Clinical & Experimental Allergy, 44, 362–370
doi: 10.1111/cea.12264
ORIGINAL ARTICLE
© 2013 John Wiley & Sons Ltd
Clinical Mechanisms
An essential role for dendritic cells in vernal keratoconjunctivitis: analysis by laser scanning confocal microscopy M. Liu, H. Gao, T. Wang, S. Wang, S. Li and W. Shi Shandong Eye Hospital, Shandong Eye Institute, Shandong Academy of Medical Sciences, Qingdao, China
Clinical & Experimental Allergy
Correspondence: W. Shi, Shandong Eye Hospital, Shandong Eye Institute, Shandong Academy of Medical Sciences, 5 Yanerdao Road, Qingdao 266071, China. E-mail: [email protected] Cite this as: M. Liu, H. Gao, T. Wang, S. Wang, S. Li and W. Shi, Clinical & Experimental Allergy, 2014 (44) 362– 370.
Summary Background CD4+ T helper type 2 cells play a central role in the pathogenesis of vernal keratoconjunctivitis (VKC), and antigen-presenting cells are required for the cell activation. In this study, we aimed to survey the density, distribution, and morphology of dendritic cells (DCs) in patients with VKC by in vivo confocal microscopy. Methods Thirty-five patients (mean, 12.4 5.3 years) affected by VKC were included. All patients were treated with 0.1% fluorometholone eye drops and 0.5% cyclosporine A eye drops. The density and morphological and distributional characteristics of DCs in each right eye were evaluated by in vivo confocal microscopy before treatment and at 1, 3, and 6 months after treatment. Thirty-five age-matched normal subjects (mean, 16.5 1.8 years) were studied as controls. Results There was significant difference in age between the VKC group and the control group (F = 18.17, P < 0.05). Compared with normal eyes, increased numbers of DCs were found in patients with VKC, with mean cell densities of 244.09 59.76 cells/mm2 at the bulbar conjunctiva, 574.53 87.34 cells/mm2 at the limbus, and 403.32 106.59 cells/ mm2 at the peripheral cornea before treatment. These DCs exhibited a typical dendritic shape. At 3 months after treatment, the DC density at the conjunctiva decreased significantly (P < 0.05), approximating that in the controls. At 3 and 6 months, the DC densities at the limbus and peripheral cornea also decreased significantly (P < 0.05), but were still statistically higher than those in the controls. These DCs, with small dendritic processes or irregular shapes, were observed to gradually locate at the epithelial basal membrane and subbasal nerve plexus. Conclusions In vivo confocal microscopy appears to be a valuable tool in evaluating the dynamic change of DCs at the conjunctiva and cornea. DCs play an essential role in VKC and therefore may constitute a target for therapeutic intervention for VKC. Keywords dendritic cells, laser scanning microscopy, vernal keratoconjunctivitis Submitted 17 April 2013; revised 22 November 2013; accepted 25 November 2013
Introduction Vernal keratoconjunctivitis (VKC) is a chronic allergic eye disease with seasonal recurrence of ocular surface inflammation; it mainly affects children and adolescents. In the past several years, many clinical and experimental studies have shown that activation of antigen-specific CD4+ T helper type 2 cells and their cytokines play a central role in the pathogenesis of VKC [1]. Dendritic cells (DCs), a type of the strongest antigen-presenting cells, are involved in processing antigens presentation and carrying Class II histocompatability antigens in the afferent arm of immunological reaction. In a mouse model of asthma, DCs were found to be attracted to the
bronchial mucosa after an inhaled allergen challenge in sensitized mice [2]. In a mouse model of allergic conjunctivitis, Ohbayashi et al. [3] indicated that conjunctival DC subsets might play an important role in the immune-regulatory processes of allergic conjunctivitis. Clinically and pathophysiologically, the mouse model of allergic conjunctivitis is most similar to human seasonal allergic conjunctivitis. As yet, no murine equivalent of VKC or atopic keratoconjunctivitis (AKC) exists [4]. Conjunctival biopsy specimens obtained from patients with VKC and controls have been usually used to analyse the expression of various molecules in VKC [5–7]. In vivo laser scanning confocal microscopy opens up a promising method to investigate the morphology of
Dendritic cells in vernal keratoconjunctivitis
conjunctiva and cornea under both normal and pathological conditions. This non-invasive, real-time, and three-dimensional observation approach could provide good-quantity images of ocular surface tissues. It was reported that in vivo laser scanning confocal microscopy was used to address the conjunctival and corneal morphological changes in patients with AKC [8] and VKC [9]. In this study, the density, distribution, and morphology of DCs in patients with VKC before and after topical treatment were revealed by in vivo laser scanning confocal microscopy to evaluate the functional role of DCs in VKC. Methods Patients The research adhered to the tenets of the Declaration of Helsinki. Informed written consent was obtained from
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all patients. The protocol was approved by the Institutional Review Board of Shandong Eye Institute. A detailed patient history was recorded for each case. Thirty-five patients (31 males and four females) affected by VKC in the active phase were included. The age ranged from 7 to 30 years (mean, 12.4 5.3 years). All were diagnosed as bilateral VKC with a median duration of 3.34 2.40 years (range, 2 to 10 years). The diagnosis of VKC was based on a history of ocular surface inflammation characterized by itching, photophobia, tearing and mucous discharge, the presence of a papillary reaction on the upper tarsal conjunctiva, and Horner–Trantas dots at the limbus. In addition, conjunctival swab and brush specimens were examined to detect eosinophils and thus to confirm the presence of an active allergic reaction. Slit-lamp microscopic examination was also performed. An activity score, according to the clinical grading of VKC [10] (Table 1), was assigned to each subject. The
Table 1. Grading of symptoms and signs Item
0
1
2
3
Symptoms Itching Tearing
No Normal tear
Occasional Sensation of fullness of the conjunctival sac Mild
Frequent Infrequent spilling of tears over the lid margin Moderate
Constant Constant spilling of tears over the lid margin Severe
Absent
Discomfort (including burning, stinging, and foreign-body sensations) Discharge
No
Small amount of mucoid discharge
Moderate amount of mucoid discharge, presence of crust upon awakening
Photophobia
No
Mild
Moderate, necessitating dark glasses
Eyelids tightly matted together on awakening, warm soaks necessary to clean eyelids during day Extreme photophobia, even with dark glasses
