Abstract
Purpose:
The immune system plays a major role in the pathogenesis of dry eye diseases (DED), and dendritic cells (DCs) are known to be important initiators of acquired immunity. Thus, the purpose of this study was to determine the contribution of DCs to the development of DED.
Methods:
Mouse dry eye model was induced by subcutaneous injections of scopolamine and was euthanized at the baseline, and 2, 4, and 7 days postinjection. The activation of the DCs was determined by the mixed leukocyte reaction (MLR), and the number of activated CD86+ DCs in the lymph nodes was determined by flow cytometry. Upregulation of cytokines in the culture supernatant of MLR was determined by ELISA.
Results:
Significantly increased superficial corneal punctate lesions and decreased number of goblet cells in the conjunctiva were observed in scopolamine-injected mice. The number of activated CD86+ DCs was significantly increased in the cervical lymph nodes but not in the inguinal lymph nodes of the dry eye mice. The stimulatory activity of the DCs derived from the cervical lymph nodes of dry eye mice was significantly higher than that of control mice, and upregulations of IL-17, IL-2, and IL-4 were observed in the culture supernatant of MLR. These results indicate that the DCs of the cervical lymph nodes were activated by the scopolamine injections.
Conclusions:
Our results indicate that DCs in our dry eye model were sufficiently activated to stimulate the T cells that participate in the onset and progression of DED.
Dry eye disease (DED) is a common ocular surface disease, and it has been reported that 17.9% women and 10.5% men over the age of 21 years in United States,
1 9.6% of women over the age of 20 years in United Kingdom,
2 and 21.8% women and 12.5% men over the age of 50 years in Japan have DED.
3 The symptoms of DED are ocular discomfort, fatigue, blurred vision, and decreased visual acuity that may interfere with daily activities. Recent studies have shown that the immune system plays a significant role in the pathogenesis of DED.
4–10 One of the important players in the immune system is the antigen-presenting cells (APCs), which activate T cells that lead to the inflammatory cascade in DED.
8 It is well known that dendritic cells (DCs), a rare population of leukocytes that are distributed throughout the lymphoid and nonlymphoid tissues in the entire body, are important for the initiation of acquired immunity by stimulating naïve T cells as APCs.
11 They also serve as regulators of immune responses by inducing self tolerance.
12
It has been suggested that Langerhans cells, a type of DCs in the cornea, can act as APCs.
11 A significant increase in the Langerhans cells density has been reported in subjects affected by dry eye.
13,14 However, it has not been definitively determined whether DCs are involved in the onset and exacerbation of DED.
Thus, the purpose of this study was to determine whether DCs are involved in the onset and exacerbation of DED. To accomplish this, we used an experimentally induced DED in mice and found that there was an activation of DCs in the cervical lymph nodes in the experimental dry eye mice.
Materials and Methods
Mouse Dry Eye Model
This research was approved by the Nara Medical University Research Institutional Animal Care and Use Committee. The maintenance of the animals and the procedures used in the in vivo experiments were performed in accordance with Institutional Guidelines and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Female, 6- to 8-week-old C57BL/6 mice were purchased from Japan SLC (Shizuoka, Japan). Experimental dry eyes were induced with slight modifications of the procedures reported in detail.
15 Briefly, 0.5 mg/0.2 mL of scopolamine hydrobromide, a muscarinic receptor blocker (Wako Pure Chemical Industries, Osaka, Japan), was injected subcutaneously in the dorsal region 3 times/day at 8 AM, 12 PM, and 5 PM. The scopolamine injections were performed daily until the time of evaluation. Control mice were injected subcutaneously with the same volume of saline on the same time schedule as the mouse dry eye model. Both the dry eye–induced mice and control mice were kept under standard environmental condition in a room at 25°C with an ambient humidity of 35% by 15 cm/sec flow of air.
