December 2012
Volume 53, Issue 13
Free
Immunology and Microbiology  |   December 2012
Profile of Local Interleukin Expression in a Cohort of Ocular Cicatricial Pemphigoid Patients
Author Affiliations & Notes
  • Ana M. Suelves
    From the Massachusetts Eye Research and Surgery Institution, Cambridge, Massachusetts; the
    Ocular Immunology and Uveitis Foundation, Cambridge, Massachusetts; and the
  • Tong Z. Zhao
    From the Massachusetts Eye Research and Surgery Institution, Cambridge, Massachusetts; the
    Ocular Immunology and Uveitis Foundation, Cambridge, Massachusetts; and the
  • Sana S. Siddique
    From the Massachusetts Eye Research and Surgery Institution, Cambridge, Massachusetts; the
    Ocular Immunology and Uveitis Foundation, Cambridge, Massachusetts; and the
  • C. Stephen Foster
    From the Massachusetts Eye Research and Surgery Institution, Cambridge, Massachusetts; the
  • Corresponding author: C. Stephen Foster, 5 Cambridge Center, 8th Floor, Cambridge, MA 02142; sfoster@mersi.com
Investigative Ophthalmology & Visual Science December 2012, Vol.53, 8112-8117. doi:10.1167/iovs.11-9322
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Ana M. Suelves, Tong Z. Zhao, Sana S. Siddique, C. Stephen Foster; Profile of Local Interleukin Expression in a Cohort of Ocular Cicatricial Pemphigoid Patients. Invest. Ophthalmol. Vis. Sci. 2012;53(13):8112-8117. doi: 10.1167/iovs.11-9322.

      Download citation file:


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

      ×
  • Supplements
Abstract

Purpose.: We investigated the expression of IL-1, IL-6, IL-12, IL-13, and IL-17 in the conjunctiva of patients with ocular cicatricial pemphigoid (OCP), also labeled as ocular mucous membrane pemphigoid (MMP).

Methods.: A retrospective case–control study was done on 5 biopsy-proven OCP subjects and 6 healthy volunteers. Conjunctival specimens were obtained, and the local expression of IL-1, IL-6, IL-12, IL-13, and IL-17 was studied by immunohistochemistry. Clinical and therapeutic features were collected during follow-up.

Results.: No remarkable IL-1, IL-6, IL-12, IL-13, or IL-17 expression was observed in normal conjunctival specimens. All OCP samples had remarkable amounts of IL-12 and IL-17 expression especially in the epithelium and stroma; there also was stromal overexpression of IL-6. The mean follow-up after the biopsy was 13 months (range 9–15 months).

Conclusions.: Our results demonstrated, for the first time to our knowledge, a local overexpression of IL-6, IL-12, and IL-17 in conjunctiva of OCP compared to controls.