No
One quadrant
Two quadrants
Three or more
Absent
Mild
Moderate
Severe
No
Mild
Moderate
No
One quadrant
Two quadrants, macro erosion
Severe, obscuring the visualization of the deep tarsal vessels Three or more quadrants, vernal ulcer
No new Vessel formation No
Neovascularization in 1 quadrant of cornea
Neovascularization in 2 Quadrants of cornea
Neovascularization in 3 or more quadrants of cornea
Presence of subepithelial fibrosis
Presence of fornix foreshortening
Symblepharon formation
Mild redness and oedema of the eyelid with meibomian gland dysfunction
Moderate inflammation with hyperaemia, scales and scurf of eyelid skin
Severe inflammation, with cracks in the eyelid skin, loss of eyelashes, and lid oedema
Signs Limbal hypertrophy Bulbar conjunctival Hyperaemia Tarsal conjunctival Papillary hypertrophy
Keratitis (superficial epithelial keratitis, punctate staining of the cornea with fluorescein, erosions, ulcer) Neovascularization of cornea
Cicatrizing conjunctivitis (superficial scarring of the conjunctiva) Blepharitis
No
© 2013 John Wiley & Sons Ltd, Clinical & Experimental Allergy, 44 : 362–370
364 M. Liu et al clinical score (0–3: 0 = absent; 3 = severe) was given on the basis of the severity of eye symptoms, including itching, tearing, discomfort, mucous discharge, and photophobia, as well as the severity of clinical signs, including bulbar conjunctival hyperaemia, upper tarsal conjunctival papillae, punctate keratitis, corneal neovascularization, cicatrizing conjunctivitis, and blepharitis. All patients were scored weekly for 4 weeks, monthly for a minimum of 6 months, and at different intervals thereafter. In vivo laser scanning confocal microscopy Each patient was examined with a digital corneal confocal laser scanning microscope (Heidelberg retina tomograph/Rostock cornea module, Heidelberg Engineering GmbH, Dossenheim, Germany). This laser scanning microscope could allow for an automatic z-scan determination of focus depth within the cornea, and provide the collection and storage of high-contrast digital images of all corneal layers, derived from manual frame acquisition or from automatic z-scan of sequential images for a depth range of 80 lm beginning from the reference layer. The right eyes of all patients were chosen for a standard procedure of examination. Briefly, one drop of 0.4% oxybuprocaine hydrochloride (Benoxil; Santen, Osaka, Japan) was instilled into the lower conjunctival sac of each eye. The patient was then required to put his head in the headrest and fixate at a target. Sequential images derived from automatic scans and manual frame acquisitions were obtained from each examined eye. The ‘section mode’ function of the instrument helped to search for DCs within the conjunctival and corneal epithelial layer, enabling instantaneous imaging of a single area of the cornea at a desired depth. The areas evaluated were superior (12 o’clock) bulbar conjunctiva, superior limbus, and peripheral cornea before treatment and at 1, 3, and 6 months after treatment. The images were analysed by a masked observer. Density, morphology, and distribution of DCs at the bulbar conjunctiva, limbus, and peripheral cornea were observed. The cells were morphologically identified as bright cellular images with a branching dendritic shape. The cell density was calculated, using the analysis software of the instrument, by averaging numbers of cells from five images in each position (randomly selected among the recorded images), counted manually within a region of 0.15 mm2 interest of standardized dimensions. The characteristics of DC density, morphology, and distribution at the bulbar conjunctiva, limbus, and peripheral cornea were also evaluated in 35 age-matched healthy subjects (21 males and 14 females; aged 13–20 years, mean 16.5 1.8 years). These subjects
were absent of current or previous local and systemic disease that could have affected the conjunctiva and cornea, and had no history of contact lens use. Topical medications Topical 0.1% fluorometholone (flumetholon, Santen, Osaka, Japan) was administered four times a day, then twice daily when the score of bulbar conjunctiva hyperaemia was one or less, and finally stopped when the total scores of signs and symptoms were less than 5. Topical 0.5% cyclosporine A (CsA) (Northern China Pharmaceutical, Zhengzhou, China) was given four times daily and then tapered to twice daily until the total score of signs and symptoms was < 5 for at least 6 months. Statistical analysis Statistical analysis was performed using SPSS 17.0 for Windows. Scores for pre- and post-treatment were analysed by variance analysis. DC densities were expressed as mean standard deviations (SD). An analysis of variance was performed to compare the density of DCs. A P value of < 0.05 was considered statistically significant. Results There was significant difference in age between the VKC group and the control group (F = 18.17, P < 0.05). At 6 months after treatment, the mean total symptom score was 0.66 1.03, and the mean total sign score was 1.94 1.21 in eyes with VKC, both significantly lower than those before treatment (7.03 1.77 and 7.4 1.67, respectively, P < 0.05; Table 2). The improvement in symptoms presented earlier than that of signs (Fig. 1). High-power microscopy, which was used to evaluate the conjunctival swab and brush specimens, revealed no eosinophils at the normal conjunctiva but significant infiltration of eosinophils at the conjunctiva of eyes
Table 2. Scores of symptoms and signs
Pre-treatment 1 month post-treatment 3 months post-treatment 6 months post-treatment F value
Symptom scores
Sign scores
Total scores
7.03 1.77 4.11 1.30 2.23 1.40 0.66 1.03 F = 134.08; P < 0.05
7.4 1.67 5.63 1.99 3.46 1.72 1.94 1.21 F = 72.25; P < 0.05
14.43 3.06 9.74 2.86 5.69 2.69 2.6 1.93 F = 129.05; P < 0.05
Values are means standard deviation. © 2013 John Wiley & Sons Ltd, Clinical & Experimental Allergy, 44 : 362–370
Dendritic cells in vernal keratoconjunctivitis Symptoms scores
8
Signs scores
With respect to morphology, DCs were highly reflective cells up to about 40 lm in size, with evident dendritic processes, and frequently distributed in clusters in patients with VKC. In normal eyes, DCs were reflective corpuscular cellular objects with small dendritic processes. Moreover, DCs were imaged with small dendritic processes or irregular cells and were observed to gradually locate at the epithelial basal membrane and subbasal nerve plexus at the end of treatment (Figs 4 and 5). No significant complication related to topical drug administration or disease recurrence was observed during the follow-up period.
