Current Eye Research
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Observation of Corneal Langerhans Cells by In Vivo Confocal Microscopy in Thyroid-Associated Ophthalmopathy Lian-Qun Wu, Jin-Wei Cheng, Ji-Ping Cai, Qi-Hua Le, Xiao-Ye Ma, Lian-Di Gao & Rui-Li Wei To cite this article: Lian-Qun Wu, Jin-Wei Cheng, Ji-Ping Cai, Qi-Hua Le, Xiao-Ye Ma, Lian-Di Gao & Rui-Li Wei (2016): Observation of Corneal Langerhans Cells by In Vivo Confocal Microscopy in Thyroid-Associated Ophthalmopathy, Current Eye Research, DOI: 10.3109/02713683.2015.1133833 To link to this article: http://dx.doi.org/10.3109/02713683.2015.1133833
Published online: 06 Jan 2016.
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Date: 12 January 2016, At: 16:42
CURRENT EYE RESEARCH http://dx.doi.org/10.3109/02713683.2015.1133833
ORIGINAL ARTICLE
Observation of Corneal Langerhans Cells by In Vivo Confocal Microscopy in Thyroid-Associated Ophthalmopathy Lian-Qun Wua, Jin-Wei Chenga, Ji-Ping Caia, Qi-Hua Leb, Xiao-Ye Maa, Lian-Di Gaoa, and Rui-Li Weia Department of Ophthalmology, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai, China; bEYE and ENT Hospital of Fudan University, Shanghai, China
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a
ABSTRACT
ARTICLE HISTORY
Purpose: To examine the density and morphology of Langerhans cells (LCs) in the cornea of patients with thyroid-associated ophthalmopathy (TAO). Methods: Forty patients with TAO and 20 healthy volunteers were studied. All subjects underwent a thorough ophthalmic examination of both eyes. The ocular surface status was assessed by Ocular Surface Disease Index (OSDI) symptom questionnaires, tear break-up time (BUT), fluorescein staining and the Schirmer test. Laser scanning in vivo confocal microscopy was applied to evaluate the LC density and morphology in both central and peripheral cornea. The correlations between confocal microscopy data and clinical data were also analyzed. Results: The OSDI and fluorescein staining values were significantly higher, while BUT and Schirmer test scores were lower in both active and inactive TAO patients compared to the controls. Central LC densities of patients with active TAO (76.38 ± 67.77 cell/mm2) and inactive TAO (47.49 ± 38.58 cell/ mm2) were both significantly higher than those of the controls (21.46 ± 21.74 cell/mm2). The number of LCs in the peripheral cornea was also significantly increased in the active TAO group (131.53 ± 74.18 cell/mm2) compared to the control group (70.21 ± 37.76 cell/mm2). Central LC morphology (LCM) values were significantly higher in both active (1.77 ± 0.63) and inactive (1.51 ± 0.63) TAO groups compared to the control group (1.01 ± 0.80), whereas peripheral LCM scores of the two TAO groups were increased without statistical significance. There were significant correlations between both central LC density and central LCM scores and clinical data, including clinical activity score, OSDI and Schirmer test scores, and between peripheral LC density and OSDI and Schirmer test scores. No significant correlations were found between peripheral LCM scores and clinical data. Conclusions: The increase of corneal LCs in density and maturation in patients with TAO reflects an activated state of the local immune system, which indicates an inflammatory process in the cornea of TAO.
Received 14 September 2015 Revised 23 November 2015 Accepted 16 December 2015
Introduction Thyroid-associated ophthalmopathy (TAO) is the most common autoimmune inflammatory orbital disease in adults. TAO occurs mainly in patients with hyperthyroidism and also in patients who are euthyroid or hypothyroid.1 TAO varies in clinical presentation, which involves not only the orbital connective tissues but also the ocular surface system including eyelid, lacrimal gland, conjunctiva and cornea.2,3 The ocular surface impairment in TAO is very common, with incidence varying from 40% to 72%;3 however, the etiology of this condition is not fully understood. Mechanical factors were thought to be the main reasons for ocular surface disorders associated with TAO, such as proptosis, lagophthalmos, upper eyelid retraction, reduced blinking rate and subsequent increased tear evaporation.1,3,4 Recently, tear analysis5 and pathological examination, including impression cytology6 and incisional biopsy of bulbar conjunctiva,7 indicated that tear dysfunction and inflammation play important roles in ocular surface damage in TAO.
CONTACT Rui-Li Wei Road, Shanghai, China. © 2016 Taylor & Francis
[email protected]
KEYWORDS
In vivo confocal microscopy; inflammation; Langerhans cells; ocular surface; thyroidassociated ophthalmopathy
Langerhans cells (LCs), serving as the professional antigenpresenting cells in the ocular surface, play a major role in corneal immune responses.8 They are equipped to capture, process and present antigens, and lead to the initiation of ocular surface immunoinflammatory responses.8,9 Corneal LCs are localized in both the central and peripheral corneal epithelium under normal circumstances.8 In vivo confocal microscopy studies have identified LC density and distribution in the normal corneal epithelium9 and demonstrated changes subsequent to pathological conditions, such as corneal trauma,10 contact lens wear11 or some other immunemediated inflammatory diseases.12 TAO is an autoimmune disease affecting both the eye and the thyroid. Nevertheless, there are no reports describing changes of the LCs in the corneal epithelium of TAO patients. In this study, we evaluate the density and morphology of LCs in the cornea of TAO patients by means of in vivo laser scanning confocal microscopy, to determine whether an inflammatory reaction is involved in the pathogenesis of ocular surface disease associated with TAO.