Introduction
Ocular cicatricial pemphigoid (OCP), also known as ocular mucous membrane pemphigoid (MMP), 1 is a systemic, autoimmune disease affecting approximately 1 in 8000 to 1 in 46,000 ophthalmic patients. 25 Appropriate management poses a challenge in these patients, as stubborn inflammation with progressive subepithelial fibrosis induces a restructuring of the ocular surface, leading to a high morbidity and legal blindness secondary to keratopathy in up to 30% of patients, 2,6 sometimes despite aggressive immunomodulatory therapy. 7  
The precise pathophysiology of OCP still largely is unknown, 8 but it is clear that there is a systemic inflammatory response with production of circulating autoantibodies to multiple conjunctival basement membrane zone (BMZ) antigens. The main epitope implicated is the cytoplasmic domain of the beta4 (β4) peptide of α6β4 integrin. 2,9 Autoantibodies recognizing base pair (BP) 180 or soluble ectodomains of BP 180 (including NC16A and LAD-1) also have been identified in a small set of OCP patients. 10,11 Additionally, the complex interplay between the immune system and conjunctival cells contributes to production of proinflammatory cytokines, chemokines, and chemical factors. 1214 Studies on the natural history of this entity have shown a marked degree of clinical and immunologic variability, ranging between mild cases that progress slowly and more severe clinical forms with a progressing-relapsing course even under treatment. 15,16 The chronic, and sometimes unpredictable course of the disease makes it difficult to determine whether the favorable effects of short-term treatment will be sustained. Moreover, clinical and therapeutic heterogeneity observed in OCP patients could be the expression of different ways of immunologic activation in conjunctiva. 10,17,18 If one could establish phenotypic variants, it could allow classifying OCP patients in different subgroups, clinically and therapeutically. Additionally, it could help in differentiating patients likely to have a benign course from those with a more virulent evolution or those who do not respond to treatment. 
The relevance of Th1 and Th2 cytokines in ocular inflammatory disorders, such as dry eye disease or seasonal allergic conjunctivitis, has been studied widely. Growing evidence suggests the important role of several cytokines, including IL-1, IL-6, and more recently IL-17, in dry eye disease. Allergic conjunctivitis is considered a classic Th2 response, with an overexpression of IL-4, IL-6, and IL-13. Moreover, much pathogenic effect observed in both entities is attributed to direct effect of these cytokines on the ocular surface. 
The purpose of our study was to investigate the expression of IL-1, IL-6, IL-12, IL-13, and IL-17 in specimens of conjunctiva in OCP patients versus controls. 
Materials and Methods
Participant Selection
The study was done in OCP patients who underwent conjunctival biopsy at the Massachusetts Eye Research and Surgery Institution (MERSI). To be eligible for inclusion in this study, OCP patients required a positive result by direct immunofluorescence (DIF) or by immunoperoxidase–avidin–biotin complex (ABC) staining if the DIF had been inconclusive. OCP subjects who had any other ocular history, past ocular surgery, or involvement of mucosa other than conjunctiva were excluded to isolate better the features of ocular MMP. These eyes were age-matched with healthy subjects. The control group had no history of eye disease other than cataract, no history of contact lens wear, and were free of any systemic, autoimmune, or infectious disorder. Conjunctival samples were obtained in these subjects at the time of cataract surgery. Five patients with biopsy-proven OCP and six controls were included in this retrospective case–control study. All conjunctival specimens were obtained between March 2009 and December 2010 by a single surgeon. 
This study adhered to the tenets of the Declaration of Helsinki, and institutional review board audited and approved the protocol of our study. Informed consent for the study was obtained from all study subjects. 
Biopsy Processing and Immunohistochemistry Technique
Each conjunctival biopsy was obtained from the inferotemporal bulbar conjunctiva quadrant in an area of clinically apparent conjunctival inflammation. Specimens from control eyes were obtained at the time of surgery from the same conjunctival location. The average size of specimens was 10 × 5 mm. Tissue was stored at −80°C, embedded in OCT medium, before slide preparation and immunostaining. Conjunctival biopsies recovered from stock were sliced into 5 μm sections and placed on poly-L-lysine–coated slides for immunostaining on frozen sections. All slides were fixed in precooled acetone for 10 minutes. Nonspecific binding was blocked by 30 minutes of incubation with 5% goat serum at 37°C, except for IL-13, which was blocked with 5% rat serum. Sections were incubated overnight at −4°C with primary antibodies (goat anti-human IL-1, −6, −12, −17; RD Systems, Inc., Boston, MA; and rat anti-human IL-13; Abcam, Cambridge, MA; Table 1). After 3 washes with PBS with BSA 0.1 M, all specimens were blocked with 5% rabbit serum for 30 minutes. Next, all slides were incubated with biotinylated-secondary antibodies (Ab) directed to IL-1, IL-6, IL-12, IL-13, and IL-17 for 1 hour (Table 1), washed with PBS-BSA, and incubated for 30 minutes with streptavidin peroxidase (Vectastain ABC kit; Vector Laboratories, Burlingame, CA). The reaction products were developed with a mixture of 3,3-diaminobenzine-4 HCL (DAB). The last step was staining with Histotik for 6 seconds, stopping the reaction with distilled water. Collagen IV was used as a positive control for primary Ab, while PBS only and secondary Ab without primary Ab were used as negative controls for the internal process. Each section was mounted and examined at 30× magnification using light microscopy (model Olympus BX 41; Olympus, Center Valley, PA). For each sample, two independent observers counted stained cells and the results were scored using a semiquantitative scale 0 to 3, with 0+ indicating less than 5 cells per field, 1+ between 5 and 25 cells per field, 2+ between 25 and 50 cells per field, and 3+ more than 50 cells per field. 
Table 1. 
 
The Sources of Primary and Secondary Antibodies, with Their Dilutions
Table 1. 
 
