The Open Ophthalmology Journal




ISSN: 1874-3641 ― Volume 13, 2019

Expression of Filaggrin in Normal and Keratinized Conjunctiva



Anne Sofie Kragegaard Lund1, 3, Steffen Heegaard1, 2, Jan Ulrik Prause1, Peter Bjerre Toft2, Lone Skov*, 3
1 Institute of Neuroscience and Pharmacology, Eye Pathology Institute, University of Copenhagen, Copenhagen, Denmark
2 Department of Ophthalmology, Glostrup Hospital, University of Copenhagen, Copenhagen, Denmark
3 Department of Dermato-allergology, Gentofte Hospital, University of Copenhagen, Denmark

Abstract

Purpose:

We wanted to investigate filaggrin expression in normal conjunctiva and in conjunctiva exposed to different degrees of mechanical stress. Mechanical stress results in parakeratinization of the conjunctiva. If filaggrin is expressed in the milder forms of parakeratinization, it might be used as a sensitive marker of mechanical stress.

Methods:

Immunohistochemical staining using antibodies to filaggrin was performed on paraffin sections of normal human conjunctiva, and on conjunctiva with different degrees of mechanical conjunctival stress.

Results:

Filaggrin was not expressed in the normal conjunctiva, nor in conjunctiva with milder forms of mechanical stress. Intense staining of filaggrin was seen in the conjunctiva of a patient with Stevens-Johnson syndrome, and marked expression of filaggrin was found in the conjunctival epithelium of a patient with moderate dysplasia of the conjunctiva.

Conclusion:

Filaggrin is not a sensitive marker of mechanical stress; it is, however, expressed in some moderate and severe forms of parakeratinization of the conjunctiva.

Keywords: Filaggrin, conjunctiva, parakeratinized, mechanical stress.


Article Information


Identifiers and Pagination:

Year: 2012
Volume: 6
First Page: 137
Last Page: 140
Publisher Id: TOOPHTJ-6-137
DOI: 10.2174/1874364101206010137

Article History:

Received Date: 23/6/2012
Revision Received Date: 22/10/2012
Acceptance Date: 24/10/2012
Electronic publication date: 28/12/2012
Collection year: 2012

Article Metrics:

CrossRef Citations:
0

Total Statistics:

Full-Text HTML Views: 821
Abstract HTML Views: 592
PDF Downloads: 160
Total Views/Downloads: 1573

Unique Statistics:

Full-Text HTML Views: 441
Abstract HTML Views: 382
PDF Downloads: 107
Total Views/Downloads: 930
Geographical View

© Lund et al.; Licensee Bentham Open.

open-access license: This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.


* Address correspondence to this author at the Department of Dermato-allergology, Gentofte Hospital, Niels Andersens Vej 65, 2900 Hellerup, Denmark; Tel: +45 39773204; Fax: +45 39777615; E-mail: lone.skov.02@regionh.dk




INTRODUCTION

Filaggrin is a barrier protein that is found in the outer keratinized layer of the epidermis. It is also expressed in parakeratinized areas of human oral epithelium [1 De Benedetto A, Qualia CM, Baroody FM, Beck LA. Filaggrin expression in oral, nasal, and esophageal mucosa J Invest Dermatol 2008; 128: 1594-7.] and the exocervix epithelium [2 Cintorino M, Syrjanen S, Leoncini P, et al. Altered expression of filaggrin in human papillomavirus (HPV) lesions of the uterine cervix Arch Gynecol Obstet 1988; 241: 235-47.]. Previous studies have shown no or very weak expression of filaggrin in the human corneal epithelium [3 Tong L, Corrales RM, Chen Z, et al. Expression and regulation of cornified envelope proteins in human corneal epithelium Invest Ophthalmol Vis Sci 2006; 47: 1938-46., 4 Saika S, Minamide A, Tanaka T, et al. Expression of involucrin by ocular surface epithelia of patients with benign and malignant disorders Curr Eye Res 2000; 21: 877-5.] and no or weak expression in the healthy human conjunctiva [4 Saika S, Minamide A, Tanaka T, et al. Expression of involucrin by ocular surface epithelia of patients with benign and malignant disorders Curr Eye Res 2000; 21: 877-5.-6 Nakamura T, Nishida K, Dota A, Matsuki M, Yamanishi K, Kinoshita S. Elevated expression of transglutaminase 1 and keratinization-related proteins in conjunctiva in severe ocular surface disease Invest Ophthalmol Vis Sci 2001; 42: 549-6.]. In one study, filaggrin was found to be expressed in severe ocular surface diseases such as alkali burns and Stevens-Johnson syndrome, at places where the epithelium had keratinized [6 Nakamura T, Nishida K, Dota A, Matsuki M, Yamanishi K, Kinoshita S. Elevated expression of transglutaminase 1 and keratinization-related proteins in conjunctiva in severe ocular surface disease Invest Ophthalmol Vis Sci 2001; 42: 549-6.].