Department of Ophthalmology, Shanghai Changzheng Hospital, Second Military Medical University, 415 Fengyang
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Materials and methods
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Subjects A total of 40 patients with TAO (10 men and 30 women; mean age 41.40 ± 6.86 years; range 30–58 years) were enrolled in this study from the Department of Ophthalmology, Shanghai Changzheng Hospital, from April 2014 to January 2015. The diagnosis of TAO was made based on Bartley criteria.13 TAO activity was evaluated by the clinical activity score (CAS), with active TAO being defined as CAS≥3/7 and inactive TAO defined as CAS≤2/7.14 Patients were excluded from the study if, before the onset of TAO, they had previously suffered from other systemic autoimmune diseases or anterior segment disorders, such as dry-eye syndrome, meibomian gland dysfunction, allergic ocular surface disease, pterygium, corneal dystrophy, and uveitis or had received either systemic or local treatment that might affect ocular surface parameters. The criteria also excluded persons who wore contact lenses or had undergone ophthalmic surgery. During the examination period, none of the patients with TAO required specific ophthalmic assistance. Twenty healthy volunteers (seven men and 13 women; mean age 44.10 ± 7.28 years; range 27–56 years) were included as normal controls. None of the normal controls wore contact lenses, used any topical eye drops or had a history of anterior segment disorders, ocular trauma or surgery. The study was approved by the Ethics Committee of Shanghai Changzheng Hospital, affiliated to the Second Military Medical University, and adhered to the tenets of the Declaration of Helsinki. Informed consent was obtained from each participant.
Clinical evaluation All subjects received a full ophthalmic examination of both eyes, which included visual acuity, intraocular pressure, proptosis, lagophthalmos, slit-lamp examination and fundus examination. The ocular surface damage was assessed by both subjective Ocular Surface Disease Index (OSDI) symptom questionnaires15 and objective examinations, including tear break-up time (BUT), fluorescein staining and Schirmer test I. The corneal fluorescein staining score was evaluated based on grades 0–3 in each of the four quadrants for a total score of 12, according to the grading scale previously described.16 All ophthalmic examinations were performed by the same researcher (J.-W.C.). In addition, all of the participants were evaluated for their smoking habits.
Bausch & Lomb, Rochester, NY) was used as a coupling medium between the applanating lens and the cornea. In vivo confocal microscopy was performed on each cornea in two different areas: central and peripheral (2 mm from the corneoscleral limbus) at 12:00. In vivo laser scanning corneal confocal microscopy was performed bilaterally on each subject by the same researcher (Q.-H.L.) at EYE and ENT Hospital of Fudan University. Three images with a maximum number of target cells from both central and peripheral cornea were evaluated in a masked fashion by the same investigator (X.-Y.M.). Bright corpuscular cells with or without dendrite-like protrusions within the corneal epithelium at the level of basal epithelial cells to subbasal nerve plexus were considered to be LCs.9 After identification of LCs, the cell density in each image (400 × 400 mm2) was calculated using open professional image analysis software Image J (Image J, Version 1.45, National Institutes of Health, Bethesda, MD) plus Counter Cell plugin and recorded as cells per square millimetre. LC morphology (LCM) was evaluated on a 0–3 scale according to the methods previously described.17 A score of 0 described the condition when the cornea was devoid of LCs; a score of 1 was given when cells lacked processes; a score of 2 (small processes) was given if the length of the processes did not exceed the longest diameter of the cell body; and a score of 3 (long processes) was given if the processes were longer than the largest diameter of the cell body. The mean LCM score was calculated and used to describe the maturation of the LCs at both regions of the cornea (Figure 1). Statistical analysis The data of the eye with the higher fluorescein staining score of each participant were included for analysis as previously described by Villani.18 In the event that the two eyes had equal fluorescein staining scores, the sequential discriminating criteria were the lower Schirmer test score and the lower BUT score.18 Data distribution and homogeneity were analyzed. Normally distributed variables were expressed as the mean ±standard deviation, and Student’s t-test was used to compare the mean values between active TAO and inactive TAO groups, and one-way ANOVA with the LSD post hoc test or Tamhane’s T2 test was used to compare the mean scores of the control subjects and patients with active TAO or inactive TAO. Skewed variables were expressed as median values and percentiles (Q1, Q3), and the Mann–Whitney U nonparametric test was applied. Fisher’s exact test was used to analyze the categorical variable. The correlations between the variables were analyzed by using Spearman’s rank correlation test. P < 0.05 was considered statistically significant (SPSS 19.0; IBM, Chicago, IL).
Confocal microscopy In vivo corneal confocal microscopy was performed on all patients with a Heidelberg Retina Tomograph (HRT3) equipped with a Rostock Corneal Module (RCM) (Heidelberg Engineering GmbH, Heidelberg, Germany). The ocular surface was anesthetized with topical anesthetic eye drops (0.5% proxymetacaine hydrochloride, Alcaine; Alcon Laboratories, Fort Worth, TX). Carbomer gel (Vidisic;
Results Clinical data The demographic and clinical features of the active and inactive TAO groups are presented in Table 1. CAS was significantly higher in active TAO compared with inactive TAO (P = 0.001, Student’s t-test). There were no significant
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CURRENT EYE RESEARCH
(A)
(B)
(C)
(D)
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Figure 1. Representative confocal microscopic images of corneal Langerhans cells (LCs) in patients with thyroid-associated ophthalmopathy (TAO) and control subjects. (A) Central cornea of a healthy volunteer. No LC is visible in this area at a depth of 48 μm [LC morphology (LCM) score = 0]. (B) Image of the central cornea of a TAO patient at a depth of 46 μm. The density of LCs is increased compared to that of normal subjects. Most of the LCs are of LCM score 1 (white arrow) and 2 (black arrow). (C) Peripheral cornea of a healthy volunteer with mixed populations of LCs at a depth of 43 μm (empty arrow shows an LCM score 3 LC). (D) Peripheral cornea of a TAO patient with gathering LCs arranged in a network fashion at a depth of 56 μm. Most of the LCs are with processes. Table 2. Comparison of ocular surface parameters of TAO patients and control subjects.
