Author Affiliations: Research Center for Inflammation and Regenerative Medicine, Faculty of Life and Medical Sciences, Doshisha University (Dr Ueta) and Department of Ophthalmology, Kyoto Prefectural University of Medicine (Drs Ueta, Sotozono, Yokoi, Inatomi, and Kinoshita), Kyoto, Japan.
Prostanoids are a group of lipid mediators that form in response to various stimuli, including prostaglandin (PG) D2 (PGD2), PGE2, PGF2α, PGI2, and thromboxane A2. There are 8 types of prostanoid receptors that are conserved in mammals ranging from mice to humans: the PGD receptor, 4 subtypes of the PGE receptor (EP1, EP2, EP3, and EP4), the PGF receptor, the PGI receptor, and the thromboxane A receptor.1 In regard to PGE receptor subtype EP4, it was reported that EP4 messenger RNA was present in the intestinal epithelium2 and that EP4 maintained intestinal homeostasis and downregulated immune response.3 Like the intestine, the ocular surface is also one of the mucosa that are in contact with commensal bacteria. In this study, we examined the expression of EP4 in human conjunctival epithelium and compared its expression between various ocular surface diseases.
This study was approved by the Institutional Review Board of Kyoto Prefectural University of Medicine, Kyoto, Japan. For reverse transcription–polymerase chain reaction assay, we obtained human conjunctival epithelial cells from healthy volunteers by brush cytology using previously described methods.4 The primers were (forward) 5′-TCA ACC ATG CCT ATT TCT ACA GCC ACT ACG-3′ and (reverse) 5′-AGG TCT CTG ATA TTC GCA AAG TCC TCA GTG-3′ for human PTGER4 and (forward) 5′-CCA TCA CCA TCT TCC AGG AG-3′ and (reverse) 5′-CCT GCT TCA CCA CCT TCT TG-3′ for human GAPDH. For immunohistochemistry, we used nearly normal bulbar conjunctival tissues obtained during surgery for conjunctivochalasis as a control, and human conjunctival tissues were also prepared from samples obtained during surgery to reconstruct the ocular surface such as treatment for various ocular surface diseases including Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN), ocular cicatricial pemphigoid (OCP), and pterygium. For EP4 staining, we used the rabbit polyclonal antibody to EP4 (Cayman Chemical Co, Ann Arbor, Michigan).
The presence of PTGER4 messenger RNA and EP4 protein in human conjunctival epithelium was examined by reverse transcription–polymerase chain reaction and immunohistological analysis, respectively. The PTGER4 messenger RNA was detected in normal human conjunctival epithelium (Figure, A). The sequences obtained from these polymerase chain reaction products were identical to the human PTGER4 complementary DNA sequence. The EP4 protein was also detected in the nearly normal conjunctival epithelium obtained from the patients with conjunctivochalasis (Figure, B). Next, we examined the conjunctival tissues with various ocular surface diseases. The EP4 protein was detected in conjunctival epithelium from patients with pterygium as well as in the conjunctival epithelium from control patients with conjunctivochalasis. However, we did not detect EP4 immunoreactivity in the conjunctival epithelium from patients with SJS/TEN or OCP (Figure, C). Our results showed that EP4 is strongly downregulated in the conjunctival epithelium of tissues with devastating ocular surface disorders such as SJS/TEN and OCP, although it is usually expressed in human conjunctival epithelium.
Polymerase chain reaction and immunohistological analysis results. A, Reverse transcription (RT)–polymerase chain reaction analyses of the expression of PTGER4 -specific messenger RNA in human conjunctival epithelium derived from 4 volunteers. bp indicates base pairs. Immunohistological analysis for prostaglandin E receptor 4 (EP4) in human conjunctival epithelium in nearly normal conjunctival tissues with conjunctivochalasis (B) and in conjunctival tissues with various ocular surface diseases such as pterygium, Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN), and ocular cicatricial pemphigoid (OCP) (C). Scale bars indicate 100 μm. For EP4 staining, we used the rabbit polyclonal antibody to EP4 (Cayman Chemical Co, Ann Arbor, Michigan) and Biotin-SP–conjugated AffiniPure F(ab′)2 fragment donkey antirabbit IgG (H + L) (Jackson Immuno Research, Baltimore, Maryland) as the secondary antibody; then, Vectastain ABC reagents (Vector Laboratories, Inc, Burlingame, California) were used for increased sensitivity with peroxidase substrate solution (DAB substrate kit; Vector Laboratories, Inc) as a chromogenic substrate.