Long-term mechanical stress to the conjunctival epithelium leads to atrophy or metaplasia, in which there is loss of goblet cells, conversion from stratified columnar to stratified squamous configuration, and keratinization [7Spencer WH. Ophthalmic pathology, an atlas and textbook. 3. ed. Philadelphia: W.B. Saunders 1985.]. Until now, the expression of filaggrin has only been studied in normal human conjunctiva and in conjunctiva that is severely damaged and keratinized.

Our aim was to study the expression of filaggrin in condition with different degrees of mechanical stress and parakeratinization of the conjunctiva. To do this, we investigated the expression in normal conjunctiva, in the conjunctiva of patients with pinguecula, pterygium or in moderate dysplasia of the conjunctiva as well as in Stevens-Johnson syndrome. Under these conditions thickening of the conjunctiva causes rubbing by the eyelids and mechanical stress. If filaggrin is expressed in milder forms of mechanical stress and parakeratinization, it might be used as a marker of mechanical stress in the conjunctiva.

MATERIALS AND METHODS

Formalin-fixed, paraffin-embedded blocks of five orbital exenterations including normal human eyes and eyelids from the same patients were used to investigate filaggrin expression in normal conjunctiva, and in skin as a positive control. We investigated tissue blocks from three middle-aged patients (mean age 55 years) and two elderly patients (mean age 76 years). The orbital exenterations were performed due to cancer or Wegener’s granulomatosis. The diseases did not involve the conjunctiva being investigated or the skin of the eyelid.

In addition, conjunctiva specimens from two patients with pterygium, two patients with pinguecula, one patient with moderate dysplasia of the conjunctiva due to human papilloma virus (HPV), and one patient with Stevens-Johnson syndrome were studied for filaggrin expression. All specimens were obtained from the Eye Pathology Institute, University of Copenhagen, Denmark.

The study was approved by the Danish Data Protection Agency and the local ethics committee (H-1-2011-154).

Immunohistochemistry

Two 4.5μm sections per patient were cut from the paraffin-embedded specimens. The sections were deparaffinized, boiled for 20 min in TrisEDTA buffer (pH=9) for antigen retrieval, and incubated for 25 min with the primary antibody to filaggrin, diluted 1:50 (clone no. 15C10, code no. NCL-Filaggrin; Novocastra Laboratories Ltd, Newcastle upon Tyne, UK). Then the sections were incubated with a secondary antibody (kit code K5003; Dako A/S) for 25 min followed by a peroxidase blocking solution (1% H2O2) for eight min and with a dilution of streptavidin peroxidase (kit code K5003; Dako A/S). Before and after each incubation with antibody or streptavidin peroxidase, the sections were washed with PBS. They were visualized using AEC chromogen (kit code K5003; Dako A/S). All sections, except for the conjunctiva from the patient with moderate dysplasia of the conjunctiva, were stained simultaneously to ensure that samples for comparison were treated in exactly the same way. Positive and negative controls were included to ensure good antigen retrieval and to prevent false positives.

RESULTS

Expression of Filaggrin in Normal Human Conjunctiva

No expression of filaggrin was seen in the healthy conjunctivas (Fig. 1F). Positive controls tissue, i.e. skin from the eyelids of the same patients, showed strong filaggrin expression in the outer, keratinized epithelium (Fig. 1G).

Fig. (1)

A. Nasal pterygium. B. Micrograph of pterygium stained with anti-filaggrin. Bar = 100 µm. Note the complete lack of filaggrin expression. C. Late stage of Stevens-Johnson syndrome. D. Micrograph of sample from (C). Bar = 100 µm. Note the marked binding of anti-filaggrin to the parakeratinized superficial layers of the epithelium. E. Micrograph of conjunctiva with moderate dysplasia. Bar = 100 µm. Only focal filaggrin expression was found. F. Micrograph of normal conjunctiva, which does not express filaggrin. Bar = 100 µm. G. Micrograph of normal skin from same patient as in (F). Bar = 100 µm. Note the marked expression of filaggrin in the keratinized layers of the epithelium.



Expression of Filaggrin in Abnormal Conjunctiva

Pinguecula

The two patients with pinguecula were men aged 56 and 81 years. By staining, there was no evidence of filaggrin expression in the conjunctivas of these patients.