Table 1. Comparison of disease profile of active and inactive TAO patients. Number Gender (male/female) Age (years) Clinical activity score Proptosis (mm) Lagophthalmos (mm) FT3 (pmol/L) FT4 (pmol/L) TSH (mU/L)
Active TAO 15 5/10 43.60 ± 5.51 5.80 ± 1.37 21.13 ± 2.39 1 (0, 3) 5.32 ± 1.83 16.78 ± 4.99 3.66 ± 6.11
Inactive TAO 25 5/20 40.08 ± 7.35 1.16 ± 1.03 19.78 ± 2.14 0 (0, 1.5) 4.59 ± 0.63 15.15 ± 3.70 2.69 ± 2.72
P a
0.457 0.118b 0.000c 0.071d 0.154e 0.155f 0.244g 0.568h
FT3 = free triiodothyronine; FT4 = free thyroxine; TSH = thyroid stimulating hormone. a: Fisher’s exact test. b, c, d, f, g, h: Student’s t-test. e: Mann–Whitney U nonparametric tests.
differences in gender, age, proptosis, lagophthalmos or the levels of free tri-iodothyronine (FT3), free thyroxine (FT4) and thyroid stimulating hormone (TSH) between the active and inactive TAO groups (Table 1). The ocular surface parameters of the three groups are summarized in Table 2. All the patients with TAO included
Age (years) OSDI (score) BUT (seconds) Fluorescein staining (score) Schirmer test (mm)
Active TAO 43.60 ± 5.51 50.00 (41.67, 56.25) 5.11 ± 2.15 3.53 ± 4.05
Inactive TAO 40.08 ± 7.35 27.08 (20.83, 39.58) 6.30 ± 2.99 1.88 ± 2.05
Control P* 44.10 ± 7.28 0.117 0 (0, 1.56) 0.000a
5.50 ± 2.15
6.46 ± 3.02
12.65 ± 1.95 0.000d
11.62 ± 1.67 0.000b 0 0.000c
* ANOVA. a: Active TAO, Inactive TAO vs. control; P = 0.000. Active TAO vs. inactive TAO; P = 0.000. Tamhane’s T2 test. b: Active TAO, Inactive TAO vs. control; P = 0.000. Active TAO vs. inactive TAO; P = 0.138. LSD post hoc test. c: Active TAO vs. control; P = 0.013. Inactive TAO vs. control; P = 0.000. Active TAO vs. inactive TAO; P = 0.403. Tamhane’s T2 test. d: Active TAO, Inactive TAO vs. control; P = 0.000. Active TAO vs. inactive TAO; P = 0.245. LSD post hoc test.
in this study had dry eye.19 OSDI and fluorescein staining scores were significantly higher and BUT and Schirmer test values were significantly lower in patients with active and inactive TAO compared to the normal subjects.
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Smoking habits of the participants were as follows: four (26.7%) smokers, two (13.3%) ex-smokers and nine (60.0%) nonsmokers in the active TAO group; six (24.0%) smokers, two (8.0%) ex-smokers and 17 (68.0%) nonsmokers in the inactive TAO group; seven (35.0%) smokers, one (5.0%) exsmoker and 12 (60.0%) nonsmokers in the control group. There was no statistically significant difference for smoking status among these three groups (P = 0.872).
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Confocal microscopy data Table 3 demonstrates our confocal microscopy results with regard to the different groups. All the active TAO patients presented with LCs in the central cornea as opposed to 19 out of 20 inactive TAO patients and 13 out of 20 control subjects. LC densities dramatically increased in the central cornea in both active and inactive TAO groups compared to the control group (P = 0.024, P = 0.020, respectively). The numbers of LCs in the peripheral cornea were significantly increased in the active TAO group compared to the control group (P = 0.025), whereas they were only slightly increased in the inactive TAO group compared to the control group (P = 0.101). There were no significant differences in LC density in the central or peripheral cornea between the active TAO and inactive TAO groups (Table 3). Morphology is believed to represent maturation of LCs. Central LCM values were significantly higher in both active and inactive TAO groups compared to the control group (P = 0.002, P = 0.019, respectively). Peripheral LCM scores were increased in the active and inactive TAO groups, without statistical significance, compared to the control group (P = 0.112, P = 0.314, respectively). There were no significant differences in central or peripheral LCM scores between the active TAO and inactive TAO groups (Table 3).
Correlations There were significant positive correlations between CAS and OSDI and fluorescein staining scores (r = 0.666, P = 0.000; r = 0.332, P = 0.037, respectively), and significant inverse correlations between CAS and BUT and Schirmer test scores (r = – 0.406, P = 0.009; r = –0.414, P = 0.008, respectively). No Table 3. Comparison of confocal microscopy data of TAO patients and control subjects. LC center (cell/mm2) LC periphery (cell/mm2) LCM center LCM periphery
Active TAO 76.38 ± 67.77
Inactive TAO 47.49 ± 38.58
Control 21.46 ± 21.74
P* 0.002a
131.53 ± 74.18
103.25 ± 62.80
70.21 ± 37.76
0.013b
1.77 ± 0.63 2.66 ± 0.45
1.51 ± 0.63 2.54 ± 0.52
1.01 ± 0.80 2.39 ± 0.46
0.006c 0.270d
* ANOVA. a: Active TAO vs. control; P = 0.024. Inactive TAO vs. control; TAO vs. inactive TAO; P = 0.379. Tamhane’s T2 test. b: Active TAO vs. control; P = 0.025. Inactive TAO vs. control; TAO vs. inactive TAO; P = 0.540. Tamhane’s T2 test. c: Active TAO vs. control; P = 0.002. Inactive TAO vs. control; TAO vs. inactive TAO; P = 0.257. LSD post hoc test. d: Active TAO vs. control; P = 0.112. Inactive TAO vs. control; TAO vs. inactive TAO; P = 0.453. LSD post hoc test.
P = 0.020. Active P = 0.101. Active P = 0.019. Active P = 0.314. Active
significant correlations were found between CAS and FT3, FT4 and TSH levels. There were significant positive correlations between central LC density and CAS and OSDI score (r = 0.330, P = 0.037; r = 0.343, P = 0.007, respectively) and significant inverse correlations between central LC density and the Schirmer test score (r = –0.367, P = 0.004). The central LCM score was positively correlated with CAS and the OSDI score (r = 0.344, P = 0.030; r = 0.340, P = 0.008, respectively) and inversely correlated with the Schirmer test score (r = –0.397, P = 0.002). Peripheral LC density was positively correlated with the OSDI score (r = 0.335, P = 0.009) and inversely correlated with the Schirmer test score (r = –0.291, P = 0.024). No significant correlations were found between the peripheral LCM value and clinical data.