The Sources of Primary and Secondary Antibodies, with Their Dilutions
Primary Antibody Working Solution Secondary Antibody Working Solution
Goat anti-human IL 1 F8/FIL1 eta polyclonal antibody (catalog number: AF1099) (1:100) Rabbit polyclonal secondary Ab to goat IgG- H &L (biotin) (catalog number: ab6740) (1:750)
Goat anti-human IL 6 polyclonal antibody (catalog number: AF-206-NA) (1:400)
Goat anti-human IL 12 polyclonal antibody (catalog number: AF-219-NA) (1:100)
Rat anti-IL 13 monoclonal Ab, clone JES 10-5A2 (catalog number: ab16219) (1:100) Rabbit polyclonal secondary Ab to rat IgG- H&L (biotin) (catalog number: ab6733)
Goat anti-human IL 17 polyclonal Ab (catalog number: AF-317-NA) (1:1000) Rabbit polyclonal secondary Ab to goat IgG- H &L (biotin) (catalog number: ab6740)
Conjunctival specimens also were stained with hematoxylin and eosin, periodic acid-Schiff and Giemsa, and analyzed by light microscopy. Histologic characteristics were noted for the presence or absence of inflammatory cells and microangiopathy. Microangiopathy was defined as ultrastructural changes, such as reduction of the vascular space, vascular basement membrane thickening, change in conjunctival vessel architecture, and perivascular inflammatory infiltrate. 
Clinical Assessment of Patients
Data collection included demographics and clinical data in both groups. In the OCP group, data collection also included the method used to diagnose OCP, grade of conjunctival clinical activity at the time of biopsy, OCP stage at the time of biopsy, histologic features, IL expression profile, and the therapy received. The response to therapy in OCP patients was noted prospectively during follow-up after biopsy. 
OCP stage was defined by the Foster classification, as has been described previously. 2 Conjunctival clinical activity was categorized at the time of biopsy and at each visit, according to the clinician's judgment. A standard grading from 0 to 4+ was used to record data, where 0+ reflected a quiescent eye, with increments of 1+ activity. The response to therapy was defined by analyzing the clinical course of the conjunctival inflammation (improvement of conjunctival injection). A complete response was defined as resolution of the baseline clinical conjunctival inflammation without relapse. A partial response was defined as resolution of clinical conjunctival inflammation, but with one or more recurrences. All patients who were not classified as having a partial or complete response were classified as nonresponders. 
Statistical Analysis
Baseline differences in sex and age between OCP patients and control patients were examined for statistical significance using the Mann-Whitney U test. Analysis was done using commercial software (SPSS version 15.0; SPSS, Chicago, IL). Results are presented with 95% confidence intervals (CIs) and P level of significance was set at 0.05. 
Results
Patient Characteristics, Histologic Features, and IL Expression
Five patients with biopsy-proven OCP and six controls were included. OCP patients (4 women and 1 man) ranged from 59 to 79 years old, the average being 67.6 years, SD 10.01. Mean age of controls (5 women and 1 men) was 65 years (SD 6.93), range 58 to 75 years. There was no statistically significant difference between the age and sex in groups (P = 0.662 and P = 1, respectively; Mann-Whitney U test). At the time of tissue sampling, all OCP patients had clinically active disease. Four OCP patients (80%) had stage III OCP according to the Foster classification. Patient 1 started azathioprine after immunohistologic confirmation of OCP diagnoses. Patient 2 was initiated with mycophenolate mofetil after biopsy, while patients 3 to 5 went on to receive treatment with methotrexate (MTX). The mean of follow-up after biopsy was 13 months (range 9–15 months). A complete response was observed in 3 patients (60%), while the other 2 (40%) had a partial response to therapy at the endpoint of follow-up. These data are summarized in Table 2. OCP was diagnosed by DIF, except in patient 2. DIF in this patient was inconclusive, but further analysis of the sample by ABC staining was unequivocal for OCP. None of the patients had surface shrinkage, progression of fornix foreshortening, symblepharon, or any significant complication resulting from biopsy during follow-up. 
Table 2. 
 
Demographic, Baseline Patient Characteristic, and Response to Therapy of OCP Patients
Table 2. 
 
Demographic, Baseline Patient Characteristic, and Response to Therapy of OCP Patients
Patient No. Age, y Sex Foster Classification Clinical Activity Treatment Received Response to Therapy
1 59 M Stage III 1+ AZA Partial
2 60 F Stage II 1+ MPM Partial
3 78 F Stage III 1+ MTX Complete
4 62 F Stage III 2+ MTX Complete
5 79 F Stage III 1+ MTX Complete
The epithelium of OCP specimens disclosed squamous metaplasia in 2 patients (40%). All OCP patients presented in our study had a variable mononuclear infiltrate, mostly in the stroma. Presence of plasma cells was observed in all OCP samples. Two pemphigoid specimens presented with decreased or absent goblet cells. Microangiopathy was observed in patients 2, 3, and 4, which was 60% of OCP subjects. 
No remarkable IL-1, IL-6, IL-12, IL-13, or IL-17 expression was observed in epithelium, stroma, or vessels in control specimens; two samples had a 0.5+ IL-1 expression in epithelium, one had 1+ IL-1 expression in stroma, and another sample had 0.5+ IL-6 expression in epithelium. All OCP samples had a very remarkable amount of IL-17 expression (+2 or more) in epithelium, stroma, and vessels. Additionally, IL-12 was overexpressed in 4 patients (80%) in the OCP series. Four patients had a mild expression of IL-1 and IL-6, mainly in stroma. Only patient 4 presented notable IL-13 expression with involvement of vasculature (see Figure). 
Figure. 
 
Histogram showing the relative expression of IL-1 (A), IL-6 (B), IL-12 (C), IL-13 (D), and IL-17 (E) in conjunctival specimens obtained from OCP subjects compared to controls.
Figure. 
 