Pterygium

One patient with pterygium was a man aged 46 years and the other was a woman aged 64 years. We did not find any expression of filaggrin in the conjunctiva specimens of these two patients (Fig. 1A, B).

Stevens-Johnson Syndrome

We investigated three different samples from a male patient with Stevens-Johnson syndrome: one biopsy taken from the conjunctiva of the right eye and one taken from the conjunctiva of the left eye, when the patient was 23 years old. The third biopsy was from the left conjunctiva when the patient was 29 years old (Fig. 1C). Intense staining of filaggrin was seen in the superficial layers of all three samples (Fig. D).

Moderate Dysplasia of the Conjunctiva

The patient with moderate dysplasia was 70 years old at the time of resection. Marked expression of filaggrin was found in the parakeratinized areas of the conjunctiva (Fig. 1E).

DISCUSSION

Filaggrin is part of the normal skin barrier, where it plays an essential role in the regulation of epidermal homeostasis [8 Sandilands A, Sutherland C, Irvine AD, McLean WH. Filaggrin in the frontline: role in skin barrier function and disease J Cell Sci 2009; 122: 1285-94., 9 O'Regan GM, Sandilands A, McLean WH, Irvine AD. Filaggrin in atopic dermatitis J Allergy Clin Immunol 2009; 124(3 )(Suppl 2): R2-6.]. It is expressed in keratinocytes during the terminal differentiation, and is therefore involved in keratinization of the skin [10 Dale BA, Holbrook KA, Kimball JR, Hoff M, Sun TT. Expression of epidermal keratins and filaggrin during human fetal skin development J Cell Biol 1985; 101: 1257-69.]. Several mutations in the gene that encodes filaggrin (FLG) have been identified, and linked to skin diseases [11 Smith FJ, Irvine AD, Terron-Kwiatkowski A, et al. Loss-offunction mutations in the gene encoding filaggrin cause ichthyosis vulgaris Nat Genet 2006; 38: 337-42., 12 Sandilands A, O'Regan GM, Liao H, et al. Prevalent and rare mutations in the gene encoding filaggrin cause ichthyosis vulgaris and predispose individuals to atopic dermatitis J Invest Dermatol 2006; 126: 1770-5.]. Homozygosity results in no production of filaggrin. In the present study, normal conjunctiva from the control group did not express any filaggrin. This is consistent with results of previous studies [5 Krenzer KL, Freddo TF. Cytokeratin expression in normal human bulbar conjunctiva obtained by impression cytology Invest Ophthalmol Vis Sci 1997; 38: 142-52., 6 Nakamura T, Nishida K, Dota A, Matsuki M, Yamanishi K, Kinoshita S. Elevated expression of transglutaminase 1 and keratinization-related proteins in conjunctiva in severe ocular surface disease Invest Ophthalmol Vis Sci 2001; 42: 549-6.]. To ensure that was not caused by a mutation in the filaggrin gene, we tested both conjunctiva and skin from the same patient at the same time. They all showed expression of filaggrin in the skin and no expression of filaggrin in the conjunctival epithelium, thereby eliminating the possibility that filaggrin was not detected in the conjunctiva because of a mutation in the gene that encodes the protein.

One study [4 Saika S, Minamide A, Tanaka T, et al. Expression of involucrin by ocular surface epithelia of patients with benign and malignant disorders Curr Eye Res 2000; 21: 877-5.] found that paraformaldehyde destroyed the antigenicity of the filaggrin molecule in biopsy specimens of human skin. No information on the chemicals used to unmask the epitope was given, but since trypsin was used to pre-treat cytokeratins, it might have been used for filaggrin. We used a Tris-EDTA buffer, and there was a positive result for filaggrin in the controls.

One study has also shown that severe damage to the conjunctiva - in the form of Stevens-Johnson syndrome, ocular cicatricial pemphigoid, or chemical injuries in the chronic cicatricial phase - induces the upregulation of filaggrin [6 Nakamura T, Nishida K, Dota A, Matsuki M, Yamanishi K, Kinoshita S. Elevated expression of transglutaminase 1 and keratinization-related proteins in conjunctiva in severe ocular surface disease Invest Ophthalmol Vis Sci 2001; 42: 549-6.].

In the present study, we found filaggrin expression in the conjunctival epithelium of a patient with moderate dysplasia of the conjunctiva. In addition, we found filaggrin expression in two specimens taken six years apart in the conjunctiva of a patient with Stevens-Johnson syndrome. This confirms that filaggrin is expressed when the conjunctiva becomes parakeratinized. Our findings also show that in severely parakeratinized conjunctiva, the expression of filaggrin may be stable over many years. Filaggrin was also expressed in conjunctival dysplasia, which is characterized by mild parakeratinization.