Discussion TAO may cause many ocular surface problems, such as conjunctival chemosis, conjunctival hyperaemia, superficial punctate keratopathy, exposure keratopathy, dry eye, superior limbic keratoconjunctivitis and corneal ulcer.3 Dry eye is one of the leading ocular surface diseases of TAO.3 In this study, all the patients with TAO had clinical signs and symptoms of dry eye. The Schirmer test results were significantly lower and the BUT values were significantly shorter in patients compared to normal subjects, which indicated both a hyposecretory and evaporative mechanism of dry eye associated with TAO. These findings are consistent with those reported by Villani et al.18 and Gürdal et al.7 Lacrimal gland dysfunction has been reported in TAO patients.20 An increase in the palpebral fissure width associated with TAO was thought to lead to accelerated tear evaporation.21 Decreased aqueous tear production and excessive tear evaporation would increase the tear film osmolarity and lead to ocular surface inflammation and damage.19,21 Furthermore, lid retraction and proptosis can result in lid friction on the ocular surface during blinking, leading to mechanical damage.3,22 Fluorescein staining scores were significantly higher in patients with TAO compared to healthy volunteers in this study. Similar results were found previously by Ismailova et al.4 and Huang et al.23 In patients with active TAO, ocular adnexa inflammation is often more severe than in nonspecific dry-eye patients, which indicates that orbital inflammation may also be involved in the ocular surface damage of TAO. In addition, the expression of thyroid hormone receptor was detected in the epithelia of lachrymal gland acini, cornea and conjunctiva, which verified that ocular surface tissue might be the direct target organs for thyroid hormone.3,20,24 However, few studies have reported the corneal changes associated with TAO.18 Confocal microscopy provides a noninvasive method to investigate different cell populations on the ocular surface in vivo at microscopic resolution. Previous studies have demonstrated the changes in corneal inflammatory cells in cases of dry eye,25 rheumatoid arthritis (RA),26 ankylosing spondylitis (AS)27 and systemic lupus erythematosus.17 Those results indicated the corneal involvement in local or systemic inflammatory conditions. TAO is an autoimmune inflammatory disease.
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CURRENT EYE RESEARCH
Villani et al.18 showed that the densities of corneal epitheliums and stromal keratocytes were significantly different between Graves’ orbitopathy (GO) patients and control subjects, and the density of activated keratocytes was significantly higher in active GO than inactive GO. Wei et al.22 reported increased LC density and reduced goblet cell density in the GO group compared to control patients. In this study, in vivo laser scanning confocal microscopy was used to evaluate inflammatory cells, especially on the LCs in the cornea of patients with TAO and healthy volunteers. Results showed that LCs had a markedly higher density in the periphery than in the central cornea in all participants. Compared to the control group, central LC density increased to about 3.5-fold in the active TAO group, whereas the increase in the inactive group was about 2.2-fold. In the peripheral cornea, the LC density increased to about 1.9fold and 1.5-fold in patients with active TAO and inactive TAO, respectively. In addition, the study demonstrated increased LCM values and maturation of LCs in the cornea, especially in the central part, in patients with TAO. Corneal LCs are deeply involved in corneal immunoregulatory processes. They have an extraordinary capacity to stimulate naive T cells and are recognized as essential regulators of both the innate and acquired arms of the immune system.25,28 Under normal conditions, LCs are scarce in the central cornea. Upon different stimuli, LCs migrate centrally and transform into the mature type by the formation of dendrite-like processes and expression of co-stimulatory molecules.28 There are two possible reasons attributed to the centripetal migration and maturation of LCs in the corneal epithelium of TAO patients in this study. The first possible reason is dry eye in patients with TAO. It has been clearly shown in previous studies that dry eye could induce overexpression of the inflammatory cytokines, chemokines and adhesion molecules,29 which are considered to have the capacity to activate LCs.30 In this study, central and peripheral LC density and the central LCM score correlated negatively with tear secretion that was lower in TAO patients than that of normal subjects. Lin et al.25 reported the elevation of LC density in both the central and peripheral parts of the cornea in patients with aqueous tear-deficient dry eye, and this supports the participation of LCs in dryeye pathogenesis.31 The second possible reason responsible for the activation of corneal LCs is the inflammatory reaction associated with TAO. Huang et al.23 reported that interleukin (IL)-1β, IL-6 and IL-8 concentrations are significantly higher in active TAO than inactive TAO and the controls, indicating that orbital inflammation may be involved in ocular surface damage of TAO. In this study, central LC density and the central LCM score correlated positively with the inflammatory activity of the disease as measured by the CAS. Marsovszky et al.27 reported greater corneal LC density and LCM in AS, which correlated with the systemic activity of AS even without ocular symptoms. Another study by Marsovszky et al.26 showed that the prevalence of central and peripheral LCs and the central LCM were higher than normal in RA, even in inactive stages of RA and without ocular symptoms. These data support the theory that these alterations of the corneal LCs are associated with the systemic inflammatory effect of the disease.
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However, the mechanisms of the migration and maturation of corneal LCs in patients with TAO are not yet fully understood. Further evaluations and comparisons of the corneal LCs by in vivo confocal microscopy in dry eye associated with TAO and in nonspecific dry eye are needed. In summary, this study provides evidence of LC participation and corneal inflammation in TAO. Further investigation of the pathogenesis underlying the corneal involvement in TAO at the molecular level is needed.
Funding This study was supported by the National Natural Science Foundation of China (Grant No. 81371056).