Histogram showing the relative expression of IL-1 (A), IL-6 (B), IL-12 (C), IL-13 (D), and IL-17 (E) in conjunctival specimens obtained from OCP subjects compared to controls.
Comparing the interleukin expression in both groups, it clearly differed for stromal expression of IL-6, and for IL-12 and IL-17 expression in epithelium and stroma (Table 3). Further details about OCP diagnosis, histologic features, and local IL expression of conjunctival OCP specimens are summarized in Tables 3 and 4
Table 3. 
 
Histologic Findings, IF Profile, IL Profile in Conjunctival Biopsy in OCP Patients
Table 3. 
 
Histologic Findings, IF Profile, IL Profile in Conjunctival Biopsy in OCP Patients
Pt. No. IF Profile Histology IL Expression
1 IgG, IgA BMZ positive at DIF Epithelium thickened
High mononuclear infiltrate
Presence of plasma cells
Normal goblet cells
No microangiopathy
IL-1* (epithelium & stroma)
IL-6* (epithelium & stroma)
IL-12* (epithelium & stroma)
IL-13 (epithelium & stroma)
IL-17* (epithelium & stroma)
2 DIF inconclusive, IgA BMZ positive at ABC staining Squamous metaplasia
Mononuclear infiltrate
Presence of plasma cells
Decreased goblet cells
Microangiopathy
IL-1 (stroma)
IL-6 (stroma)
IL 12* (epithelium & stroma)
IL-13 (stroma)
IL 17* (epithelium, stroma, vessels)
3 IgG, IgA, IgM, IgD BMZ positive at DIF Epithelium thickened, irregular
Mononuclear infiltrate
Presence of plasma cells
Normal goblet cells
Microangiopathy
IL-1 (epithelium, stroma, vessels)
IL-6* (stroma)
IL-12 (stroma)
IL 17* (stroma, vessels)
4 IgG, IgA, and IgM BMZ positive at DIF Epithelium thickened
Impressive mononuclear infiltrate
Presence of plasma cells
Increased goblet cells
Microangiopathy
IL-1* (epithelium & stroma)
IL-6* (epithelium, stroma, vessels)
IL-12* (epithelium, stroma, vessels)
IL-13* (epithelium, stroma, vessels)
IL-17* (epithelium, stroma, vessels)
5 IgG, IgA, and C4 BMZ positive at DIF Squamous metaplasia
Small number of mononuclear cells Absence of goblet cells
Presence of plasma cells
No microangiopathy
IL-12* (epithelium & stroma)
IL-17* (epithelium & stroma)
Table 4. 
 
Local Cytokine Expression
Table 4. 
 
