LABORATORY SCIENCES Inhibition of Histamine-Induced Human Conjunctival Epithelial Cell Responses by Ocular Allergy Drugs John M. Yanni, PhD; Lori K. Weimer, BA; Najam A. Sharif, PhD; Shou X. Xu, MS; Daniel A. Gamache, PhD; Joan M. Spellman, MS Objective: To evaluate the effects of topical ocular drugs secretion (50% inhibitory concentration, 1.7-5.5 nmol/L) with histamine H1-antagonist activity on histamine- than predicted from binding data, while antazoline and stimulated phosphatidylinositol turnover and interleu- pheniramine were far less potent (20- to 140-fold) in func- kin (IL) 6 and IL-8 secretion from human conjunctival tional assays. Levocabastine (dissociation constant, 52.6 epithelial cells. nmol/L) exhibited greater functional activity (50% in- hibitory concentration, 8-25 nmol/L) than either antazo- Methods: Primary human conjunctival epithelial cell cul- line or pheniramine. tures were stimulated with histamine in the presence or ab- sence of test drugs. Phosphatidylinositol turnover was quan- Conclusions: Histamine-stimulated phosphatidylino- tified by ion exchange chromatography and cytokine con- sitol turnover and cytokine secretion by human conjunc- tentofsupernatantsbyenzyme-linkedimmunosorbentassay. tival epithelial cells are attenuated by compounds with H1-antagonist activity. However, antihistaminic po- Results: Antazoline hydrochloride, emedastine difuma- tency alone does not predict anti-inflammatory poten- rate, levocabastine hydrochloride, olopatadine hydro- tial. Olopatadine, emedastine, and levocabastine were no- chloride, and pheniramine maleate attenuated histamine- tably more potent than pheniramine and antazoline. stimulated phosphatidylinositol turnover and IL-6 and IL-8 secretion. Emedastine was the most potent in li- Clinical Relevance: Selected topical ocular drugs with gand binding, phosphatidylinositol turnover, and IL-6 se- antihistaminic activity may offer therapeutic advan- cretion, with dissociation constant and 50% inhibitory tages to patients with allergic conjunctivitis by inhibit- concentrations of 1-3 nmol/L. Olopatadine, antazoline, ing proinflammatory cytokine secretion from human con- and pheniramine exhibited similar H1-binding affinities junctival epithelial cells. (32-39 nmol/L). However, olopatadine was approxi- mately 10-fold more potent as an inhibitor of cytokine Arch Ophthalmol. 1999;117:643-647 UMAN CONJUNCTIVAL epi- enhanced by exposure to 1-µmol/L hista- thelial cells (HCEs) se- mine.2 In addition, histamine caused a crete cytokines after dose-dependent stimulation of IL-6, IL-8, stimulation by various and GM-CSF release by normal and trans- cell-activating agents. formed human bronchial epithelial cells 1 Gamache et al showed that primary cul- that appeared to occur via histamine H1- H 3 tures of HCEs secrete tumor necrosis fac- receptor activation. The proinflamma- tor a, interleukin (IL) 6, IL-8, and granu- tory properties of these cytokines are well locyte-macrophage colony-stimulating documented. Elevated IL-6 levels have factor (GM-CSF) after IL-1a, phorbol my- been reported to be associated with a va- ristate acetate, and calcium ionophore riety of inflammatory conditions, includ- A23187 treatment. The authors suggested ing asthma, psoriasis, uveitis, and aller- that this capability of the conjunctival epi- gic rhinitis.4-7 Intravitreal injection of IL-6 thelium indicates a possible effector func- has been shown to produce uveitis in rats tion for the tissue in allergic conjunctivi- and rabbits.8 Interleukin 8 is a potent mem- tis. Other investigators have reported that ber of the C-X-C family of chemokines. It histamine, a major mediator of allergic dis- promotes integrin expression, neutro- eases, induces the production of cyto- phil degranulation, and chemotaxis of ba- 9-13 From Ophthalmic Products kines by airway epithelium. For in- sophils and eosinophils. Research, Alcon Laboratories stance, GM-CSF release from human The presence of these polymorpho- Inc, Fort Worth, Tex. tracheal epithelial cells was significantly nuclear leukocytes (primarily eosino- ARCH OPHTHALMOL / VOL 117, MAY 1999 643 ©1999 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 MATERIALS AND METHODS performed as previously described21 with minor modifica- tions. The HCEs were incubated with tritiated ([3H]) myoinositol (0.037 MBq/0.5 mL; 55.5-62.9 3 1010 Bq/ CELL CULTURES mmol; Amersham Life Science, Inc, Arlington Heights, Ill) in Dulbecco modified Eagle medium (GIBCO, Gaithers- Methods detailing the preparation of primary epithelial cell burg, Md) for 24 hours in 5% carbon dioxide at 37°C to cultures and cytokine release studies with the use of these cells label the cell membrane lipids. Cells were then exposed to have been described.1 