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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24. Dias AC, Módulo CM, Jorge AG, Braz AM, Jordão AA Jr, Filho RB, et al. Influence of thyroid hormone on thyroid hormone receptor-1 expression and lachrymal gland and ocular surface morphology. Invest Ophthalmol Vis Sci 2007;48:3038–3042. 25. Lin H, Li W, Dong N, Chen W, Liu J, Chen L, et al. Changes in corneal epithelial layer inflammatory cells in aqueous tear-deficient dry eye. Invest Ophthalmol Vis Sci 2010;51:122–128. 26. Marsovszky L, Resch MD, Németh J, Toldi G, Medgyesi E, Kovács L, et al. In vivo confocal microscopic evaluation of corneal Langerhans cell density, and distribution and evaluation of dry eye in rheumatoid arthritis. Innate Immun 2013;19:348–354. 27. Marsovszky L, Németh J, Resch MD, Toldi G, Legány N, Kovács L, et al. Corneal Langerhans cell and dry eye examinations in ankylosing spondylitis. Innate Immun 2014;20: 471–477. 28. Hamrah P, Dana MR. Corneal antigen-presenting cells. Chem Immunol Allergy 2007;92:58–70. 29. Hessen M, Akpek EK. Dry eye: an inflammatory ocular disease. J Ophthalmic Vis Res 2014;9:240–250. 30. Dana MR, Dai R, Zhu S, Yamada J, Streilein JW. Interleukin-1 receptor antagonist suppresses Langerhans cell activity and promotes ocular immune privilege. Invest Ophthalmol Vis Sci 1998;39:70–77. 31. Schaumburg CS, Siemasko KF, De Paiva CS, Wheeler LA, Niederkorn JY, Pflugfelder SC, et al. Ocular surface APCs are necessary for autoreactive T cell-mediated experimental autoimmune lacrimal keratoconjunctivitis. J Immunol 2011;187: 3653–3662.
ISSN: 0271-3683 (Print) 1460-2202 (Online) Journal homepage: http://www.tandfonline.com/loi/icey20
Observation of Corneal Langerhans Cells by In Vivo Confocal Microscopy in Thyroid-Associated Ophthalmopathy Lian-Qun Wu, Jin-Wei Cheng, Ji-Ping Cai, Qi-Hua Le, Xiao-Ye Ma, Lian-Di Gao & Rui-Li Wei To cite this article: Lian-Qun Wu, Jin-Wei Cheng, Ji-Ping Cai, Qi-Hua Le, Xiao-Ye Ma, Lian-Di Gao & Rui-Li Wei (2016): Observation of Corneal Langerhans Cells by In Vivo Confocal Microscopy in Thyroid-Associated Ophthalmopathy, Current Eye Research, DOI: 10.3109/02713683.2015.1133833 To link to this article: http://dx.doi.org/10.3109/02713683.2015.1133833
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Date: 12 January 2016, At: 16:42
CURRENT EYE RESEARCH http://dx.doi.org/10.3109/02713683.2015.1133833
ORIGINAL ARTICLE
Observation of Corneal Langerhans Cells by In Vivo Confocal Microscopy in Thyroid-Associated Ophthalmopathy Lian-Qun Wua, Jin-Wei Chenga, Ji-Ping Caia, Qi-Hua Leb, Xiao-Ye Maa, Lian-Di Gaoa, and Rui-Li Weia Department of Ophthalmology, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai, China; bEYE and ENT Hospital of Fudan University, Shanghai, China
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a
ABSTRACT
ARTICLE HISTORY
Purpose: To examine the density and morphology of Langerhans cells (LCs) in the cornea of patients with thyroid-associated ophthalmopathy (TAO). Methods: Forty patients with TAO and 20 healthy volunteers were studied. All subjects underwent a thorough ophthalmic examination of both eyes. The ocular surface status was assessed by Ocular Surface Disease Index (OSDI) symptom questionnaires, tear break-up time (BUT), fluorescein staining and the Schirmer test. Laser scanning in vivo confocal microscopy was applied to evaluate the LC density and morphology in both central and peripheral cornea. The correlations between confocal microscopy data and clinical data were also analyzed. Results: The OSDI and fluorescein staining values were significantly higher, while BUT and Schirmer test scores were lower in both active and inactive TAO patients compared to the controls. Central LC densities of patients with active TAO (76.38 ± 67.77 cell/mm2) and inactive TAO (47.49 ± 38.58 cell/ mm2) were both significantly higher than those of the controls (21.46 ± 21.74 cell/mm2). The number of LCs in the peripheral cornea was also significantly increased in the active TAO group (131.53 ± 74.18 cell/mm2) compared to the control group (70.21 ± 37.76 cell/mm2). Central LC morphology (LCM) values were significantly higher in both active (1.77 ± 0.63) and inactive (1.51 ± 0.63) TAO groups compared to the control group (1.01 ± 0.80), whereas peripheral LCM scores of the two TAO groups were increased without statistical significance. There were significant correlations between both central LC density and central LCM scores and clinical data, including clinical activity score, OSDI and Schirmer test scores, and between peripheral LC density and OSDI and Schirmer test scores. No significant correlations were found between peripheral LCM scores and clinical data. Conclusions: The increase of corneal LCs in density and maturation in patients with TAO reflects an activated state of the local immune system, which indicates an inflammatory process in the cornea of TAO.
Received 14 September 2015 Revised 23 November 2015 Accepted 16 December 2015
Introduction Thyroid-associated ophthalmopathy (TAO) is the most common autoimmune inflammatory orbital disease in adults. TAO occurs mainly in patients with hyperthyroidism and also in patients who are euthyroid or hypothyroid.1 TAO varies in clinical presentation, which involves not only the orbital connective tissues but also the ocular surface system including eyelid, lacrimal gland, conjunctiva and cornea.2,3 The ocular surface impairment in TAO is very common, with incidence varying from 40% to 72%;3 however, the etiology of this condition is not fully understood. Mechanical factors were thought to be the main reasons for ocular surface disorders associated with TAO, such as proptosis, lagophthalmos, upper eyelid retraction, reduced blinking rate and subsequent increased tear evaporation.1,3,4 Recently, tear analysis5 and pathological examination, including impression cytology6 and incisional biopsy of bulbar conjunctiva,7 indicated that tear dysfunction and inflammation play important roles in ocular surface damage in TAO.
CONTACT Rui-Li Wei Road, Shanghai, China. © 2016 Taylor & Francis
[email protected]
KEYWORDS
In vivo confocal microscopy; inflammation; Langerhans cells; ocular surface; thyroidassociated ophthalmopathy
Langerhans cells (LCs), serving as the professional antigenpresenting cells in the ocular surface, play a major role in corneal immune responses.8 They are equipped to capture, process and present antigens, and lead to the initiation of ocular surface immunoinflammatory responses.8,9 Corneal LCs are localized in both the central and peripheral corneal epithelium under normal circumstances.8 In vivo confocal microscopy studies have identified LC density and distribution in the normal corneal epithelium9 and demonstrated changes subsequent to pathological conditions, such as corneal trauma,10 contact lens wear11 or some other immunemediated inflammatory diseases.12 TAO is an autoimmune disease affecting both the eye and the thyroid. Nevertheless, there are no reports describing changes of the LCs in the corneal epithelium of TAO patients. In this study, we evaluate the density and morphology of LCs in the cornea of TAO patients by means of in vivo laser scanning confocal microscopy, to determine whether an inflammatory reaction is involved in the pathogenesis of ocular surface disease associated with TAO.