To our knowledge, this is the first documentation regarding downregulation of EP4 expression in human conjunctival epithelium in tissues with devastating ocular surface inflammatory disorders, although there were reports of expression of EP receptors in ocular tissues.5 Kabashima et al3 reported that EP4 deficiency impaired mucosal barrier function and aggregation of neutrophils and lymphocytes in the colon and that administration of an EP4-selective agonist to wild-type mice ameliorated severe colitis; they concluded that EP4 maintains intestinal homeostasis. On the other hand, Yao et al6 recently reported that PGE2 acts on its receptor EP4 on T cells and dendritic cells and promotes immune inflammation.
In human conjunctival tissues, the EP4 protein was detected in only epithelial cells but not infiltrating cells into subconjunctival tissues. Because there is mucosal inflammation on the ocular surface even in patients with chronic-phase SJS/TEN or OCP, we suspect that the downregulation of EP4 expression in conjunctival epithelium might be associated with the ocular surface inflammation in patients with SJS/TEN or OCP and that there is a possibility that EP4 in human normal conjunctival epithelium suppresses the ocular surface inflammation.
Correspondence: Dr Ueta, Department of Ophthalmology, Kyoto Prefectural University of Medicine, 465 Kajiicho, Hirokoji, Kawaramachi, Kamigyoku, Kyoto 602-0841, Japan (firstname.lastname@example.org).
Financial Disclosure: None reported.
Funding/Support: This work was supported in part by grants-in-aid for scientific research from the Japanese Ministry of Health, Labour, and Welfare (Research on Intractable Diseases), the Japanese Ministry of Education, Culture, Sports, Science, and Technology, a research grant from the Kyoto Foundation for the Promotion of Medical Science, the Intramural Research Fund of Kyoto Prefectural University of Medicine, a Shiseido Female Researcher Science Grant, and a research grant from the Japan Allergy Foundation.
Additional Contributions: Chikako Endo provided technical assistance.
Thank you for submitting a comment on this article. It will be reviewed by JAMA Ophthalmology editors. You will be notified when your comment has been published. Comments should not exceed 500 words of text and 10 references.
Do not submit personal medical questions or information that could identify a specific patient, questions about a particular case, or general inquiries to an author. Only content that has not been published, posted, or submitted elsewhere should be submitted. By submitting this Comment, you and any coauthors transfer copyright to the journal if your Comment is posted.
* = Required Field
Disclosure of Any Conflicts of Interest*
Indicate all relevant conflicts of interest of each author below, including all relevant financial interests, activities, and relationships within the past 3 years including, but not limited to, employment, affiliation, grants or funding, consultancies, honoraria or payment, speakers’ bureaus, stock ownership or options, expert testimony, royalties, donation of medical equipment, or patents planned, pending, or issued. If all authors have none, check "No potential conflicts or relevant financial interests" in the box below. Please also indicate any funding received in support of this work. The information will be posted with your response.
Some tools below are only available to our subscribers or users with an online account.
Download citation file:
Web of Science® Times Cited: 2
Customize your page view by dragging & repositioning the boxes below.
Enter your username and email address. We'll send you a link to reset your password.
Enter your username and email address. We'll send instructions on how to reset your password to the email address we have on record.
Athens and Shibboleth are access management services that provide single sign-on to protected resources. They replace the multiple user names and passwords necessary to access subscription-based content with a single user name and password that can be entered once per session. It operates independently of a user's location or IP address. If your institution uses Athens or Shibboleth authentication, please contact your site administrator to receive your user name and password.