To our knowledge, filaggrin expression in conjunctivae with milder degrees of parakeratinization has not previously been described. Filaggrin was not detected in the conjunctiva of patients with pterygium and pinguecula. Our hypothesis was that even mild parakeratinization may lead to filaggrin expression. However, we found no filaggrin expression in patients suffering from pterygium or pinguecula. We did not have skin samples from these patients, and the lack of filaggrin expression might have been caused by a mutation in filaggrin gene. However, since only 9% of the population of European origin are carriers of the most common filaggrin mutations [13 Palmer CN, Irvine AD, Terron-Kwiatkowski A, et al. Common loss-of-function variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis Nat Genet 2006; 38: 441-6.], it is unlikely that the two patients with pterygium and the two patients with pinguecula would all be homozygotes for a mutation. More patients would have to be tested to make sure that these results was not caused by a mutation. Other more sensitive methods of measuring filaggrin in the conjunctiva could also be used to confirm the results.

In conclusion, filaggrin is not a sensitive marker of mechanical stress to the conjunctiva. However, our results show that filaggrin is expressed in Stevens-Johnson syndrome with severe parakeratinization. Filaggrin may also be expressed in conjunctival dysplasia.

CONFLICT OF INTEREST

The authors confirm that this article content has no conflict of interest.

ACKNOWLEDGEMENTS

This study was supported by Lundbeck Foundation, Copenhagen.

REFERENCES

[1] De Benedetto A, Qualia CM, Baroody FM, Beck LA. Filaggrin expression in oral, nasal, and esophageal mucosa J Invest Dermatol 2008; 128: 1594-7.
[2] Cintorino M, Syrjanen S, Leoncini P, et al. Altered expression of filaggrin in human papillomavirus (HPV) lesions of the uterine cervix Arch Gynecol Obstet 1988; 241: 235-47.
[3] Tong L, Corrales RM, Chen Z, et al. Expression and regulation of cornified envelope proteins in human corneal epithelium Invest Ophthalmol Vis Sci 2006; 47: 1938-46.
[4] Saika S, Minamide A, Tanaka T, et al. Expression of involucrin by ocular surface epithelia of patients with benign and malignant disorders Curr Eye Res 2000; 21: 877-5.
[5] Krenzer KL, Freddo TF. Cytokeratin expression in normal human bulbar conjunctiva obtained by impression cytology Invest Ophthalmol Vis Sci 1997; 38: 142-52.
[6] Nakamura T, Nishida K, Dota A, Matsuki M, Yamanishi K, Kinoshita S. Elevated expression of transglutaminase 1 and keratinization-related proteins in conjunctiva in severe ocular surface disease Invest Ophthalmol Vis Sci 2001; 42: 549-6.
[7] Spencer WH. Ophthalmic pathology, an atlas and textbook. 3. ed. Philadelphia: W.B. Saunders 1985.
[8] Sandilands A, Sutherland C, Irvine AD, McLean WH. Filaggrin in the frontline: role in skin barrier function and disease J Cell Sci 2009; 122: 1285-94.
[9] O'Regan GM, Sandilands A, McLean WH, Irvine AD. Filaggrin in atopic dermatitis J Allergy Clin Immunol 2009; 124(3 )(Suppl 2): R2-6.
[10] Dale BA, Holbrook KA, Kimball JR, Hoff M, Sun TT. Expression of epidermal keratins and filaggrin during human fetal skin development J Cell Biol 1985; 101: 1257-69.
[11] Smith FJ, Irvine AD, Terron-Kwiatkowski A, et al. Loss-offunction mutations in the gene encoding filaggrin cause ichthyosis vulgaris Nat Genet 2006; 38: 337-42.
[12] Sandilands A, O'Regan GM, Liao H, et al. Prevalent and rare mutations in the gene encoding filaggrin cause ichthyosis vulgaris and predispose individuals to atopic dermatitis J Invest Dermatol 2006; 126: 1770-5.
[13] Palmer CN, Irvine AD, Terron-Kwiatkowski A, et al. Common loss-of-function variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis Nat Genet 2006; 38: 441-6.

Endorsements



"Open access will revolutionize 21st century knowledge work and accelerate the diffusion of ideas and evidence that support just in time learning and the evolution of thinking in a number of disciplines."