Mice were euthanatized on days 0 (baseline), and 2, 4, and 7 days after the beginning of the scopolamine, and the eyes including the eyelids, the cervical lymph nodes, the inguinal lymph nodes, and spleens were removed immediately.
A total of 76 mice were used in this study with 3 to 4 mice for each group. For histologic analyses, four mice/group for the dry eye model and control on days 0, 2, 4, 7, totaling 32 mice; three mice/group for the fluorescence activated cell sorter (FACS) analysis for the dry eye models on days 2, 4, 7, and 14, totaling 12 mice; four mice/group for MLR for the dry eye models and controls on days 2, 4, 7, totaling 24 mice; four mice/group for the dry eye model and controls for corneal epithelial damage evaluation to avoid histologic and immunologic changes by corneal fluorescein staining.
Corneal Fluorescein Staining
The corneas were stained with fluorescein to determine the condition of the corneal epithelium at the baseline, days 2, 4, and 7. For this, 0.6 μL of 2.5% fluorescein (Alcon Laboratories, Fort Worth, TX, USA) was dropped into the lateral conjunctival sac. After 3 minutes, the staining of the ocular surface was evaluated by slit-lamp biomicroscopy under cobalt blue light and photographed. The degree of punctate staining was assessed in a masked fashion with a standard grading system (National Eye Institute, Bethesda, MD, USA) of 0 to 3 for the central, superior, inferior, nasal, and temporal areas of the cornea.
16
Histopathologic Analyses
The eyes including the lids were excised and fixed in 10% buffered formalin and embedded in methacrylate (Wako Pure Chemical Industries, Osaka, Japan). The eyes were sectioned in the vertical plane at a thickness of 20 μm. The sections were stained with hematoxylin & eosin (H&E) or with periodic acid-Schiff (PAS). Sections passing through the optic disc were examined and photographed with a conventional light microscope (Olympus, Tokyo, Japan). One masked observer evaluated whether a gross inflammation was present in the cornea, conjunctiva, and lacrimal gland. The number of goblet cells in the superior and inferior conjunctiva was determined under a microscope with a 20× objective. The number of conjunctival goblet cells was counted manually by two independent masked observers. The concordance rate of two observers was excellent. Three sections were selected for counting, and the average was used for the statistical analyses.
Isolation of Dendritic Cells (DCs) From Lymph Nodes
The excised cervical and inguinal lymph nodes were gently compressed with a syringe over a nylon mesh to obtain single-cell suspensions. The single-cell suspensions were filtered through the nylon mesh and centrifuged for 15 minutes at 700g at 4°C. The cell pellets were placed on ice for 15 minutes, and then placed in a medium containing 200 μL of fetal calf serum (FCS; HyClone, Logan, UT, USA) and 10 mL of Rosewell Park Memorial Institute (RPMI; Sigma Aldrich Corp., St. Louis, MO, USA) medium. The cell suspensions were filtered through the nylon mesh, then centrifuged for 8 minutes at 700g at 4°C. The cells were resuspended in 4 mL of 40% Percoll (GE Healthcare UK Ltd., Amersham Place, Little Chalfont, Buckinghamshire England) and overlaid onto 4 mL of 70% Percoll. The 40%/70% discontinuous Percoll gradient was centrifuged at 40g at 4°C for 25 minutes. Cells from the interface were collected, washed, and resuspended in RPMI supplement with 1% FCS. These prepared cells were further incubated with CD11c microbeads (Miltenyi Biotec K.K., Tokyo, Japan) and CD11c+ cells were isolated with a magnetic column. These CD11c+ cells were used as the cervical and inguinal lymph node–derived DCs.
Isolation of Splenic Dendritic Cells
Spleen cells were prepared after the injection of collagenase type IV (100 units/mL; Sigma Aldrich Corp.) into the spleen, and the spleen cells were overlaid onto 4 mL of 50% Percoll. The Percoll gradient was centrifuged at 40g at 4°C for 25 minutes, and the cells from the interface were collected, washed, and resuspended in PBS supplemented with 2% FCS. These cells were incubated with CD11c microbeads as above and CD11c+ cells were isolated and used as splenic DCs in MLR analyses. It is noted that CD11c is a specific marker for DCs in mice.