Local Cytokine Expression
Variable OCP (n = 5) Controls (n = 6)
IL-1 epithelium 3 (60%) 2 (33.3%)
IL-1 stroma 4 (80%) 1 (16.7%)
IL-1 vessels 1 (20%) 0 (0%)
IL-6 epithelium 2 (40%) 1 (16.7%)
IL-6 stroma 4 (80%) 0 (0%)
IL-6 vessels 1 (20%) 0 (0%)
IL-12 epithelium 4 (80%) 0 (0%)
IL-12 stroma 5 (100%) 0 (0%)
IL-12 vessels 1 (20%) 0 (0%)
IL-13 epithelium 2 (40%) 0 (0%)
IL-13 stroma 3 (60%) 0 (0%)
IL-13 vessels 1 (20%) 0 (0%)
IL-17 epithelium 4 (80%) 0 (0%)
IL-17 stroma 5 (100%) 0 (0%)
IL-17 vessels 3 (60%) 0 (0%)
Discussion
In our study, we found meaningful differences in the stromal expression of IL-6, IL-12, and IL-17, along with epithelial upregulation of IL-12 and IL-17 in the conjunctiva of active ocular pemphigoid patients compared to healthy patients. 
Evidence suggests that OCP develops as a consequence of the loss of immunologic tolerance to a hemidesmosome-associated structural component in the conjunctival BMZ (mainly β4 integrin and much more infrequently BP 180 or its soluble ectodomains). 911 Loss of tolerance results in development of circulating IgG and IgA autoantibodies, which binds to the conjunctival BMZ complex. Antibody deposition, with subsequent alterations in signal transduction, elicits a cascade of events, including cytokine and chemokine generation. This results in recruitment of a panoply of inflammatory cell types, with subsequent activation of fibroblasts, which multiply and secrete new (Type 3) collagen, causing subepithelial fibrosis and “shrinkage” of the conjunctiva. 19,20 Even though circulating autoantibodies binding to the conjunctival BMZ complex are considered the very heart of immunopathogenesis of OCP, some investigators state that an antibody-mediated hypothesis may fail to explain fully some characteristic features observed in this and other autoimmune blistering diseases, 21,22 such as the strong susceptibility to the disease linked with major histocompatibility complex (MHC) class II allele (HLA-DQβ1*0301). 2326  
To explore the underlying microenvironment in OCP patients, the role of different cytokines has been determined in serum and conjunctiva. Expression and upregulation of IL-1, 27 IL-5, 28 and tumor necrosis factor-alpha (TNF-α) 29 in OCP peripheral blood was demonstrated by Foster et al., while IL-6 was found to be decreased in blood serum of OCP patients compared to controls. Other inflammatory molecules, such as TNF-α, 30,31 macrophage migration inhibitory factor, 32 macrophage colony-stimulating factor, 33 and IL-4, 34 also have been shown by Foster et al. to be involved in the activation of the immune system limited to the conjunctival biopsy. Saw et al. hypothesized that IL-13 35 had a key role in such patients, inducing activation of fibroblasts and T cells, which results in conjunctival fibrosis. More recently, Lambiase et al. pointed out an increased recruitment of T helper 17 (Th17) lymphocytes in OCP conjunctiva. 36 Our findings were in line with previous results reported by Saw and Lambiase, but also suggested that the conjunctival autoimmunity in ocular pemphigoid also is driven by other cytokines, such as IL-6 and IL-12. 
The upregulation of IL-6, IL-12, and IL-17 that was displayed in the conjunctival specimens of our patients supports the hypothesis that T cell response might have a role in the autoimmunity in OCP. Razzaque et al. postulated that T cells may recognize BMZ epitopes and bound DQβ1*0301, resulting in an activation of B cells that would elicit production of antiBMZ antibodies. 37 The disparate IL-6 observations decreased in serum and increased in conjunctiva of OCP patients emphasizes a local autoimmune expression of this autoimmune systemic disease. The belief that circulating autoantibodies are the key immunopathologic feature of OCP has been supported by the effectiveness of therapies targeting these specific cells, such as rituximab and/or intravenous immunoglobulin; both of these modalities have been shown to stop the progression of disease in stubborn cases. 3841 Our histologic results, which show the presence of plasma cells in 100% of conjunctival OCP specimens, also are consistent with these observations. 
Note should be made of the remarkable ineffectiveness of anti-T cell agents, such as calcineurin inhibitors, in the care of patients with OCP. 42 Further investigations will be needed to determine the cells responsible for the local production of these cytokines, the relevance of T cells in disease pathogenesis, and the precise mechanism by which scarring in conjunctiva occurs. 