7 6 5 4 3 2 1 0
Pretreatment
1 month
3 month
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6 month
Fig. 1. Changes in scores for symptoms and signs.
Discussion
with VKC before treatment. At 1 month after treatment, infiltrated eosinophils disappeared. Under the in vivo laser scanning confocal microscope, DCs appeared as bright dense images with dendritic processes, distributing between a zone of 20 and 35 lm at the level of the basal epithelial cells with a mean density of 18.06 5.18 cells/mm2 at the bulbar conjunctiva in normal eyes and 244.09 59.76 cells/mm2 in eyes with VKC. At the limbus and peripheral cornea, DCs were observed between a zone of 30 and 65 lm, extending from the basal epithelial cells, epithelial basal membrane, and sub basal nerve plexus. The mean density of DCs at the limbus was 183.16 37.38 cells/ mm2 in normal eyes and 574.53 87.34 cells/mm2 in eyes with VKC, while at the peripheral cornea, it was 83.37 19.06 cells/mm2 and 403.32 106.59 cells/ mm2, respectively. There was significant difference in the mean DCs density between the VKC group and the control group. The mean density of DCs in patients with VKC decreased significantly at the bulbar conjunctiva at 1 month after treatment compared with that before treatment (244.09 59.76 cells/mm2 vs. 43.79 9.15 cells/mm2, P < 0.05). At 3 months, the mean cell density in patients with VKC was similar to normal subjects. However, no significant difference was detected in the number of DCs between before and at 1 month after treatment at the limbus (574.53 87.34 cells/mm2 vs. 536.22 82.17 cells/mm2, P > 0.05) and at the peripheral cornea (403.32 106.59 cells/mm2 vs. 355.91 93.02 cells/mm2, P > 0.05). The mean DC density was found to decrease significantly at the limbus and peripheral cornea at 3 and 6 months (all P < 0.05). The limbal and peripheral DC densities at 6 months were still greater than that in normal eyes (P < 0.05). The mean densities of DCs before and after treatment period at the bulbar conjunctiva, limbus, and peripheral cornea are shown in Tables 3, 4 and 5; Fig. 2. Trends are also observable in DC density categories (Tables 3, 4 and 5; Fig. 3).
VKC is a common allergic disease with intense ocular surface symptoms and corneal involvement. DCs are crucial in determining the nature of allergic response [4]. Klein et al. [11] found that patients with perennial allergic rhinitis had a higher number of CD1a+ and CD11c+ MHC class II+ DCs in the epithelium and in the lamina propria of their nasal mucosa than did healthy controls. Substantial numbers of Langerhans’ cells (LCs) were also found in the skin of patients with atopic dermatitis [12]. Conjunctival biopsy specimens taken from AKC and VKC showed higher counts for DCs markers, including HLA-DR (MHC class II), FceRI, and CD1a than healthy control subjects [5]. Mastropasqua and coworkers [13] investigated DC distribution in the inflamed cornea, showing that patients with VKC, when compared with normal controls, had much more DC expression. In our large series of patients with VKC, there were increased numbers of DCs in the lamina propria and epithelium compared with those seen in healthy subjects. Using similar devices, the distribution of DCs within the bulbar conjunctiva, limbus, and peripheral cornea of normal human eyes was previously
© 2013 John Wiley & Sons Ltd, Clinical & Experimental Allergy, 44 : 362–370
Fig. 2. Mean densities of dendritic cells at the bulbar conjunctiva, limbus, and peripheral cornea.
366 M. Liu et al Table 3. Dendritic cell density level category at the limbus
Dendritic cell density Values Mean SD (cells/mm2) Categories < 200 cells/mm2, n (%) 200–400 cells/mm2, n (%) 400–600 cells/mm2, n (%) ≥ 600 cells/mm2, n (%)
Normal
Pre-treatment
1 month post-treatment
3 months post-treatment
6 months post-treatment
183.16 37.38
574.53 87.34
536.22 82.17
329.72 79.95
292.10 88.76
23 (66%) 12 (34%) 0 0
0 4 (11%) 25 (72%) 6 (17%)
0 6 (17%) 25 (72%) 4 (11%)
0 33 (94%) 2 (6%) 0
3 (9%) 32 (91%) 0 0
SD, standard deviation.
Table 4. Dendritic cell density level category at the peripheral cornea
Dendritic cell density Values Mean SD (cells/mm2) Categories < 200 cells/mm2, n (%) 200–400 cells/mm2, n (%) 400–600 cells/mm2, n (%)
Normal
Pre-treatment
1 month post-treatment
3 months post-treatment
6 months post-treatment
83.37 19.06
403.32 106.59
355.91 93.02
294.47 65.75
225.73 72.67
35 (100%) 0 0
0 18 (51%) 17 (49%)
0 26 (74%) 09 (26%)
2 (6%) 33 (34%) 0
5 (14%) 30 (86%) 0
SD, standard deviation.
Table 5. Dendritic cell density level category at the bulbar conjunctiva
Dendritic cell density Values Mean SD (cells/mm2) Categories < 50 cells/mm2, n (%) 50–100 cells/mm2, n (%) 100–150 cells/mm2, n (%) 150–200 cells/mm2, n (%) ≥ 200 cells/mm2, n (%)
Normal
Pre-treatment
1 month post-treatment
3 months post-treatment
6 months post-treatment
18.06 5.18
244.09 59.76
43.79 9.15
22.56 7.78
19.43 6.23
23 (66%) 12 (34%) 0 0 0
35 (100%) 0 0 0 0
35 (100%) 0 0 0 0
35 (100%) 0 0 0 0
0 0 0 9 (26%) 26 (74%)
SD, standard deviation.