Analyses of Surface Markers on DCs Derived From Lymph Nodes
DCs derived from the cervical and inguinal lymph nodes were stained with various monoclonal antibodies (mAbs) to determine their surface immunophenotypes. Additionally, cervical lymph nodes of mice that had been injected with scopolamine or vehicle for 7 days were evaluated similarly on day 14. The mAbs used for the staining were anti-CD11c and anti-CD86. The CD11c antigen is present on DCs in the lymphoid organs and blood (known as a specific marker to determine mouse DCs), and CD86 is expressed on activated antigen-presenting cells including DCs. These mAbs (purchased from BD Biosciences, San Jose, CA, USA) were conjugated with phycoerythrin (PE) or fluoresceinisothiocyanate (FITC). FITC- or PE-conjugated isotype matched mAb (IgG; BD Biosciences) was used as a negative control. Samples were preincubated with anti-CD16/CD32 mAb to block the nonspecific binding of mAbs through Fc receptors (FC Block; BD Biosciences). The cells were analysed with a FACS Calibur HG (Becton–Dickinson and Company, Franklin Lakes, NJ, USA). The shifts in the mean fluorescent intensity (MFI) of CD86 was calculated as follows:
MFI shift = MFI of CD86 on DCs with scopolamine injection / MFI of CD86 on DCs without scopolamine injection.
Mixed Leukocyte Reaction (MLR)
The MLR was used to examine the stimulatory activities of the cervical lymph node–derived DCs. For MLR, CD4+ T cells were prepared from allogeneic BALB/c spleen cells with a CD4+ T cell isolation 2 kit (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The responder splenic CD4+ T cells (2 × 105 cells) were cultured with graded numbers of stimulator DCs (2 × 103, 6 × 103, 2 × 104, and 6 × 104 cells) obtained from the cervical lymph nodes of scopolamine-injected mice or from control mice for 96 hours. Splenic DCs, isolated as CD11c+ cells, were used as a positive control. The proliferative activity of CD4+T cells was estimated by a MTT cell proliferation assay kit according to the manufacture's instruction (Cayman Chemical Co., Ann Arbor, MI, USA). A stimulation index for the MLR was calculated as follows:
Stimulation index = T cells stimulated with allogeneic DCs / T cells alone.
Measurement of Cytokine Production
The amount of IL-2 in the supernatants of MLR was determined by an ELISA kit for IL-2 (IL-2 Mouse IL-2 ELISA Ready-SET Go!; eBioscience, Thermo Fisher Scientific K.K., Yokohama, Kanagawa, Japan), and the production of IL-4 or IL-17 was also examined by an ELISA assay (Mouse IL-4 ELISA Kit and Mouse IL-17 ELISA Kit; Invitrogen, Vienna, Austria).
Statistical Analyses
The differences in the cornea scores, conjunctival goblet cell counts, stimulation indexes of the MLR and cytokine production after injection between scopolamine group and saline group were statistically analyzed by Welch's t-tests. The differences between the cervical lymph nodes and inguinal lymph nodes of the MFI shift in FACS after injection were statistically analyzed by Welch's t-tests. The annual change of MFI shift and corneal scores after scopolamine injection was evaluated by single factor one-way ANOVA with Tukey post hoc testing. A P < 0.05 was considered statistically significant. These tests were performed using MS Excel 2013 (Microsoft Corporation, Redmond, WA, USA).
