Downregulation of IL-8, ECP, and total IgE in the tears of patients with atopic keratoconjunctivitis treated with rebamipide eyedrops

Rebamipide was developed as a gastroprotective drug; it increases gastric mucus production [1, 2] and suppresses gastric mucosal inflammation [3, 4]. Urashima et al. reported that rebamipide up-regulated the secretion and production of mucin of the ocular surface [5].

Rebamipide eyedrops are approved in Japan for the treatment of dry eye disease. Our group documented that the administration of 2% rebamipide ophthalmic suspension was well-tolerated by patients and that it effectively improved the objective signs and subjective symptoms of dry eye [6‐8].

Some patients with allergic conjunctival diseases present with dry eye, which is the type of a decrease in the tear break-up time (BUT) [9, 10]. We found that rebamipide suppressed polyI:C-induced inflammatory cytokines in human conjunctival epithelial cells [11] and suggested that the combination of rebamipide eyedrops and conventional anti-allergic treatments might be effective in patients with vernal/atopic keratoconjunctivitis (VKC, AKC) refractory to conventional treatment with anti-allergic- and/or immunosuppressive/steroid eyedrops [12].

We prescribed rebamipide eyedrops to patients with AKC who presented with dry eye and examined their effect on the level of IL-8, eosinophil cationic protein (ECP), and total IgE in their tears. We now report that in patients with AKC, the tear level of IL-8, ECP, and total IgE was decreased at 4–6 weeks after starting the administration of rebamipide eyedrops.

Our study protocol was approved by the ethical review board of Kyoto Prefectural University of Medicine; all patients provided prior written informed consent.

Our study included 4 patients (6 eyes) with AKC and dry eye whom we treated with rebamipide in 2013; their tear BUT was decreased and the 6 eyes had not been treated with any topical steroids or immunosuppressants. They were 3 males and 1 female; their age ranged from 15–49 years (mean 29.8). They were instructed to instill rebamipide eyedrops four times a day. The details of the six eyes of the four patients were shown in Table 1.

We started the administration of rebamipide eyedrops in patients with dry eye and itching of the ocular surface who were or were not treated earlier with anti-allergic eyedrops. Tear samples were collected on Schirmer’s measurement strips (Schirmer Tear Production Measuring Strips, Showa Yakuhin Kako, Tokyo, Japan) according to the method previously reported [13]. The Schirmer filter papers with collected tears were immersed in 100 ul Tris-buffered saline with Tween 20 (TBST) (DAKO, Japan) for 10 min at room temperature and 50 μl of TBST containing the tears were used for measuring IL-8 and total IgE with BD™ CBA flex sets; 5 μl of TBST containing tears were diluted 20 times with TBST and used a total of 100 μl for measurements with the human eosinophil cationic protein ELISA Kit. The tear volume on the Schirmer filter papers was calculated based on a standard curve obtained from 0 to 25 μl of distilled water at 1-μl intervals.

The concentration of IL-8 and total IgE in tears was measured with BD™ CBA flex sets and BD™ human soluble protein master buffer kits according to the manufacturer’s instructions (BD Bioscience-PharMingen, San Diego, CA). The amount of ECP in the tears was measured using the human eosinophil cationic protein ELISA Kit according to the manufacturer’s instructions (Aviscera Bioscience, Inc., CA, USA).

Subjective symptoms associated with AKC, i.e. itching, foreign body sensation, and eye mucus, were evaluated with a patient questionnaire. Before each clinical examination, the 4 patients marked the severity of their ocular symptoms on a scale from 0 (none) to 10 (most severe). Data were expressed as the mean and the individual values and evaluated by Steel’s test using JMP (version 10.0.2 software; SAS Institute Japan Ltd).

In case 1, case 2 and case 4, IL-8 levels in their tears were lower 2 and 4–6 weeks after- than before the start of rebamipide treatment (Figure 1a). In case 3, the level of IL-8 in his tears was reduced at 4 weeks after the start of rebamipide treatment (Figure 1a), but not 2 weeks. We performed statistical analysis of the change in the tear level of IL-8 in the six eyes. The controls were 14 eyes without atopy. Before rebamipide treatment, the tear level of IL-8 was higher in the six eyes than in 14 control eyes (mean ± SD, 28.0 ± 37.0 ng/ml vs. 1.0 ± 0.6 ng/ml). As shown in Figure 1b, rebamipide eyedrops produced a significant decrease in the IL-8 levels of all treated eyes 2 and 4–6 weeks after the start of therapy (p <0.005 and p <0.0001, respectively, by Dunnett’s test).

In case 1, ECP was not measured. In the right eye of case 2 and the left eye of case 4, the level of ECP in their tears was lower 2 and 4–6 weeks after- than before the start of rebamipide treatment (Figure 2a). In the left eye of case 2 and the right eye of case 3, the level of ECP in their tears was reduced at 4 weeks after the start of rebamipide treatment, but not 2 weeks (Figure 2a). We assessed the changes in the tear ECP level in these 4 eyes. The controls were 10 eyes without atopy. The ECP level in the four eyes was significantly higher than in the controls (1840 ± 585 ng/ml vs. 77 ± 40 ng/ml, mean + SD; p < 0.01; Student’s t-test). The ECP levels were significantly lower after 4–6 weeks of rebamipide treatment (p <0.05; Dunnett’s test) but not after 2 weeks (Figure 2b).

In case 1, case 4, and the right eye of case 2, total IgE level in their tears were lower 2 and 4–6 weeks after- than before the start of rebamipide treatment (Figure 3a). In case 3 and the left eye of case 2, the level of total IgE in their tears was reduced at 4 weeks after the start of rebamipide treatment, but not 2 weeks (Figure 3a). We determined the changes in the tear total IgE level in the six eyes. The controls were 14 eyes without atopy. These levels were significantly higher in the patient- than the control eyes (2990 ± 1920 ng/ml vs. 0.9 ± 2 ng/ml, mean + SD, p < 0.05; Student’s t-test). The total IgE level was significantly lower after 4–6 weeks (p < 0.05; Dunnett’s test) but not after 2 weeks of rebamipide treatment (Figure 3b).

In all cases, the addition of rebamipide eyedrops to treat their eye reduced their subjective symptoms. The statistical analysis of the scale evaluated with a patient questionnaire showed that the patients’ subjective symptoms associated with AKC were significantly improved 2 and 4–6 weeks after starting rebamipide therapy (Table 2).