detected, with mean cellular densities of 22 25 cells/ mm2, 288 102 cells/mm2, and 98 8 cells/mm2, respectively [14–16]. It has been known that DCs in the limbus can be recruited into the cornea by appropriate inflammatory or immunogenic stimuli [17]. The greater density of DCs in patients with VKC may be secondary to cell migration into the cornea from the limbus stimulated by immunogenic stimuli. By far, no studies have determined the role of DCs in the development of VKC before and after treatment. In this study, the density of DCs in VKC decreased significantly at the bulbar conjunctiva, limbus, and peripheral cornea after topical treatment, but till the end of the follow-up, the densities of DCs at the limbus and peripheral cornea of patients with VKC remained higher than that in normal eyes. Moreover, we found that
these DCs were small in size, with small dendritic processes or no processes, which may reflect the immaturities of DCs. Two populations of DCs, MHC class II positive (mature) cells and MHC class II negative (immature) cells, were observed to be in the normal murine corneal epithelium by immunofluorescence [18, 19]. Guthoff et al. [14] discovered by in vivo confocal microscopy that LCs in normal human eyes presented as either large cells bearing long processes or smaller cells lacking cell dendrites, presumably indicating mature and immature phenotypes, respectively. Mature DCs may have a slender nucleated cell body, from which extends a maze of long processes resembling dendrites of nerve cells. Immature DCs may have a large cell body with fewer and shorter processes, able to capture and process antigens but unable to present © 2013 John Wiley & Sons Ltd, Clinical & Experimental Allergy, 44 : 362–370
Dendritic cells in vernal keratoconjunctivitis
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(a)
(b)
(c)
Fig. 3. The density level category of dendritic cells at the bulbar conjunctiva (a), limbus (b), and peripheral cornea (c) in patients with VKC.
antigens to lymphocytes. With appropriate stimuli derived from an inflamed microenvironment, they can change into the mature phenotype capable of presenting antigens and costimulatory signals to lymphocytes. Therefore, DCs remaining on the ocular surface of patients with VKC may be more sensitive to future reactivation of the disease and change into the mature © 2013 John Wiley & Sons Ltd, Clinical & Experimental Allergy, 44 : 362–370
phenotype, which can lead to the recurrence of VKC. Further investigations are needed to support this hypothesis. To date, precise characterization of ocular surface, including epithelial DCs, has been performed mainly in vitro by means of immuno-histochemical staining of tissues. Conversely, these techniques have the limitation
368 M. Liu et al (a)
(b)
(c)
Fig. 4. In vivo confocal microscopy images. Dendritic cells at the superior bulbar conjunctiva (a), limbus (b), and peripheral cornea (c) in normal eyes.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
(k)
(l)
Fig. 5. In vivo confocal microscopy images of dendritic cells at the bulbar conjunctiva, limbus, and peripheral cornea in patients with VKC. Numerous dendritic-shaped cells with typical branching dendrites are visible at the bulbar conjunctiva (a), limbus (b), and peripheral cornea (c) before the treatment. At 1 month after treatment, the dendritic cell density at the bulbar conjunctiva decreased significantly (d); however, at the limbus (e) and peripheral cornea (f), no significant reduction is detected. At 3 months, dendritic cells with small branching dendrites are visible at the limbus (h) and peripheral cornea (i), and their densities decrease significantly. At 6 months, the cell densities at the limbus (k) and peripheral cornea (l) further decrease, but the branching dendritic shape is maintained.
that the full morphology of the cells is rarely seen in tissue sections. Confocal microscopy is a rapid, non-invasive, high-resolution technique used to investigate in vivo the morphologic features of normal and pathologic human ocular surface. It provides a universal tool to assess ocular architecture and morphology with properties similar to those provided by conventional histopathology, without the invasive preparation and need for biopsy. Previous reports confirmed that
DCs could be evaluated on the morphological basis by laser scanning confocal microscopy [20], with a characteristic morphology distinguishing them from other cells and structures in the epithelial and subbasal layers [20–23]. Recent advances of laser in vivo confocal microscopes have made it possible to image, with high magnification and resolution, the microscopic architecture of the transparent corneal tissues and non-transparent structures such as limbal and conjunctival epithelium. Although mature DC morphology is typical, and immature cells featuring a mild branching dendritic shape might also be interpreted as macrophages, this was unlikely in the present study, as the latter are known to be present in lower densities and almost exclusively restricted to the posterior stroma of the cornea [13]. Only cells in the epithelium and subbasal nerve plexus were involved in our study. Topical corticosteroids remain the mainstay of treatment for VKC, producing significant improvement in acute symptoms, for its dramatic inhibition of DC differentiation and maturation [24]. However, prolonged use of steroids may result in many complications. In this study, corticosteroids were only administered in the initially acute stage. Topical CsA in different concentrations has been shown to be beneficial in the management of several allergic eye diseases [25–28]. It is an immunomodulator that inhibits CD4 T lymphocyte proliferation by inhibiting interleukin-2 receptor expression. Akpek et al. [29] reported favourable results of topical CsA in the treatment of patients with AKC, without any side-effects. The longest follow-up for topical CsA was 6 months [10], while in our study, eight patients were observed for over 6 months. We found that the densities of DCs in patients with VKC continued to decrease, with density and morphology differing from the normal eyes. DCs remaining at the patients may be more responsive to lower doses of therapy. Hence, topical CsA may be long-term used for patients with VKC. Further studies with more patients and longer follow-up periods are needed to confirm our opinion and to determine the long-term safety of topical CsA in this setting. © 2013 John Wiley & Sons Ltd, Clinical & Experimental Allergy, 44 : 362–370
Dendritic cells in vernal keratoconjunctivitis
In conclusion, in vivo confocal microscopy appears to be a valuable tool in evaluating the dynamic change in DCs at the conjunctiva and cornea. The number and maturation state of DCs are increased at the conjunctiva and cornea of patients with VKC, which can be controlled by topical use of corticosteroids and CsA. Therefore, DCs can play a crucial role in the pathogenesis of VKC. Targeting the function of DCs might be useful for therapeutic intervention. Acknowledgement The authors thank Ms. Ping Lin for her editorial assistance.
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Sources of funding Supported by grants from the National Natural Science Foundation of China (81170815), the Natural Science Foundation of Shandong Province (ZR2012HQ041), the National Basic Research Program of China (2013CB967004), and the Taishan Scholar Program (20081148). Conflict of interest The authors report no conflict of interests. The authors alone are responsible for the content and writing of the article.