The gold standard (and really, the only definitive) method for diagnosing OCP remains the immunohistochemical analysis of conjunctival biopsy specimens, with demonstration of one or more immunoreactants at the epithelial basement membrane zone. To our knowledge there are no clinical, analytical, or histologic data that correlate with prognosis, disease evolution, or therapy response in OCP patients. Previous reports have shown that the presence of autoantibodies does not correlate with activity of disease. 43 The different spectrum of cytokine expression observed in our study emphasizes that there might be different patterns of local activation of systemic autoimmunity. Moreover, a better comprehension of the molecular pathways of conjunctival activation could help to understand better the variable clinical-therapeutic response observed in clinical practice, which would allow individualize therapeutic approaches. 
We recognize that the small number of subjects included in our pilot study limit the possibility of performing a fair statistical comparison regarding the local expression of IL, as well as the correlation between conjunctival biopsy and clinical behavior or response to therapy. Long-term studies in larger populations are warranted to evaluate the validity of our observations. In summary, our study has demonstrated for the first time to our knowledge that IL-6, IL-12, and IL-17 are overexpressed in OCP conjunctival tissue compared to controls, which underlines that T cells may have a role in OCP pathogenesis. Conjunctival biopsy analysis may provide a better understanding of the biomolecular activation pathways that exist in OCP. Ultimately, this may lead to the categorization of specific OCP phenotypes and allow for greater personalization of treatment strategies. 
References
Chan LS Ahmed AR Anhalt GJ The first international consensus on mucous membrane pemphigoid: definition, diagnostic criteria, pathogenic factors, medical treatment, and prognostic indicators. Arch Dermatol . 2002;138:370–379. [PubMed]
Foster CS. Cicatricial pemphigoid. Trans Am Ophthalmol . 1986;84:527–663.
Bettelheim H Kraft D Zehetbauer G. Uber den sogenanneten Augenpemphigus (pemphigus ocularis; pemphigus conjunctivae). Klin Auggenheilkd . 1972;160:65–75.
Smith RC Myers EA Lamb HD. Ocular and oral pemphigus: report of case with anatomic findings in eyeball. Arch Ophthalmol . 1934;11:635–640. [CrossRef]
Bedell AJ. Ocular pemphigus: a clinical presentation. Trans Am Ophthalmnol Soc . 1964;62:109–122.
Hardy KM Perry HO Pingree GC Benign mucous membrane pemphigoid. Arch Dermatol . 1971;104:467–475. [CrossRef] [PubMed]
Sami N Letko E Androudi S Daoud Y Foster CS Ahmed AR. Intravenous immunoglobulin therapy in patients with ocular-cicatricial pemphigoid: a longterm follow-up. Ophthalmology . 2004;111:1380–1382. [CrossRef] [PubMed]
Moll R Moll I. Epidermal adhesion molecules and basement membrane components as target structures of autoimmunity. Virchows Arch . 1998;432:487–504. [CrossRef] [PubMed]
Bhol KC Dans MJ Simmons RK Foster CS Giancotti FG Ahmed AR. The autoantibodies to alpha 6 beta 4 integrin of patients affected by ocular cicatricial pemphigoid recognize predominantly epitopes within the large cytoplasmic domain of human beta 4. J Immunol . 2000;165:2824–2829. [CrossRef] [PubMed]
Oyama N Setterfield JF Powell AM Bullous pemphigoid antigen II (BP180) and its soluble extracellular domains are major autoantigens in mucous membrane pemphigoid: the pathogenic relevance to HLA class II alleles and disease severity. Br J Dermatol . 2006;154:90–98. [CrossRef] [PubMed]
Schmidt E Skrobek C Kromminga A Cicatricial pemphigoid: IgA and IgG autoantibodies target epitopes on intra- and extracellular domains of bullous pemphigoid antigen 180. Br J Dermatol . 2001;145:778–783. [CrossRef] [PubMed]
Ahmed M Zein G Khawaja F Foster CS. Ocular cicatricial pemphigoid: pathogenesis, diagnosis and treatment. Prog Retin Eye Res . 2004;23:579–592. [CrossRef] [PubMed]
Eschle-Meniconi ME Ahmad SR Foster CS. Mucous membrane pemphigoid: an update. Curr Opin Ophthalmol . 2005;16:303–307. [CrossRef] [PubMed]
Razzaque MS Ahmed BS Foster CS Ahmed AR. Effects of IL-4 on conjunctival fibroblasts: possible role in ocular cicatricial pemphigoid. Invest Ophthalmol Vis Sci . 2003;44:3417–3423. [CrossRef] [PubMed]
Bruch-Gerharz D Hertl M Ruzicka T. Mucous membrane pemphigoid: clinical aspects, immunopathological features and therapy. Eur J Dermatol . 2007;17:191–200. [PubMed]
Yancey KB Egan CA. Pemphigoid: clinical, histologic, immunopathologic, and therapeutic considerations. JAMA . 2000;284:350–356. [CrossRef] [PubMed]
Tyagi S Bhol K Natarajan K Livir-Rallatos C Foster CS Ahmed AR. Ocular cicatricial pemphigoid antigen: partial sequence and biochemical characterization. Proc Natl Acad Sci U S A . 1996;93:1471–1479.
Smith EP Taylor TB Meyer LJ Zone JJ. Identification of a basement membrane zone antigen reactive with circulating IgA antibody in ocular cicatricial pemphigoid. J Invest Dermatol . 1993;101:619–623. [CrossRef] [PubMed]