Daniel Pesut
(Indiana University School of Nursing, USA)

"It is important that students and researchers from all over the world can have easy access to relevant, high-standard and timely scientific information. This is exactly what Open Access Journals provide and this is the reason why I support this endeavor."


Jacques Descotes
(Centre Antipoison-Centre de Pharmacovigilance, France)

"Publishing research articles is the key for future scientific progress. Open Access publishing is therefore of utmost importance for wider dissemination of information, and will help serving the best interest of the scientific community."


Patrice Talaga
(UCB S.A., Belgium)

"Open access journals are a novel concept in the medical literature. They offer accessible information to a wide variety of individuals, including physicians, medical students, clinical investigators, and the general public. They are an outstanding source of medical and scientific information."


Jeffrey M. Weinberg
(St. Luke's-Roosevelt Hospital Center, USA)

"Open access journals are extremely useful for graduate students, investigators and all other interested persons to read important scientific articles and subscribe scientific journals. Indeed, the research articles span a wide range of area and of high quality. This is specially a must for researchers belonging to institutions with limited library facility and funding to subscribe scientific journals."


Debomoy K. Lahiri
(Indiana University School of Medicine, USA)

"Open access journals represent a major break-through in publishing. They provide easy access to the latest research on a wide variety of issues. Relevant and timely articles are made available in a fraction of the time taken by more conventional publishers. Articles are of uniformly high quality and written by the world's leading authorities."


Robert Looney
(Naval Postgraduate School, USA)

"Open access journals have transformed the way scientific data is published and disseminated: particularly, whilst ensuring a high quality standard and transparency in the editorial process, they have increased the access to the scientific literature by those researchers that have limited library support or that are working on small budgets."


Richard Reithinger
(Westat, USA)

"Not only do open access journals greatly improve the access to high quality information for scientists in the developing world, it also provides extra exposure for our papers."


J. Ferwerda
(University of Oxford, UK)

"Open Access 'Chemistry' Journals allow the dissemination of knowledge at your finger tips without paying for the scientific content."


Sean L. Kitson
(Almac Sciences, Northern Ireland)

"In principle, all scientific journals should have open access, as should be science itself. Open access journals are very helpful for students, researchers and the general public including people from institutions which do not have library or cannot afford to subscribe scientific journals. The articles are high standard and cover a wide area."


Hubert Wolterbeek
(Delft University of Technology, The Netherlands)

"The widest possible diffusion of information is critical for the advancement of science. In this perspective, open access journals are instrumental in fostering researches and achievements."


Alessandro Laviano
(Sapienza - University of Rome, Italy)

"Open access journals are very useful for all scientists as they can have quick information in the different fields of science."


Philippe Hernigou
(Paris University, France)

"There are many scientists who can not afford the rather expensive subscriptions to scientific journals. Open access journals offer a good alternative for free access to good quality scientific information."


Fidel Toldrá
(Instituto de Agroquimica y Tecnologia de Alimentos, Spain)

"Open access journals have become a fundamental tool for students, researchers, patients and the general public. Many people from institutions which do not have library or cannot afford to subscribe scientific journals benefit of them on a daily basis. The articles are among the best and cover most scientific areas."


M. Bendandi
(University Clinic of Navarre, Spain)

"These journals provide researchers with a platform for rapid, open access scientific communication. The articles are of high quality and broad scope."


Peter Chiba
(University of Vienna, Austria)

"Open access journals are probably one of the most important contributions to promote and diffuse science worldwide."


Jaime Sampaio
(University of Trás-os-Montes e Alto Douro, Portugal)

"Open access journals make up a new and rather revolutionary way to scientific publication. This option opens several quite interesting possibilities to disseminate openly and freely new knowledge and even to facilitate interpersonal communication among scientists."


Eduardo A. Castro
(INIFTA, Argentina)

"Open access journals are freely available online throughout the world, for you to read, download, copy, distribute, and use. The articles published in the open access journals are high quality and cover a wide range of fields."


Kenji Hashimoto
(Chiba University, Japan)

"Open Access journals offer an innovative and efficient way of publication for academics and professionals in a wide range of disciplines. The papers published are of high quality after rigorous peer review and they are Indexed in: major international databases. I read Open Access journals to keep abreast of the recent development in my field of study."


Daniel Shek
(Chinese University of Hong Kong, Hong Kong)

"It is a modern trend for publishers to establish open access journals. Researchers, faculty members, and students will be greatly benefited by the new journals of Bentham Science Publishers Ltd. in this category."


Jih Ru Hwu
(National Central University, Taiwan)


Browse Contents



Webmaster Contact: info@benthamopen.net
Copyright © 2019 Bentham Open