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© 2013 John Wiley & Sons Ltd, Clinical & Experimental Allergy, 44 : 362–370
doi: 10.1111/cea.12264
ORIGINAL ARTICLE
© 2013 John Wiley & Sons Ltd
Clinical Mechanisms
An essential role for dendritic cells in vernal keratoconjunctivitis: analysis by laser scanning confocal microscopy M. Liu, H. Gao, T. Wang, S. Wang, S. Li and W. Shi Shandong Eye Hospital, Shandong Eye Institute, Shandong Academy of Medical Sciences, Qingdao, China
Clinical & Experimental Allergy
Correspondence: W. Shi, Shandong Eye Hospital, Shandong Eye Institute, Shandong Academy of Medical Sciences, 5 Yanerdao Road, Qingdao 266071, China. E-mail: [email protected] Cite this as: M. Liu, H. Gao, T. Wang, S. Wang, S. Li and W. Shi, Clinical & Experimental Allergy, 2014 (44) 362– 370.
Summary Background CD4+ T helper type 2 cells play a central role in the pathogenesis of vernal keratoconjunctivitis (VKC), and antigen-presenting cells are required for the cell activation. In this study, we aimed to survey the density, distribution, and morphology of dendritic cells (DCs) in patients with VKC by in vivo confocal microscopy. Methods Thirty-five patients (mean, 12.4 5.3 years) affected by VKC were included. All patients were treated with 0.1% fluorometholone eye drops and 0.5% cyclosporine A eye drops. The density and morphological and distributional characteristics of DCs in each right eye were evaluated by in vivo confocal microscopy before treatment and at 1, 3, and 6 months after treatment. Thirty-five age-matched normal subjects (mean, 16.5 1.8 years) were studied as controls. Results There was significant difference in age between the VKC group and the control group (F = 18.17, P < 0.05). Compared with normal eyes, increased numbers of DCs were found in patients with VKC, with mean cell densities of 244.09 59.76 cells/mm2 at the bulbar conjunctiva, 574.53 87.34 cells/mm2 at the limbus, and 403.32 106.59 cells/ mm2 at the peripheral cornea before treatment. These DCs exhibited a typical dendritic shape. At 3 months after treatment, the DC density at the conjunctiva decreased significantly (P < 0.05), approximating that in the controls. At 3 and 6 months, the DC densities at the limbus and peripheral cornea also decreased significantly (P < 0.05), but were still statistically higher than those in the controls. These DCs, with small dendritic processes or irregular shapes, were observed to gradually locate at the epithelial basal membrane and subbasal nerve plexus. Conclusions In vivo confocal microscopy appears to be a valuable tool in evaluating the dynamic change of DCs at the conjunctiva and cornea. DCs play an essential role in VKC and therefore may constitute a target for therapeutic intervention for VKC. Keywords dendritic cells, laser scanning microscopy, vernal keratoconjunctivitis Submitted 17 April 2013; revised 22 November 2013; accepted 25 November 2013
Introduction Vernal keratoconjunctivitis (VKC) is a chronic allergic eye disease with seasonal recurrence of ocular surface inflammation; it mainly affects children and adolescents. In the past several years, many clinical and experimental studies have shown that activation of antigen-specific CD4+ T helper type 2 cells and their cytokines play a central role in the pathogenesis of VKC [1]. Dendritic cells (DCs), a type of the strongest antigen-presenting cells, are involved in processing antigens presentation and carrying Class II histocompatability antigens in the afferent arm of immunological reaction. In a mouse model of asthma, DCs were found to be attracted to the
bronchial mucosa after an inhaled allergen challenge in sensitized mice [2]. In a mouse model of allergic conjunctivitis, Ohbayashi et al. [3] indicated that conjunctival DC subsets might play an important role in the immune-regulatory processes of allergic conjunctivitis. Clinically and pathophysiologically, the mouse model of allergic conjunctivitis is most similar to human seasonal allergic conjunctivitis. As yet, no murine equivalent of VKC or atopic keratoconjunctivitis (AKC) exists [4]. Conjunctival biopsy specimens obtained from patients with VKC and controls have been usually used to analyse the expression of various molecules in VKC [5–7]. In vivo laser scanning confocal microscopy opens up a promising method to investigate the morphology of
Dendritic cells in vernal keratoconjunctivitis
conjunctiva and cornea under both normal and pathological conditions. This non-invasive, real-time, and three-dimensional observation approach could provide good-quantity images of ocular surface tissues. It was reported that in vivo laser scanning confocal microscopy was used to address the conjunctival and corneal morphological changes in patients with AKC [8] and VKC [9]. In this study, the density, distribution, and morphology of DCs in patients with VKC before and after topical treatment were revealed by in vivo laser scanning confocal microscopy to evaluate the functional role of DCs in VKC. Methods Patients The research adhered to the tenets of the Declaration of Helsinki. Informed written consent was obtained from
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all patients. The protocol was approved by the Institutional Review Board of Shandong Eye Institute. A detailed patient history was recorded for each case. Thirty-five patients (31 males and four females) affected by VKC in the active phase were included. The age ranged from 7 to 30 years (mean, 12.4 5.3 years). All were diagnosed as bilateral VKC with a median duration of 3.34 2.40 years (range, 2 to 10 years). The diagnosis of VKC was based on a history of ocular surface inflammation characterized by itching, photophobia, tearing and mucous discharge, the presence of a papillary reaction on the upper tarsal conjunctiva, and Horner–Trantas dots at the limbus. In addition, conjunctival swab and brush specimens were examined to detect eosinophils and thus to confirm the presence of an active allergic reaction. Slit-lamp microscopic examination was also performed. An activity score, according to the clinical grading of VKC [10] (Table 1), was assigned to each subject. The
Table 1. Grading of symptoms and signs Item
0
1
2
3
Symptoms Itching Tearing
No Normal tear
Occasional Sensation of fullness of the conjunctival sac Mild
Frequent Infrequent spilling of tears over the lid margin Moderate
Constant Constant spilling of tears over the lid margin Severe
Absent
Discomfort (including burning, stinging, and foreign-body sensations) Discharge
No
Small amount of mucoid discharge
Moderate amount of mucoid discharge, presence of crust upon awakening
Photophobia
No
Mild
Moderate, necessitating dark glasses