Results
Corneal Fluorescein Staining
The number of punctate corneal fluorescein staining was minimal (score 0.5) on the corneas at the baseline in both groups. The degree of corneal staining was significantly increased following the scopolamine injections compared with the baseline or controls on days 2 (2.8 ± 1.0,
P < 0.05), 4 (3.5 ± 1.3,
P < 0.01), and 7 (6.8 ± 1.3,
P <0.01;
Fig. 1). These results indicated that scopolamine injections induced dry eye in the mice.
Histopathologic Study
Conjunctiva
In the control group, the conjunctiva consisted of a basal cuboidal layer with three to four overlying flattened but not cornfield epithelial cell layers (
Figs. 2A,
2C). In the scopolamine-injected mice, the conjunctiva consisted of four or more epithelial cell layers with a marked thickening and a marked thickening of the lamina propria on days 2, 4, and 7 (
Figs. 2B,
2D). The number of stained goblet cells in the scopolamine-injected mice was fewer than that of control mice. An infiltration of inflammatory cells was not observed in both groups.
Corneas
In the control group, the corneal surface consisted of four to five layers of epithelial cells, and in the scopolamine-injected mice, the corneal surface also showed thickening of the epithelial lining. An infiltration of inflammatory cells was not observed in both groups (
Figs. 3A,
3B).
Lacrimal Gland
The morphology of the lacrimal gland in the scopolamine-injected mice did not differ significantly from that of the control mice. An infiltration of inflammatory cells was not observed in both groups (
Figs. 3C,
3D).
Eyelid
The morphology of the eyelid rim including the Meibomian glands in the scopolamine-injected mice did not differ significantly from that of the control mice. An infiltration of inflammatory cells was not observed in both groups (
Figs. 3E,
3F).
Number of Goblet Cells
The mean number of goblet cells in the scopolamine-injected mice on day 7 was 47.5 ± 8, which was significantly fewer than that in the control mice at 67.5 ± 11.0 (
P < 0.01), but the difference was not statistically significant on days 2 (64.5 ± 7.7,
P = 0.60) and 4 (66.2 ± 8.6,
P = 0.82;
Fig. 4).
Expression of CD86 on DCs
CD86 is a well-known costimulatory molecule related to the stimulatory activity of DCs. Thus, we next examined the expression of CD86 on the DCs from the cervical lymph nodes (draining lymph node), and compared it with that on the DCs from the inguinal lymph node by flow cytometry. As shown in
Figure 5D (histogram), there was a higher expression of CD86 on the DCs prepared from the cervical lymph nodes of scopolamine-injected mice, but not on DCs from the cervical lymph nodes in PBS-injected mice, or on the DCs from the inguinal lymph node in the PBS-injected mice and in the DCs from the inguinal lymph node in the scopolamine-injected mice (
Figs. 5C,
5E,
5F). This was the case when the MFI shifts were examined; MFI shifts of the DCs from the inguinal lymph nodes were not significant, that is, 0.97 on day 2, 1.01 on day 4, and 1.1 on day 7, after the scopolamine injections (closed circles in
Fig. 6). These findings suggest that the DCs of the inguinal lymph nodes were not activated (
Fig. 6). Thus, the increase in the expression of CD86 was not observed in DCs from the inguinal lymph nodes indicating that DC are not activated in the nondraining lymph nodes. On the other hand, the shifts in the MFI of the DCs from the cervical lymph nodes were increased to 2.12 on day 2, to 2.25 on day 4, and to 1.54 on day 7 after the scopolamine injections (closed diamonds in
Fig. 6). There were significant differences between cervical lymph nodes and inguinal lymph nodes on day 2 (
P < 0.01) and 7 (
P < 0.05), clearly indicating that the DCs in the cervical lymph nodes were activated by the scopolamine injections. The MFI shift decreased to 1.21 at 14 days in the DCs from the cervical lymph nodes obtained from the mice that were treated with scopolamine for 7 days and no injections for 7 days (closed diamond in
Fig. 6). There was a significant difference of MFI shift between day 4 and day 14 (injection for 7 days and followed by no injection for 7 days) (
P < 0.05).