Rice BA Foster CS. Immunopathology of cicatricial pemphigoid affecting the conjunctiva. Ophthalmology . 1990;97:1476–1483. [CrossRef] [PubMed]
Bernauer W Wright P Dart JK Leonard JN Lightman S. The conjunctiva in acute and chronic mucous membrane pemphigoid. An immunohistochemical analysis. Ophthalmology . 1993;100:339–346. [CrossRef] [PubMed]
Black AP Seneviratne SL Jones L Rapid effector function of circulating NC16A-specific T cells in individuals with mucous membrane pemphigoid. Br J Dermatol . 2004;151:1160–1164. [CrossRef] [PubMed]
Hertl M Eming R Veldman C. T cell control in autoimmune bullous skin disorders. J Clin Invest . 2006;116:1159–1166. [CrossRef] [PubMed]
Zakka LR Reche P Ahmed AR. Role of MHC class II genes in the pathogenesis of pemphigoid. Autoimmun Rev . 2011;11:40–47. [CrossRef] [PubMed]
Yunis JJ Mobini N Yunis EJ Common major histocompatibility complex class II markers in clinical variants of cicatricial pemphigoid. Proc Natl Acad Sci U S A . 1994;91:7747–7751. [CrossRef] [PubMed]
Chan LS Hammerberg C Cooper KD. Significantly increased occurrence of HLADQB1* 0301 allele in patients with ocular cicatricial pemphigoid. J Invest Dermatol . 1997;108:129–132. [CrossRef] [PubMed]
Ahmed AR Foster S Zaltas M Association of DQw7 (DQB1*0301) with ocular cicatricial pemphigoid. Proc Natl Acad Sci U S A . 1991;88:11579–11582. [CrossRef] [PubMed]
Kumari S Bhol KC Rehman F Foster CS Ahmed AR. Interleukin 1 components in cicatricial pemphigoid. Role in intravenous immunoglobulin therapy. Cytokine . 2001;14:218–224. [CrossRef] [PubMed]
Letko E Bhol K Colon J Foster CS Ahmed AR. Biology of interleukin-5 in ocular cicatricial pemphigoid. Grafes Arch Clin Exp Opthalmol . 2002;240:565–569. [CrossRef]
Lee SJ Li Z Sherman B Foster CS. Serum levels of tumor necrosis factor-alpha and interleukin-6 in ocular cicatricial pemphigoid. Invest Ophthalmol Vis Sci . 1993;34:3522–3525. [PubMed]
Saw VP Dart RJ Galatowicz G Daniels JT Dart JK Calder VL. Tumor necrosis factor-alpha in ocular mucous membrane pemphigoid and its effect on conjunctival fibroblasts. Invest Ophthalmol Vis Sci . 2009;50:5310–5317. [CrossRef] [PubMed]
Cordero Coma M Yilmaz T Foster CS . Tumour necrosis factor-alpha in conjunctivae affected by ocular cicatricial pemphigoid. Acta Ophthalmol Scand . 2007;85:753–755. [CrossRef] [PubMed]
Razzaque MS Foster CS Ahmed AR. Role of macrophage migration inhibitory factor in conjunctival pathology in ocular cicatricial pemphigoid. Invest Ophthalmol Vis Sci . 2004;45:1174–1181. [CrossRef] [PubMed]
Razzaque MS Foster CS Ahmed AR. Role of enhanced expression of m-CSF in conjunctiva affected by cicatricial pemphigoid. Invest Ophthalmol Vis Sci . 2002;43:2977–2983. [PubMed]
Razzaque MS Ahmed BS Foster CS Ahmed AR. Effects of IL-4 on conjunctival fibroblasts: possible role in ocular cicatricial pemphigoid. Invest Ophthalmol Vis Sci . 2003;44:3417–3423. [CrossRef] [PubMed]
Saw VP Offiah I Dart RJ Conjunctival interleukin-13 expression in mucous membrane pemphigoid and functional effects of interleukin-13 on conjunctival fibroblasts in vitro. Am J Pathol . 2009;175:2406–2415. [CrossRef] [PubMed]
Lambiase A Micera A Mantelli F T-helper 17 lymphocytes in ocular cicatricial pemphigoid. Mol Vis . 2009;15:1449–1455. [PubMed]
Zakka LR Reche P Ahmed AR. Role of MHC Class II Genes in the pathogenesis of pemphigoid. Autoimmun Rev . 2011;11:40–47. [CrossRef] [PubMed]
Foster CS Chang PY Ahmed AR. Combination of rituximab and intravenous immunoglobulin for recalcitrant ocular cicatricial pemphigoid: a preliminary report. Ophthalmology . 2010;117:861–869. [CrossRef] [PubMed]
Foster CS Ahmed AR. Intravenous immunoglobulin therapy for ocular cicatricial pemphigoid: a preliminary study. Ophthalmology . 1999;106:2136–2143. [CrossRef] [PubMed]
Sami N Letko E Androudi S Intravenous immunoglobulin therapy in patients with ocular-cicatricial pemphigoid: a long-term follow-up. Ophthalmology . 2004;111:1380–1382. [CrossRef] [PubMed]
Ross AH Jaycock P Cook SD The use of rituximab in refractory mucous membrane pemphigoid with severe ocular involvement. Br J Ophthalmol . 2009;93:421–422. [CrossRef] [PubMed]
Kaçmaz RO Kempen JH Newcomb C Cyclosporine for ocular inflammatory diseases. Ophthalmology . 2010;117:576–584. [CrossRef] [PubMed]
Yeh SW Usman AQ Ahmed AR. Profile of autoantibody to basement membrane zone proteins in patients with mucous membrane pemphigoid: long-term follow-up and influence of therapy. Clin Immunol . 2004;112:268–272. [CrossRef] [PubMed]
Footnotes
 Presented in part at the 2nd World Congress on Controversies in Ophthalmology, Barcelona, Spain, 2011.
Footnotes
 The authors alone are responsible for the content and writing of this paper.
Footnotes
 Disclosure: A.M. Suelves, None; T.Z. Zhao, None; S.S. Siddique, None; C.S. Foster, None
Figure. 
 