Eyelids tightly matted together on awakening, warm soaks necessary to clean eyelids during day Extreme photophobia, even with dark glasses
No
One quadrant
Two quadrants
Three or more
Absent
Mild
Moderate
Severe
No
Mild
Moderate
No
One quadrant
Two quadrants, macro erosion
Severe, obscuring the visualization of the deep tarsal vessels Three or more quadrants, vernal ulcer
No new Vessel formation No
Neovascularization in 1 quadrant of cornea
Neovascularization in 2 Quadrants of cornea
Neovascularization in 3 or more quadrants of cornea
Presence of subepithelial fibrosis
Presence of fornix foreshortening
Symblepharon formation
Mild redness and oedema of the eyelid with meibomian gland dysfunction
Moderate inflammation with hyperaemia, scales and scurf of eyelid skin
Severe inflammation, with cracks in the eyelid skin, loss of eyelashes, and lid oedema
Signs Limbal hypertrophy Bulbar conjunctival Hyperaemia Tarsal conjunctival Papillary hypertrophy
Keratitis (superficial epithelial keratitis, punctate staining of the cornea with fluorescein, erosions, ulcer) Neovascularization of cornea
Cicatrizing conjunctivitis (superficial scarring of the conjunctiva) Blepharitis
No
© 2013 John Wiley & Sons Ltd, Clinical & Experimental Allergy, 44 : 362–370
364 M. Liu et al clinical score (0–3: 0 = absent; 3 = severe) was given on the basis of the severity of eye symptoms, including itching, tearing, discomfort, mucous discharge, and photophobia, as well as the severity of clinical signs, including bulbar conjunctival hyperaemia, upper tarsal conjunctival papillae, punctate keratitis, corneal neovascularization, cicatrizing conjunctivitis, and blepharitis. All patients were scored weekly for 4 weeks, monthly for a minimum of 6 months, and at different intervals thereafter. In vivo laser scanning confocal microscopy Each patient was examined with a digital corneal confocal laser scanning microscope (Heidelberg retina tomograph/Rostock cornea module, Heidelberg Engineering GmbH, Dossenheim, Germany). This laser scanning microscope could allow for an automatic z-scan determination of focus depth within the cornea, and provide the collection and storage of high-contrast digital images of all corneal layers, derived from manual frame acquisition or from automatic z-scan of sequential images for a depth range of 80 lm beginning from the reference layer. The right eyes of all patients were chosen for a standard procedure of examination. Briefly, one drop of 0.4% oxybuprocaine hydrochloride (Benoxil; Santen, Osaka, Japan) was instilled into the lower conjunctival sac of each eye. The patient was then required to put his head in the headrest and fixate at a target. Sequential images derived from automatic scans and manual frame acquisitions were obtained from each examined eye. The ‘section mode’ function of the instrument helped to search for DCs within the conjunctival and corneal epithelial layer, enabling instantaneous imaging of a single area of the cornea at a desired depth. The areas evaluated were superior (12 o’clock) bulbar conjunctiva, superior limbus, and peripheral cornea before treatment and at 1, 3, and 6 months after treatment. The images were analysed by a masked observer. Density, morphology, and distribution of DCs at the bulbar conjunctiva, limbus, and peripheral cornea were observed. The cells were morphologically identified as bright cellular images with a branching dendritic shape. The cell density was calculated, using the analysis software of the instrument, by averaging numbers of cells from five images in each position (randomly selected among the recorded images), counted manually within a region of 0.15 mm2 interest of standardized dimensions. The characteristics of DC density, morphology, and distribution at the bulbar conjunctiva, limbus, and peripheral cornea were also evaluated in 35 age-matched healthy subjects (21 males and 14 females; aged 13–20 years, mean 16.5 1.8 years). These subjects
were absent of current or previous local and systemic disease that could have affected the conjunctiva and cornea, and had no history of contact lens use. Topical medications Topical 0.1% fluorometholone (flumetholon, Santen, Osaka, Japan) was administered four times a day, then twice daily when the score of bulbar conjunctiva hyperaemia was one or less, and finally stopped when the total scores of signs and symptoms were less than 5. Topical 0.5% cyclosporine A (CsA) (Northern China Pharmaceutical, Zhengzhou, China) was given four times daily and then tapered to twice daily until the total score of signs and symptoms was < 5 for at least 6 months. Statistical analysis Statistical analysis was performed using SPSS 17.0 for Windows. Scores for pre- and post-treatment were analysed by variance analysis. DC densities were expressed as mean standard deviations (SD). An analysis of variance was performed to compare the density of DCs. A P value of < 0.05 was considered statistically significant. Results There was significant difference in age between the VKC group and the control group (F = 18.17, P < 0.05). At 6 months after treatment, the mean total symptom score was 0.66 1.03, and the mean total sign score was 1.94 1.21 in eyes with VKC, both significantly lower than those before treatment (7.03 1.77 and 7.4 1.67, respectively, P < 0.05; Table 2). The improvement in symptoms presented earlier than that of signs (Fig. 1). High-power microscopy, which was used to evaluate the conjunctival swab and brush specimens, revealed no eosinophils at the normal conjunctiva but significant infiltration of eosinophils at the conjunctiva of eyes
Table 2. Scores of symptoms and signs
Pre-treatment 1 month post-treatment 3 months post-treatment 6 months post-treatment F value
Symptom scores
Sign scores
Total scores
7.03 1.77 4.11 1.30 2.23 1.40 0.66 1.03 F = 134.08; P < 0.05
7.4 1.67 5.63 1.99 3.46 1.72 1.94 1.21 F = 72.25; P < 0.05
14.43 3.06 9.74 2.86 5.69 2.69 2.6 1.93 F = 129.05; P < 0.05
Values are means standard deviation. © 2013 John Wiley & Sons Ltd, Clinical & Experimental Allergy, 44 : 362–370
Dendritic cells in vernal keratoconjunctivitis Symptoms scores
8
Signs scores
With respect to morphology, DCs were highly reflective cells up to about 40 lm in size, with evident dendritic processes, and frequently distributed in clusters in patients with VKC. In normal eyes, DCs were reflective corpuscular cellular objects with small dendritic processes. Moreover, DCs were imaged with small dendritic processes or irregular cells and were observed to gradually locate at the epithelial basal membrane and subbasal nerve plexus at the end of treatment (Figs 4 and 5). No significant complication related to topical drug administration or disease recurrence was observed during the follow-up period.