Briefly, cultures of HCEs were initiated histamine (10 nmol/L to 1 mmol/L) for 60 minutes at 23°C. from donor tissues obtained by various eye banks within 8 To determine the potencies of the antagonists, the drugs hours post mortem. The tissues were enzymatically digested were added to the cells 20 minutes before the addition of overnight. Epithelial cells were gently scraped from the tis- histamine (100 µmol/L). The assay was terminated by the sue surface, dissociated into a single cell suspension, and cul- addition of ice-cold 0.1-mol/L formic acid. With ion ex- tured in Clonetics keratinocyte growth medium (Biowhittaker change columns containing 1 mL of AG1-X8 resin in for- Corp, Walkersville, Md). Cells were used only through pas- mate form, free [3H]myoinositol was removed from the cell sage 6. Cultures were maintained in a preconfluent state to lysates with deionized water; the water-soluble [3H]inosi- prevent differentiation. Cells were identified as epithelial by tol phosphates were then eluted with 1.2-mol/L ammo- positive keratin staining, as described previously.1 nium formate. The [3H]inositol phosphates were quanti- fied by liquid scintillation spectrometry. CYTOKINE ASSAYS DATA ANALYSIS Several compounds with histamine H1-antagonist activity were evaluated for their ability to inhibit secretion of cy- The antagonist potency (IC50) was defined as the concen- tokines (IL-6 and IL-8) from cultured HCEs in response tration of the drug required to produce 50% inhibition of to histamine stimulation. Cells were plated at 2 3 104 cells the agonist-stimulated functional response. Data derived per well and cultured overnight in 5% carbon dioxide at from the cytokine assays were calculated as mean and SEM 37°C. The following day, keratinocyte growth medium con- values that represent the variability among identically treated taining test compound was added directly to wells and the culture wells. The dose-dependent effect of pharmacologi- cells were incubated for 30 minutes before 24-hour stimu- cal agents and IC50s were determined by linear regression. lation with histamine (30 µmol/L). Three culture wells were Data obtained in the PI turnover assays were analyzed by used for each treatment group. At harvest, cell monolay- means of a nonlinear, iterative curve fitting program as pre- ers were examined microscopically to confirm viability and viously described.18,20 Data are expressed as mean ± SEM supernatants were collected, centrifuged at 200g, and stored from 3 to 5 independent experiments. at −20°C. Samples were analyzed for IL-6 and IL-8 by en- zyme-linked immunosorbent assay (R&D Systems, Min- TEST COMPOUNDS neapolis, Minn) as directed by the manufacturer. The sen- sitivities of each enzyme-linked immunosorbent assay are Compounds were obtained as follows: antazoline hydro- 0.7 pg/mL for IL-6 and 3.0 pg/mL for IL-8. chloride and pheniramine maleate (Sigma-Aldrich Corp, St Louis, Mo); emedastine difumarate (Kanebo Ltd, Osaka, HISTAMINE-INDUCED Japan); olopatadine hydrochloride (Kyowa Hakko Kogyo PHOSPHATIDYLINOSITOL TURNOVER Co Ltd, Tokyo, Japan); and levocabastine hydrochloride (Livostin; Ciba Vision Ophthalmics, Atlanta, Ga). Hista- The determination of phosphatidylinositol (PI) turnover mine dihydrochloride was obtained from Research Bio- induced by stimulation of phospholipase C in HCEs was chemicals International, Natick, Mass. phils) contributes to the pathogenesis of the late-phase HCEs. These receptors were coupled to inositol phos- allergic response. Degranulation of the eosinophilic leu- phate generation, which mobilized intracellular cal- kocytes leads to the release of major basic protein, eo- cium. Calcium mobilization peaked within 10 seconds sinophil peroxidase, eosinophil cationic protein, and eo- and was sustained for 20 minutes after stimulation with sinophil-derived neurotoxin.14 Reports have demonstrated histamine. Pharmacological studies indicated that his- that the eosinophil-derived proinflammatory mediator, tamine H1-receptor antagonists potently antagonized these major basic protein, has the ability to activate neutro- effects. Recently, Weimer et al19 demonstrated that phils as well as stimulating additional histamine re- histamine stimulation of HCEs induced the secretion of lease, further amplifying the allergic response.15 IL-6, IL-8, and GM-CSF in a concentration- and time- Elevated levels of histamine in tears of patients with dependent manner. They also presented evidence that the allergic ocular diseases have been reported. Tears col- potent H1-antagonist emedastine inhibited cytokine se- lected after ocular antigen challenge contained signifi- cretion at concentrations consistent with its published 20 cantly more histamine than tears collected after