Department of Ophthalmology, Shanghai Changzheng Hospital, Second Military Medical University, 415 Fengyang
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Materials and methods
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Subjects A total of 40 patients with TAO (10 men and 30 women; mean age 41.40 ± 6.86 years; range 30–58 years) were enrolled in this study from the Department of Ophthalmology, Shanghai Changzheng Hospital, from April 2014 to January 2015. The diagnosis of TAO was made based on Bartley criteria.13 TAO activity was evaluated by the clinical activity score (CAS), with active TAO being defined as CAS≥3/7 and inactive TAO defined as CAS≤2/7.14 Patients were excluded from the study if, before the onset of TAO, they had previously suffered from other systemic autoimmune diseases or anterior segment disorders, such as dry-eye syndrome, meibomian gland dysfunction, allergic ocular surface disease, pterygium, corneal dystrophy, and uveitis or had received either systemic or local treatment that might affect ocular surface parameters. The criteria also excluded persons who wore contact lenses or had undergone ophthalmic surgery. During the examination period, none of the patients with TAO required specific ophthalmic assistance. Twenty healthy volunteers (seven men and 13 women; mean age 44.10 ± 7.28 years; range 27–56 years) were included as normal controls. None of the normal controls wore contact lenses, used any topical eye drops or had a history of anterior segment disorders, ocular trauma or surgery. The study was approved by the Ethics Committee of Shanghai Changzheng Hospital, affiliated to the Second Military Medical University, and adhered to the tenets of the Declaration of Helsinki. Informed consent was obtained from each participant.
Clinical evaluation All subjects received a full ophthalmic examination of both eyes, which included visual acuity, intraocular pressure, proptosis, lagophthalmos, slit-lamp examination and fundus examination. The ocular surface damage was assessed by both subjective Ocular Surface Disease Index (OSDI) symptom questionnaires15 and objective examinations, including tear break-up time (BUT), fluorescein staining and Schirmer test I. The corneal fluorescein staining score was evaluated based on grades 0–3 in each of the four quadrants for a total score of 12, according to the grading scale previously described.16 All ophthalmic examinations were performed by the same researcher (J.-W.C.). In addition, all of the participants were evaluated for their smoking habits.
Bausch & Lomb, Rochester, NY) was used as a coupling medium between the applanating lens and the cornea. In vivo confocal microscopy was performed on each cornea in two different areas: central and peripheral (2 mm from the corneoscleral limbus) at 12:00. In vivo laser scanning corneal confocal microscopy was performed bilaterally on each subject by the same researcher (Q.-H.L.) at EYE and ENT Hospital of Fudan University. Three images with a maximum number of target cells from both central and peripheral cornea were evaluated in a masked fashion by the same investigator (X.-Y.M.). Bright corpuscular cells with or without dendrite-like protrusions within the corneal epithelium at the level of basal epithelial cells to subbasal nerve plexus were considered to be LCs.9 After identification of LCs, the cell density in each image (400 × 400 mm2) was calculated using open professional image analysis software Image J (Image J, Version 1.45, National Institutes of Health, Bethesda, MD) plus Counter Cell plugin and recorded as cells per square millimetre. LC morphology (LCM) was evaluated on a 0–3 scale according to the methods previously described.17 A score of 0 described the condition when the cornea was devoid of LCs; a score of 1 was given when cells lacked processes; a score of 2 (small processes) was given if the length of the processes did not exceed the longest diameter of the cell body; and a score of 3 (long processes) was given if the processes were longer than the largest diameter of the cell body. The mean LCM score was calculated and used to describe the maturation of the LCs at both regions of the cornea (Figure 1). Statistical analysis The data of the eye with the higher fluorescein staining score of each participant were included for analysis as previously described by Villani.18 In the event that the two eyes had equal fluorescein staining scores, the sequential discriminating criteria were the lower Schirmer test score and the lower BUT score.18 Data distribution and homogeneity were analyzed. Normally distributed variables were expressed as the mean ±standard deviation, and Student’s t-test was used to compare the mean values between active TAO and inactive TAO groups, and one-way ANOVA with the LSD post hoc test or Tamhane’s T2 test was used to compare the mean scores of the control subjects and patients with active TAO or inactive TAO. Skewed variables were expressed as median values and percentiles (Q1, Q3), and the Mann–Whitney U nonparametric test was applied. Fisher’s exact test was used to analyze the categorical variable. The correlations between the variables were analyzed by using Spearman’s rank correlation test. P < 0.05 was considered statistically significant (SPSS 19.0; IBM, Chicago, IL).
Confocal microscopy In vivo corneal confocal microscopy was performed on all patients with a Heidelberg Retina Tomograph (HRT3) equipped with a Rostock Corneal Module (RCM) (Heidelberg Engineering GmbH, Heidelberg, Germany). The ocular surface was anesthetized with topical anesthetic eye drops (0.5% proxymetacaine hydrochloride, Alcaine; Alcon Laboratories, Fort Worth, TX). Carbomer gel (Vidisic;
Results Clinical data The demographic and clinical features of the active and inactive TAO groups are presented in Table 1. CAS was significantly higher in active TAO compared with inactive TAO (P = 0.001, Student’s t-test). There were no significant
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(A)
(B)
(C)
(D)
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Figure 1. Representative confocal microscopic images of corneal Langerhans cells (LCs) in patients with thyroid-associated ophthalmopathy (TAO) and control subjects. (A) Central cornea of a healthy volunteer. No LC is visible in this area at a depth of 48 μm [LC morphology (LCM) score = 0]. (B) Image of the central cornea of a TAO patient at a depth of 46 μm. The density of LCs is increased compared to that of normal subjects. Most of the LCs are of LCM score 1 (white arrow) and 2 (black arrow). (C) Peripheral cornea of a healthy volunteer with mixed populations of LCs at a depth of 43 μm (empty arrow shows an LCM score 3 LC). (D) Peripheral cornea of a TAO patient with gathering LCs arranged in a network fashion at a depth of 56 μm. Most of the LCs are with processes. Table 2. Comparison of ocular surface parameters of TAO patients and control subjects.
