Inhibition of Histamine-Induced Human Conjunctival Epithelial Cell Responses by Ocular Allergy Drugs

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% inhibitory 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 approximately 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 ␣, 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-1␣, 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-

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©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 ϫ 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 quantified 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 ϫ 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 enzyme-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 selected after ocular antigen challenge contained signifi- cretion at concentrations consistent with its published 20 cantly more histamine than tears collected after sterile affinity for the H1 receptor. saline exposure.16 The concentration of histamine in tears The current experiments were conducted to com- collected from patients with vernal conjunctivitis was in- pare the inhibitory effects of compounds with H1- creased more than 3 times compared with the concen- antagonist activity currently available for topical ocular tration in normal subjects.17 Sharif et al18 demonstrated use on histamine-stimulated activation of the second mes- the presence of functional histamine H1-receptors on senger system and cytokine release from HCEs.

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©1999 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 Emedastine Difumarate (r 2 = 0.982) 2 100 Olopatadine Hydrochloride (r = 0.886) Levocabastine Hydrochloride (r 2 = 0.992) Antazoline Phosphate (r 2 = 0.962) Pheniramine Maleate (r 2 = 0.999)

80 100

50 % Inhibition

25 40 % Maximal PI Response

0 Emedastine Difumerate Olopatadine –11 –10 –9 –8–7 –6 –5 –4 20 Hydrochloride Dose Log, mol/L Levocabastine Hydrochloride Figure 2. Effect of histamine H1-antagonists on histamine-induced Antazoline Phosphate interleukin 6 secretion from human conjunctival epithelial cells. Cells were Pheniramine Maleate incubated with test compound for 30 minutes before stimulation with 0 histamine (30 µmol/L) for 24 hours. Supernatants were analyzed for –11–10 –9 –8–7 –6 –5 –4 cytokines by specific enzyme-linked immunosorbent assay. Log [Antagonist], mol/L

Figure 1. Effect of histamine H -antagonists on histamine-induced 1 Emedastine Difumarate (r 2 = 0.960) phosphoinositide (PI) turnover in human conjunctival epithelial cells. Tritiated ([ 3H])myoinositol–labeled cells were exposed to drug for 20 Olopatadine Hydrochloride (r 2 = 0.953) minutes before stimulation with histamine for 60 minutes. [ 3H]inositol Levocabastine Hydrochloride (r 2 = 0.976) phosphates were quantified by ion exchange chromatography followed by Antazoline Phosphate (r 2 = 0.909) liquid scintillation spectrometry. Pheniramine Maleate (r 2 = 0.806)

100 RESULTS

Exposure of HCEs to 100-µmol/L histamine maximally 75 stimulated PI turnover (2.54 ± 0.16-fold above basal levels). Similarly, exposure of these cells to 30-µmol/L histamine increased IL-6 and IL-8 secretion 1.59 ± 0.19- and 50

1.80 ± 0.28-fold above basal levels, respectively. (Basal lev- % Inhibition els of the cytokines were 7667 ± 2110 pg/106 cells [n = 4] for IL-6 and 9857 ± 2386 pg/106 cells [n = 6] for IL-8.) 25 Treatment of HCEs with drugs possessing antihis-

taminic activity and available for topical ocular admin- 0 istration before histamine exposure resulted in concen- –11 –10 –9 –8–7 –6 –5 tration-dependent inhibition of PI turnover, IL-6 secretion, Dose Log, mol/L and IL-8 secretion. All 5 compounds tested produced con- Figure 3. Effect of histamine H1-antagonists on histamine-stimulated centration-dependent inhibition of the histamine- interleukin 8 secretion from human conjunctival epithelial cells. Assay and stimulated cell functional responses (Figure 1, Figure 2, data collection procedures are as noted in Figure 2. and Figure 3). Emedastine was the most potent compound tested (Table). The potency of emedastine in intact cells was PI turnover and IL-6 and IL-8 secretion, respectively. In consistent with its activity determined in receptor bind- fact, olopatadine inhibited histamine-stimulated secre- ing assays with the use of tissue homogenates. Its IC50 tion of IL-8 at a concentration (IC50, 1.7 nmol/L) lower values for histamine-induced PI turnover and IL-6 and than emedastine’s efficacious concentration in the same IL-8 secretion were 1.54, 2.5, and 4.0 nmol/L, respec- assay system. tively. Levocabastine and olopatadine were also potent Antazoline and pheniramine, 2 first-generation topi- inhibitors of these histamine-stimulated responses. Le- cal ocular antihistamines, were dramatically less potent vocabastine inhibited the PI turnover and IL-6 and IL-8 inhibitors of these histamine-induced cell-based re- secretion (IC50s, 8.32, 25.1, and 11.9 nmol/L, respec- sponses (PI turnover, IL-6 and IL-8 secretion) than pre- tively). Olopatadine was more potent than predicted from dicted from their histamine H1-receptor binding affini- its published histamine H1-receptor binding affinity (36 ties (Table). The calculated IC50 values for these 22 nmol/L), with IC50s of 10.03, 5.5, and 1.7 nmol/L for compounds on the parameters listed above ranged from