Mixed-Leukocyte Reaction (MLR)
The stimulatory activity of DCs derived from the cervical lymph nodes obtained from the dry eye model mice was examined for the MLR where CD4
+ T cells were stimulated by DCs. The cervical lymph node DCs were prepared from the mice treated with scopolamine for 2, 4, and 7 days. The stimulatory activity of the cervical lymph node DCs from dry eye model mice was significantly higher than that of control mice injected with PBS for 2, 4, and 7 days (
Fig. 7,
P < 0.05), especially when stimulated with the higher numbers of DCs. Furthermore, no difference was observed when the stimulatory activity of the inguinal lymph node DCs from the dry eye model was compared with that from the control mice (data not shown).
Cytokine Production
An increase in the stimulatory activity of DCs from the cervical lymph nodes of scopolamine-injected mice was also confirmed when the level of IL-2 in the culture supernatant of MLR was determined. The amount of IL-2 in the culture supernatant was significantly enhanced when CD4
+ T cells were stimulated with DCs from the cervical lymph nodes of scopolamine-injected mice compared with those from the cervical lymph nodes of PBS-injected mice (
Fig. 8). These results are in accordance with those of the MTT assay. Furthermore, we measured the amounts of IL-4 and IL-17 in the culture supernatants of MLR of CD4
+ T cells that were stimulated by purified DCs from the cervical lymph nodes (draining lymph nodes) or inguinal lymph nodes. A significant increase in the production of IL-17 was observed in MLR stimulated by DCs from the cervical lymph nodes of scopolamine-injected mice (4-days serial injection) when compared with those from the inguinal lymph nodes of the scopolamine-injected mice or those from the cervical lymph nodes of PBS-injected mice (
Fig. 9A). This was also true when IL-4 in the culture supernatants of MLR was measured (
Fig. 9B). These results indicated that the injection of scopolamine alone can activate DCs in the draining lymph nodes, and activated DCs can upregulate the production of IL-17, resulting in the inflammatory responses through the activation of various cells including macrophages. These findings clearly indicate that an increase in the stimulatory activity to CD4
+T cells was observed in the DCs obtained from the draining lymph node, the cervical lymph node, of the dry eye model mice. These changes then result in the activation of T cells that leads to the onset of DED.
Discussion
The previously reported dry eye models had severe manifestations of DED.
17–19
Clinically, the incidence of mild cases DED is comparable to that of severe DED cases. The results of a recent study showed that the most prevalent subtype of dry eye was the aqueous-deficient dry eye (35.0%), followed by the short tear break-up time (BUT)-type (26.7%). Our dry eye model is an aqueous-deficient type. Sjogren syndrome is an example of severe DED, and it makes up only 7.6% of the cases of dry eyes.
20 Thus, it is important to establish a mild DED murine model to investigate DED, which can then be used to study the characteristics of DED in humans.
A dry eye mouse model induced by scopolamine injections and desiccating stress in a controlled environmental chamber has been reported.
15,21–24 We have created a dry eye mouse model by only scopolamine injections under standard environmental conditions without desiccating stress. Our dry eye model was confirmed to be a mild dry eye model by the corneal staining scores. The recent criteria for diagnosing dry eye in Asia was determined by BUT.
25 However, to compare our results with that of previous reports, we employed the score of corneal staining, which was classically used for the evaluation of dry eye. The score of corneal fluorescein staining was increased after 2 days and then gradually increased until day 7. This time course is similar to that reported.
15
Scopolamine, a parasympathetic nerve blocker, blocks the ACh receptors (M3). It is generally believed that a subcutaneous injection of scopolamine affects the ocular surface and the lacrimal glands, and decreases the secretion of tears.
15 These changes result in DED. After daily injections of scopolamine, the tear volume gradually decreases and corneal epithelial cell disorders develop. We did not observe an infiltration of inflammatory cells into the conjunctiva, cornea, and lacrimal glands of the dry eye model mice as reported.