7 6 5 4 3 2 1 0
Pretreatment
1 month
3 month
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6 month
Fig. 1. Changes in scores for symptoms and signs.
Discussion
with VKC before treatment. At 1 month after treatment, infiltrated eosinophils disappeared. Under the in vivo laser scanning confocal microscope, DCs appeared as bright dense images with dendritic processes, distributing between a zone of 20 and 35 lm at the level of the basal epithelial cells with a mean density of 18.06 5.18 cells/mm2 at the bulbar conjunctiva in normal eyes and 244.09 59.76 cells/mm2 in eyes with VKC. At the limbus and peripheral cornea, DCs were observed between a zone of 30 and 65 lm, extending from the basal epithelial cells, epithelial basal membrane, and sub basal nerve plexus. The mean density of DCs at the limbus was 183.16 37.38 cells/ mm2 in normal eyes and 574.53 87.34 cells/mm2 in eyes with VKC, while at the peripheral cornea, it was 83.37 19.06 cells/mm2 and 403.32 106.59 cells/ mm2, respectively. There was significant difference in the mean DCs density between the VKC group and the control group. The mean density of DCs in patients with VKC decreased significantly at the bulbar conjunctiva at 1 month after treatment compared with that before treatment (244.09 59.76 cells/mm2 vs. 43.79 9.15 cells/mm2, P < 0.05). At 3 months, the mean cell density in patients with VKC was similar to normal subjects. However, no significant difference was detected in the number of DCs between before and at 1 month after treatment at the limbus (574.53 87.34 cells/mm2 vs. 536.22 82.17 cells/mm2, P > 0.05) and at the peripheral cornea (403.32 106.59 cells/mm2 vs. 355.91 93.02 cells/mm2, P > 0.05). The mean DC density was found to decrease significantly at the limbus and peripheral cornea at 3 and 6 months (all P < 0.05). The limbal and peripheral DC densities at 6 months were still greater than that in normal eyes (P < 0.05). The mean densities of DCs before and after treatment period at the bulbar conjunctiva, limbus, and peripheral cornea are shown in Tables 3, 4 and 5; Fig. 2. Trends are also observable in DC density categories (Tables 3, 4 and 5; Fig. 3).
VKC is a common allergic disease with intense ocular surface symptoms and corneal involvement. DCs are crucial in determining the nature of allergic response [4]. Klein et al. [11] found that patients with perennial allergic rhinitis had a higher number of CD1a+ and CD11c+ MHC class II+ DCs in the epithelium and in the lamina propria of their nasal mucosa than did healthy controls. Substantial numbers of Langerhans’ cells (LCs) were also found in the skin of patients with atopic dermatitis [12]. Conjunctival biopsy specimens taken from AKC and VKC showed higher counts for DCs markers, including HLA-DR (MHC class II), FceRI, and CD1a than healthy control subjects [5]. Mastropasqua and coworkers [13] investigated DC distribution in the inflamed cornea, showing that patients with VKC, when compared with normal controls, had much more DC expression. In our large series of patients with VKC, there were increased numbers of DCs in the lamina propria and epithelium compared with those seen in healthy subjects. Using similar devices, the distribution of DCs within the bulbar conjunctiva, limbus, and peripheral cornea of normal human eyes was previously
© 2013 John Wiley & Sons Ltd, Clinical & Experimental Allergy, 44 : 362–370
Fig. 2. Mean densities of dendritic cells at the bulbar conjunctiva, limbus, and peripheral cornea.
366 M. Liu et al Table 3. Dendritic cell density level category at the limbus
Dendritic cell density Values Mean SD (cells/mm2) Categories < 200 cells/mm2, n (%) 200–400 cells/mm2, n (%) 400–600 cells/mm2, n (%) ≥ 600 cells/mm2, n (%)
Normal
Pre-treatment
1 month post-treatment
3 months post-treatment
6 months post-treatment
183.16 37.38
574.53 87.34
536.22 82.17
329.72 79.95
292.10 88.76
23 (66%) 12 (34%) 0 0
0 4 (11%) 25 (72%) 6 (17%)
0 6 (17%) 25 (72%) 4 (11%)
0 33 (94%) 2 (6%) 0
3 (9%) 32 (91%) 0 0
SD, standard deviation.
Table 4. Dendritic cell density level category at the peripheral cornea
Dendritic cell density Values Mean SD (cells/mm2) Categories < 200 cells/mm2, n (%) 200–400 cells/mm2, n (%) 400–600 cells/mm2, n (%)
Normal
Pre-treatment
1 month post-treatment
3 months post-treatment
6 months post-treatment
83.37 19.06
403.32 106.59
355.91 93.02
294.47 65.75
225.73 72.67
35 (100%) 0 0
0 18 (51%) 17 (49%)
0 26 (74%) 09 (26%)
2 (6%) 33 (34%) 0
5 (14%) 30 (86%) 0
SD, standard deviation.
Table 5. Dendritic cell density level category at the bulbar conjunctiva
Dendritic cell density Values Mean SD (cells/mm2) Categories < 50 cells/mm2, n (%) 50–100 cells/mm2, n (%) 100–150 cells/mm2, n (%) 150–200 cells/mm2, n (%) ≥ 200 cells/mm2, n (%)
Normal
Pre-treatment
1 month post-treatment
3 months post-treatment
6 months post-treatment
18.06 5.18
244.09 59.76
43.79 9.15
22.56 7.78
19.43 6.23
23 (66%) 12 (34%) 0 0 0
35 (100%) 0 0 0 0
35 (100%) 0 0 0 0
35 (100%) 0 0 0 0
0 0 0 9 (26%) 26 (74%)
SD, standard deviation.