Table 1. Comparison of disease profile of active and inactive TAO patients. Number Gender (male/female) Age (years) Clinical activity score Proptosis (mm) Lagophthalmos (mm) FT3 (pmol/L) FT4 (pmol/L) TSH (mU/L)
Active TAO 15 5/10 43.60 ± 5.51 5.80 ± 1.37 21.13 ± 2.39 1 (0, 3) 5.32 ± 1.83 16.78 ± 4.99 3.66 ± 6.11
Inactive TAO 25 5/20 40.08 ± 7.35 1.16 ± 1.03 19.78 ± 2.14 0 (0, 1.5) 4.59 ± 0.63 15.15 ± 3.70 2.69 ± 2.72
P a
0.457 0.118b 0.000c 0.071d 0.154e 0.155f 0.244g 0.568h
FT3 = free triiodothyronine; FT4 = free thyroxine; TSH = thyroid stimulating hormone. a: Fisher’s exact test. b, c, d, f, g, h: Student’s t-test. e: Mann–Whitney U nonparametric tests.
differences in gender, age, proptosis, lagophthalmos or the levels of free tri-iodothyronine (FT3), free thyroxine (FT4) and thyroid stimulating hormone (TSH) between the active and inactive TAO groups (Table 1). The ocular surface parameters of the three groups are summarized in Table 2. All the patients with TAO included
Age (years) OSDI (score) BUT (seconds) Fluorescein staining (score) Schirmer test (mm)
Active TAO 43.60 ± 5.51 50.00 (41.67, 56.25) 5.11 ± 2.15 3.53 ± 4.05
Inactive TAO 40.08 ± 7.35 27.08 (20.83, 39.58) 6.30 ± 2.99 1.88 ± 2.05
Control P* 44.10 ± 7.28 0.117 0 (0, 1.56) 0.000a
5.50 ± 2.15
6.46 ± 3.02
12.65 ± 1.95 0.000d
11.62 ± 1.67 0.000b 0 0.000c
* ANOVA. a: Active TAO, Inactive TAO vs. control; P = 0.000. Active TAO vs. inactive TAO; P = 0.000. Tamhane’s T2 test. b: Active TAO, Inactive TAO vs. control; P = 0.000. Active TAO vs. inactive TAO; P = 0.138. LSD post hoc test. c: Active TAO vs. control; P = 0.013. Inactive TAO vs. control; P = 0.000. Active TAO vs. inactive TAO; P = 0.403. Tamhane’s T2 test. d: Active TAO, Inactive TAO vs. control; P = 0.000. Active TAO vs. inactive TAO; P = 0.245. LSD post hoc test.
in this study had dry eye.19 OSDI and fluorescein staining scores were significantly higher and BUT and Schirmer test values were significantly lower in patients with active and inactive TAO compared to the normal subjects.
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Smoking habits of the participants were as follows: four (26.7%) smokers, two (13.3%) ex-smokers and nine (60.0%) nonsmokers in the active TAO group; six (24.0%) smokers, two (8.0%) ex-smokers and 17 (68.0%) nonsmokers in the inactive TAO group; seven (35.0%) smokers, one (5.0%) exsmoker and 12 (60.0%) nonsmokers in the control group. There was no statistically significant difference for smoking status among these three groups (P = 0.872).
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Confocal microscopy data Table 3 demonstrates our confocal microscopy results with regard to the different groups. All the active TAO patients presented with LCs in the central cornea as opposed to 19 out of 20 inactive TAO patients and 13 out of 20 control subjects. LC densities dramatically increased in the central cornea in both active and inactive TAO groups compared to the control group (P = 0.024, P = 0.020, respectively). The numbers of LCs in the peripheral cornea were significantly increased in the active TAO group compared to the control group (P = 0.025), whereas they were only slightly increased in the inactive TAO group compared to the control group (P = 0.101). There were no significant differences in LC density in the central or peripheral cornea between the active TAO and inactive TAO groups (Table 3). Morphology is believed to represent maturation of LCs. Central LCM values were significantly higher in both active and inactive TAO groups compared to the control group (P = 0.002, P = 0.019, respectively). Peripheral LCM scores were increased in the active and inactive TAO groups, without statistical significance, compared to the control group (P = 0.112, P = 0.314, respectively). There were no significant differences in central or peripheral LCM scores between the active TAO and inactive TAO groups (Table 3).
Correlations There were significant positive correlations between CAS and OSDI and fluorescein staining scores (r = 0.666, P = 0.000; r = 0.332, P = 0.037, respectively), and significant inverse correlations between CAS and BUT and Schirmer test scores (r = – 0.406, P = 0.009; r = –0.414, P = 0.008, respectively). No Table 3. Comparison of confocal microscopy data of TAO patients and control subjects. LC center (cell/mm2) LC periphery (cell/mm2) LCM center LCM periphery
Active TAO 76.38 ± 67.77
Inactive TAO 47.49 ± 38.58
Control 21.46 ± 21.74
P* 0.002a
131.53 ± 74.18
103.25 ± 62.80
70.21 ± 37.76
0.013b
1.77 ± 0.63 2.66 ± 0.45
1.51 ± 0.63 2.54 ± 0.52
1.01 ± 0.80 2.39 ± 0.46
0.006c 0.270d
* ANOVA. a: Active TAO vs. control; P = 0.024. Inactive TAO vs. control; TAO vs. inactive TAO; P = 0.379. Tamhane’s T2 test. b: Active TAO vs. control; P = 0.025. Inactive TAO vs. control; TAO vs. inactive TAO; P = 0.540. Tamhane’s T2 test. c: Active TAO vs. control; P = 0.002. Inactive TAO vs. control; TAO vs. inactive TAO; P = 0.257. LSD post hoc test. d: Active TAO vs. control; P = 0.112. Inactive TAO vs. control; TAO vs. inactive TAO; P = 0.453. LSD post hoc test.