26
Our FACS analyses of the dry eye model mice revealed that the DCs in the inguinal lymph nodes were not activated but the DCs in the cervical lymph nodes were activated (i.e., an upregulation of a costimulatory molecule, CD86, was clearly observed). These results indicated that the immune system of the eyes was activated locally in the DED mice.
It has been reported that the decrease of immunity by scopolamine was through the blockage of the parasympathetic nerves.
26 The blockage led to a decrease in the tear volume,
15 and the ensuing desiccating stress on the ocular surface promoted the activation and maturation of immature APCs.
8 Thus, the DCs in the cervical lymph nodes were more likely activated by the drying of the ocular surface.
Interestingly, the mice that were treated with scopolamine for 7 days and then not injected for 7 days had a low degree of shift of the MFI of the DCs in the cervical lymph nodes at 14 days. This confirms that the activation of the DCs was induced by the scopolamine injections.
It is well known that not only DCs but also other APCs such as monocytes, macrophages, and B cells, can activate naïve T cells to differentiate into effector T cells.
27 In accordance, the APCs that activated naïve T cells were more likely to be the DCs in our dry eye model, the DCs from the draining lymph nodes. The cervical lymph nodes had an increased expression of CD86 and the enhanced stimulatory activity in MLR and assays for IL-2 production from stimulated CD4
+ T cells, because they are phenotypically and functionally in the activation state. Furthermore, it was noted that there was a significant increase in the production of inflammatory IL-17 in MLR stimulated with DCs purified from the cervical lymph nodes of the scopolamine-injected mice. This suggests that the injection of scopolamine alone can activate DCs in the draining lymph nodes, and thus activated DCs can enhance the differentiation of Th17 cells that upregulate the production of IL-17 resulting in the inflammatory responses. These results indicate that the activated DCs in the cervical lymph nodes can participate in the development and progression of DED as APCs.
Our results are in well accord with the recent report on effector T cells, especially the inflammatory-prone Th17 cells that are differentiated from naïve T cell at the draining lymph nodes. The nodes are where the T cells interact with the DCs that traffic from the ocular surface occurs, and the inflammatory effector T cells infiltrate the ocular surface. Furthermore, an upregulation of granulocyte macrophage colony-stimulating factor (GM-CSF) produced from these Th17 cells can activate CD11b
+ cells that contribute to the pathogenesis of DED.
28 Therefore, activation of T cells is generally restricted by the status of DCs (i.e., DCs can control the direction and magnitude of immune responses at the initial step). In our study, we have examined the activation status of the DCs in the draining lymph nodes to determine the initial step of the DED pathogenesis.
We defined the effects of scopolamine by histologic sections alone, and we did not examine the inflammatory cell populations because the degree of inflammation was very mild. Inflammatory cells were not detected by routine histologic evaluations. Therefore, when specific immunologic analysis for inflammatory cells was performed, it might be detectable. However, we detected the activation of DCs by FACS analysis, and we have also found an upregulation of IL-17 in the culture supernatant of MLR that stimulated with DCs from the cervical lymph nodes in scopolamine-injected mice. Thus, the activated DCs by scopolamine-injection can be involved in the inflammatory responses resulting in the onset of DED.
Further studies are needed to determine the different types of cytokines that are released in DED mice, which would confirm the participation of DCs in the immunity for the development of the DED in more detail. Small sample (n = 3 or 4) may be the reason that the differences were not significant. We will need further examination with larger sample sizes.
In conclusions, DED was induced by subcutaneous injections of scopolamine in mice. Our results showed that the DCs in the cervical lymph nodes of the experimental dry eye mice were more activated than in control mice. These results suggest that DCs may act as APCs and participate in the onset and the progression of DED.
Acknowledgments
Disclosure: S. Maruoka, None; M. Inaba, None; N. Ogata, None
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