Histogram showing the relative expression of IL-1 (A), IL-6 (B), IL-12 (C), IL-13 (D), and IL-17 (E) in conjunctival specimens obtained from OCP subjects compared to controls.
Figure. 
 
Histogram showing the relative expression of IL-1 (A), IL-6 (B), IL-12 (C), IL-13 (D), and IL-17 (E) in conjunctival specimens obtained from OCP subjects compared to controls.
Table 1. 
 
The Sources of Primary and Secondary Antibodies, with Their Dilutions
Table 1. 
 
The Sources of Primary and Secondary Antibodies, with Their Dilutions
Primary Antibody Working Solution Secondary Antibody Working Solution
Goat anti-human IL 1 F8/FIL1 eta polyclonal antibody (catalog number: AF1099) (1:100) Rabbit polyclonal secondary Ab to goat IgG- H &L (biotin) (catalog number: ab6740) (1:750)
Goat anti-human IL 6 polyclonal antibody (catalog number: AF-206-NA) (1:400)
Goat anti-human IL 12 polyclonal antibody (catalog number: AF-219-NA) (1:100)
Rat anti-IL 13 monoclonal Ab, clone JES 10-5A2 (catalog number: ab16219) (1:100) Rabbit polyclonal secondary Ab to rat IgG- H&L (biotin) (catalog number: ab6733)
Goat anti-human IL 17 polyclonal Ab (catalog number: AF-317-NA) (1:1000) Rabbit polyclonal secondary Ab to goat IgG- H &L (biotin) (catalog number: ab6740)
Table 2. 
 
Demographic, Baseline Patient Characteristic, and Response to Therapy of OCP Patients
Table 2. 
 
Demographic, Baseline Patient Characteristic, and Response to Therapy of OCP Patients
Patient No. Age, y Sex Foster Classification Clinical Activity Treatment Received Response to Therapy
1 59 M Stage III 1+ AZA Partial
2 60 F Stage II 1+ MPM Partial
3 78 F Stage III 1+ MTX Complete
4 62 F Stage III 2+ MTX Complete
5 79 F Stage III 1+ MTX Complete
Table 3. 
 
Histologic Findings, IF Profile, IL Profile in Conjunctival Biopsy in OCP Patients
Table 3. 
 
Histologic Findings, IF Profile, IL Profile in Conjunctival Biopsy in OCP Patients
Pt. No. IF Profile Histology IL Expression
1 IgG, IgA BMZ positive at DIF Epithelium thickened
High mononuclear infiltrate
Presence of plasma cells
Normal goblet cells
No microangiopathy
IL-1* (epithelium & stroma)
IL-6* (epithelium & stroma)
IL-12* (epithelium & stroma)
IL-13 (epithelium & stroma)
IL-17* (epithelium & stroma)
2 DIF inconclusive, IgA BMZ positive at ABC staining Squamous metaplasia
Mononuclear infiltrate
Presence of plasma cells
Decreased goblet cells
Microangiopathy
IL-1 (stroma)
IL-6 (stroma)
IL 12* (epithelium & stroma)
IL-13 (stroma)
IL 17* (epithelium, stroma, vessels)
3 IgG, IgA, IgM, IgD BMZ positive at DIF Epithelium thickened, irregular
Mononuclear infiltrate
Presence of plasma cells
Normal goblet cells
Microangiopathy
IL-1 (epithelium, stroma, vessels)
IL-6* (stroma)
IL-12 (stroma)
IL 17* (stroma, vessels)
4 IgG, IgA, and IgM BMZ positive at DIF Epithelium thickened
Impressive mononuclear infiltrate
Presence of plasma cells
Increased goblet cells
Microangiopathy
IL-1* (epithelium & stroma)
IL-6* (epithelium, stroma, vessels)
IL-12* (epithelium, stroma, vessels)
IL-13* (epithelium, stroma, vessels)
IL-17* (epithelium, stroma, vessels)
5 IgG, IgA, and C4 BMZ positive at DIF Squamous metaplasia
Small number of mononuclear cells Absence of goblet cells
Presence of plasma cells
No microangiopathy
IL-12* (epithelium & stroma)
IL-17* (epithelium & stroma)
Table 4. 
 
Local Cytokine Expression
Table 4. 
 
Local Cytokine Expression
Variable OCP (n = 5) Controls (n = 6)
IL-1 epithelium 3 (60%) 2 (33.3%)
IL-1 stroma 4 (80%) 1 (16.7%)
IL-1 vessels 1 (20%) 0 (0%)
IL-6 epithelium 2 (40%) 1 (16.7%)
IL-6 stroma 4 (80%) 0 (0%)
IL-6 vessels 1 (20%) 0 (0%)
IL-12 epithelium 4 (80%) 0 (0%)
IL-12 stroma 5 (100%) 0 (0%)
IL-12 vessels 1 (20%) 0 (0%)
IL-13 epithelium 2 (40%) 0 (0%)
IL-13 stroma 3 (60%) 0 (0%)
IL-13 vessels 1 (20%) 0 (0%)
IL-17 epithelium 4 (80%) 0 (0%)
IL-17 stroma 5 (100%) 0 (0%)
IL-17 vessels 3 (60%) 0 (0%)
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×