P = 0.020. Active P = 0.101. Active P = 0.019. Active P = 0.314. Active
significant correlations were found between CAS and FT3, FT4 and TSH levels. There were significant positive correlations between central LC density and CAS and OSDI score (r = 0.330, P = 0.037; r = 0.343, P = 0.007, respectively) and significant inverse correlations between central LC density and the Schirmer test score (r = –0.367, P = 0.004). The central LCM score was positively correlated with CAS and the OSDI score (r = 0.344, P = 0.030; r = 0.340, P = 0.008, respectively) and inversely correlated with the Schirmer test score (r = –0.397, P = 0.002). Peripheral LC density was positively correlated with the OSDI score (r = 0.335, P = 0.009) and inversely correlated with the Schirmer test score (r = –0.291, P = 0.024). No significant correlations were found between the peripheral LCM value and clinical data.
Discussion TAO may cause many ocular surface problems, such as conjunctival chemosis, conjunctival hyperaemia, superficial punctate keratopathy, exposure keratopathy, dry eye, superior limbic keratoconjunctivitis and corneal ulcer.3 Dry eye is one of the leading ocular surface diseases of TAO.3 In this study, all the patients with TAO had clinical signs and symptoms of dry eye. The Schirmer test results were significantly lower and the BUT values were significantly shorter in patients compared to normal subjects, which indicated both a hyposecretory and evaporative mechanism of dry eye associated with TAO. These findings are consistent with those reported by Villani et al.18 and Gürdal et al.7 Lacrimal gland dysfunction has been reported in TAO patients.20 An increase in the palpebral fissure width associated with TAO was thought to lead to accelerated tear evaporation.21 Decreased aqueous tear production and excessive tear evaporation would increase the tear film osmolarity and lead to ocular surface inflammation and damage.19,21 Furthermore, lid retraction and proptosis can result in lid friction on the ocular surface during blinking, leading to mechanical damage.3,22 Fluorescein staining scores were significantly higher in patients with TAO compared to healthy volunteers in this study. Similar results were found previously by Ismailova et al.4 and Huang et al.23 In patients with active TAO, ocular adnexa inflammation is often more severe than in nonspecific dry-eye patients, which indicates that orbital inflammation may also be involved in the ocular surface damage of TAO. In addition, the expression of thyroid hormone receptor was detected in the epithelia of lachrymal gland acini, cornea and conjunctiva, which verified that ocular surface tissue might be the direct target organs for thyroid hormone.3,20,24 However, few studies have reported the corneal changes associated with TAO.18 Confocal microscopy provides a noninvasive method to investigate different cell populations on the ocular surface in vivo at microscopic resolution. Previous studies have demonstrated the changes in corneal inflammatory cells in cases of dry eye,25 rheumatoid arthritis (RA),26 ankylosing spondylitis (AS)27 and systemic lupus erythematosus.17 Those results indicated the corneal involvement in local or systemic inflammatory conditions. TAO is an autoimmune inflammatory disease.
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Villani et al.18 showed that the densities of corneal epitheliums and stromal keratocytes were significantly different between Graves’ orbitopathy (GO) patients and control subjects, and the density of activated keratocytes was significantly higher in active GO than inactive GO. Wei et al.22 reported increased LC density and reduced goblet cell density in the GO group compared to control patients. In this study, in vivo laser scanning confocal microscopy was used to evaluate inflammatory cells, especially on the LCs in the cornea of patients with TAO and healthy volunteers. Results showed that LCs had a markedly higher density in the periphery than in the central cornea in all participants. Compared to the control group, central LC density increased to about 3.5-fold in the active TAO group, whereas the increase in the inactive group was about 2.2-fold. In the peripheral cornea, the LC density increased to about 1.9fold and 1.5-fold in patients with active TAO and inactive TAO, respectively. In addition, the study demonstrated increased LCM values and maturation of LCs in the cornea, especially in the central part, in patients with TAO. Corneal LCs are deeply involved in corneal immunoregulatory processes. They have an extraordinary capacity to stimulate naive T cells and are recognized as essential regulators of both the innate and acquired arms of the immune system.25,28 Under normal conditions, LCs are scarce in the central cornea. Upon different stimuli, LCs migrate centrally and transform into the mature type by the formation of dendrite-like processes and expression of co-stimulatory molecules.28 There are two possible reasons attributed to the centripetal migration and maturation of LCs in the corneal epithelium of TAO patients in this study. The first possible reason is dry eye in patients with TAO. It has been clearly shown in previous studies that dry eye could induce overexpression of the inflammatory cytokines, chemokines and adhesion molecules,29 which are considered to have the capacity to activate LCs.30 In this study, central and peripheral LC density and the central LCM score correlated negatively with tear secretion that was lower in TAO patients than that of normal subjects. Lin et al.25 reported the elevation of LC density in both the central and peripheral parts of the cornea in patients with aqueous tear-deficient dry eye, and this supports the participation of LCs in dryeye pathogenesis.31 The second possible reason responsible for the activation of corneal LCs is the inflammatory reaction associated with TAO. Huang et al.23 reported that interleukin (IL)-1β, IL-6 and IL-8 concentrations are significantly higher in active TAO than inactive TAO and the controls, indicating that orbital inflammation may be involved in ocular surface damage of TAO. In this study, central LC density and the central LCM score correlated positively with the inflammatory activity of the disease as measured by the CAS. Marsovszky et al.27 reported greater corneal LC density and LCM in AS, which correlated with the systemic activity of AS even without ocular symptoms. Another study by Marsovszky et al.26 showed that the prevalence of central and peripheral LCs and the central LCM were higher than normal in RA, even in inactive stages of RA and without ocular symptoms. These data support the theory that these alterations of the corneal LCs are associated with the systemic inflammatory effect of the disease.
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However, the mechanisms of the migration and maturation of corneal LCs in patients with TAO are not yet fully understood. Further evaluations and comparisons of the corneal LCs by in vivo confocal microscopy in dry eye associated with TAO and in nonspecific dry eye are needed. In summary, this study provides evidence of LC participation and corneal inflammation in TAO. Further investigation of the pathogenesis underlying the corneal involvement in TAO at the molecular level is needed.
Funding This study was supported by the National Natural Science Foundation of China (Grant No. 81371056).
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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