles), but not in the negative control sample. Original magnification: (A, B) ×100.
Figure 4.
 
CD40 and CD86 expression after in vitro stimulation. Magnetic bead-isolated CD45-positive cells (Unstimulated group), and cells stimulated for 3 days with DMEM containing 1% FBS (Basal medium group) and DMEM containing 1% FBS, TNF-α and LPS (TNF-α and LPS group) were immunostained using PE-conjugated anti-CD11c (red) and FITC-conjugated anti-CD40 or CD86 (green) mAbs. Unstimulated cells are not stained by anti-CD40 or CD86 mAbs. CD40 (A)- and CD86 (B)-positive cell rates in CD11c-positive cells in the TNF-α and LPS group are significantly high compared with those in the Basal medium group. Lower panels of A (CD40+CD11c+, yellow) and B (CD86+CD11c+, yellow) are representative photographs. Red cells are CD11c-single positive. Original magnification, ×200. *P < 0.001.
Figure 4.
 
CD40 and CD86 expression after in vitro stimulation. Magnetic bead-isolated CD45-positive cells (Unstimulated group), and cells stimulated for 3 days with DMEM containing 1% FBS (Basal medium group) and DMEM containing 1% FBS, TNF-α and LPS (TNF-α and LPS group) were immunostained using PE-conjugated anti-CD11c (red) and FITC-conjugated anti-CD40 or CD86 (green) mAbs. Unstimulated cells are not stained by anti-CD40 or CD86 mAbs. CD40 (A)- and CD86 (B)-positive cell rates in CD11c-positive cells in the TNF-α and LPS group are significantly high compared with those in the Basal medium group. Lower panels of A (CD40+CD11c+, yellow) and B (CD86+CD11c+, yellow) are representative photographs. Red cells are CD11c-single positive. Original magnification, ×200. *P < 0.001.
Table 1.
 
Oligonucleotide Primers for RT-PCR
Table 1.
 
Oligonucleotide Primers for RT-PCR
Gene PCR Primers Product Size (bp)
GAPDH
5′-GGTGAAGGTCGGTGTGAACGGA-3′
5′-TGTTAGTGGGGTCTCGCTCCTG-3′ 223
HLA.DRa
5′-TCGAAATGGAAAACCTGTCACC-3′
5′-CCCAATAATGATGCCCACCA-3′ 267
CD1c
5′-TGGTGACAATGCAGACGCA-3′
5′-GGAGATGGCACCCATGTTGTA-3′ 414
CD16
5′-TTCATTGACGCTGCCACAGT-3′
5′-CCGCTATCTTTGAGTGTGGCTT-3′ 281
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