detected, with mean cellular densities of 22 25 cells/ mm2, 288 102 cells/mm2, and 98 8 cells/mm2, respectively [14–16]. It has been known that DCs in the limbus can be recruited into the cornea by appropriate inflammatory or immunogenic stimuli [17]. The greater density of DCs in patients with VKC may be secondary to cell migration into the cornea from the limbus stimulated by immunogenic stimuli. By far, no studies have determined the role of DCs in the development of VKC before and after treatment. In this study, the density of DCs in VKC decreased significantly at the bulbar conjunctiva, limbus, and peripheral cornea after topical treatment, but till the end of the follow-up, the densities of DCs at the limbus and peripheral cornea of patients with VKC remained higher than that in normal eyes. Moreover, we found that
these DCs were small in size, with small dendritic processes or no processes, which may reflect the immaturities of DCs. Two populations of DCs, MHC class II positive (mature) cells and MHC class II negative (immature) cells, were observed to be in the normal murine corneal epithelium by immunofluorescence [18, 19]. Guthoff et al. [14] discovered by in vivo confocal microscopy that LCs in normal human eyes presented as either large cells bearing long processes or smaller cells lacking cell dendrites, presumably indicating mature and immature phenotypes, respectively. Mature DCs may have a slender nucleated cell body, from which extends a maze of long processes resembling dendrites of nerve cells. Immature DCs may have a large cell body with fewer and shorter processes, able to capture and process antigens but unable to present © 2013 John Wiley & Sons Ltd, Clinical & Experimental Allergy, 44 : 362–370
Dendritic cells in vernal keratoconjunctivitis
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(a)
(b)
(c)
Fig. 3. The density level category of dendritic cells at the bulbar conjunctiva (a), limbus (b), and peripheral cornea (c) in patients with VKC.
antigens to lymphocytes. With appropriate stimuli derived from an inflamed microenvironment, they can change into the mature phenotype capable of presenting antigens and costimulatory signals to lymphocytes. Therefore, DCs remaining on the ocular surface of patients with VKC may be more sensitive to future reactivation of the disease and change into the mature © 2013 John Wiley & Sons Ltd, Clinical & Experimental Allergy, 44 : 362–370
phenotype, which can lead to the recurrence of VKC. Further investigations are needed to support this hypothesis. To date, precise characterization of ocular surface, including epithelial DCs, has been performed mainly in vitro by means of immuno-histochemical staining of tissues. Conversely, these techniques have the limitation
368 M. Liu et al (a)
(b)
(c)
Fig. 4. In vivo confocal microscopy images. Dendritic cells at the superior bulbar conjunctiva (a), limbus (b), and peripheral cornea (c) in normal eyes.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
(k)
(l)
Fig. 5. In vivo confocal microscopy images of dendritic cells at the bulbar conjunctiva, limbus, and peripheral cornea in patients with VKC. Numerous dendritic-shaped cells with typical branching dendrites are visible at the bulbar conjunctiva (a), limbus (b), and peripheral cornea (c) before the treatment. At 1 month after treatment, the dendritic cell density at the bulbar conjunctiva decreased significantly (d); however, at the limbus (e) and peripheral cornea (f), no significant reduction is detected. At 3 months, dendritic cells with small branching dendrites are visible at the limbus (h) and peripheral cornea (i), and their densities decrease significantly. At 6 months, the cell densities at the limbus (k) and peripheral cornea (l) further decrease, but the branching dendritic shape is maintained.
that the full morphology of the cells is rarely seen in tissue sections. Confocal microscopy is a rapid, non-invasive, high-resolution technique used to investigate in vivo the morphologic features of normal and pathologic human ocular surface. It provides a universal tool to assess ocular architecture and morphology with properties similar to those provided by conventional histopathology, without the invasive preparation and need for biopsy. Previous reports confirmed that
DCs could be evaluated on the morphological basis by laser scanning confocal microscopy [20], with a characteristic morphology distinguishing them from other cells and structures in the epithelial and subbasal layers [20–23]. Recent advances of laser in vivo confocal microscopes have made it possible to image, with high magnification and resolution, the microscopic architecture of the transparent corneal tissues and non-transparent structures such as limbal and conjunctival epithelium. Although mature DC morphology is typical, and immature cells featuring a mild branching dendritic shape might also be interpreted as macrophages, this was unlikely in the present study, as the latter are known to be present in lower densities and almost exclusively restricted to the posterior stroma of the cornea [13]. Only cells in the epithelium and subbasal nerve plexus were involved in our study. Topical corticosteroids remain the mainstay of treatment for VKC, producing significant improvement in acute symptoms, for its dramatic inhibition of DC differentiation and maturation [24]. However, prolonged use of steroids may result in many complications. In this study, corticosteroids were only administered in the initially acute stage. Topical CsA in different concentrations has been shown to be beneficial in the management of several allergic eye diseases [25–28]. It is an immunomodulator that inhibits CD4 T lymphocyte proliferation by inhibiting interleukin-2 receptor expression. Akpek et al. [29] reported favourable results of topical CsA in the treatment of patients with AKC, without any side-effects. The longest follow-up for topical CsA was 6 months [10], while in our study, eight patients were observed for over 6 months. We found that the densities of DCs in patients with VKC continued to decrease, with density and morphology differing from the normal eyes. DCs remaining at the patients may be more responsive to lower doses of therapy. Hence, topical CsA may be long-term used for patients with VKC. Further studies with more patients and longer follow-up periods are needed to confirm our opinion and to determine the long-term safety of topical CsA in this setting. © 2013 John Wiley & Sons Ltd, Clinical & Experimental Allergy, 44 : 362–370
Dendritic cells in vernal keratoconjunctivitis
In conclusion, in vivo confocal microscopy appears to be a valuable tool in evaluating the dynamic change in DCs at the conjunctiva and cornea. The number and maturation state of DCs are increased at the conjunctiva and cornea of patients with VKC, which can be controlled by topical use of corticosteroids and CsA. Therefore, DCs can play a crucial role in the pathogenesis of VKC. Targeting the function of DCs might be useful for therapeutic intervention. Acknowledgement The authors thank Ms. Ping Lin for her editorial assistance.
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Sources of funding Supported by grants from the National Natural Science Foundation of China (81170815), the Natural Science Foundation of Shandong Province (ZR2012HQ041), the National Basic Research Program of China (2013CB967004), and the Taishan Scholar Program (20081148). Conflict of interest The authors report no conflict of interests. The authors alone are responsible for the content and writing of the article.
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