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©1999 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 sented evidence that the histamine-stimulated cytokine Histamine H Antagonists: Inhibition of IL-6 and IL-8 1 response was the result of histamine H1-receptor activa- Secretion and PI Turnover in Human Conjunctival tion in these cells. These findings are supported by data Epithelial Cells and H Receptor Binding Affinities* 1 showing that the effects of histamine on PI turnover and 2+ IL-6 IL-8 PI Turnover Binding intracellular Ca concentration in HCEs were not sig-

IC50, IC50, IC50, Ki, nificantly blocked by H2 and H3 antagonists but were dra- 18 H1 Antagonist nmol/L nmol/L nmol/L nmol/L matically reduced by H1 antagonists. Emedastine difumarate 2.5 4.0 1.54 1.22† Our data confirm histamine’s ability to stimulate PI Olopatadine hydrochloride 5.5 1.7 10.03 36.00‡ turnover and cytokine secretion from HCEs. The con- Levocabastine hydrochloride 25.1 11.9 8.32 52.60† centrations of the agonist used in the present studies (30- Antazoline hydrochloride 1014.0 652.0 4200.00 38.40† 100 µmol/L) are consistent with previous cytokine- Pheniramine maleate 4826.0 1216.0 4500.00 33.90† stimulating concentrations of the biogenic amine.2,3,19,24,25 Current data demonstrate that compounds capable IL indicates interleukin; PI, phosphoinositide; IC , 50% inhibitory * 50 of antagonizing histamine H receptors prevent concentration; and Ki, the concentration of the drug required at equilibrium to 1 inhibit the receptor binding by 50%. histamine-stimulated IL-6 and IL-8 secretion. These data †From Sharif et al.20 confirm emedastine’s ability to prevent cytokine secre- 22 ‡From Yanni et al. tion19 and characterize the effects of other compounds that possess antihistaminic activity. First-generation topical ocular antihistamines antazoline and pheni- 652 to 4200 nmol/L for antazoline and 1216 to 4826 ramine have reported affinities for the histamine H1 nmol/L for pheniramine. receptor of 38.4 and 33.9 nmol/L, respectively.20 The binding paradigms used to generate these values used COMMENT cell membranes as the receptor source. In the present studies, which used living, whole cells as the test sys- Histamine is recognized as a primary mediator of aller- tems, the 2 compounds were surprisingly less potent. gic disease. Its acute vascular effects lead to erythema and This was true not only for cytokine secretion but also for edema, and its pruritogenic effects are responsible for the histamine-stimulated PI turnover. These physiological itch characteristic of allergic conjunctivitis.23 Addi- effects are linked via increased intracellular Ca2+ concen- tional biological effects of histamine have been re- trations, which are known to facilitate secretory events. ported. The most interesting of these relative to its role Supporting data obtained with calcium ionophore A23187 as a mediator of allergic diseases is its ability to stimu- exposure of HCEs have shown a stimulation of cytokine 1 late or up-regulate proinflammatory cytokine synthesis secretion. The IC50 values for antazoline and pheni- and/or secretion. ramine ranged from 652 to 4200 nmol/L and from 1216 Delneste et al24 investigated the effect of histamine to 4826 nmol/L, respectively. These data suggest that the on adhesion molecule expression and IL-6 production first-generation antihistamines have limited ability to by human vascular endothelial cells. The authors re- interact with intact cells of the human conjunctiva. ported that histamine at concentrations ranging from 10 These findings may explain the limited clinical utility of µmol/L to 1 mmol/L increased IL-6 synthesis from these these early antihistamines when used as single-entity cells. These authors also reported that IL-8 messenger products without vasoconstrictors. RNA expression and secretion were enhanced by expo- Second-generation topical ocular antihistamines, sure of endothelial cells to histamine in concentrations levocabastine and emedastine, inhibited histamine- greater than 1 µmol/L.25 Similar findings by Tonnel et al26 stimulated PI turnover and cytokine secretion. The with the use of human umbilical vein endothelial cells potency of these molecules in these assays was consis- indicated that histamine H1 and H2 receptors play a role tent with their affinities for the H1 receptor (52.6 nmol/L in cytokine secretion. for levocabastine and 1.22 nmol/L for emedastine).20 Histamine has also been reported to stimulate cy- Levocabastine’s IC50 values ranged from 8.3 to 25.1 tokine secretion from epithelial cells. Secretion of IL-6, nmol/L. Emedastine has been reported to be the most IL-8, and GM-CSF by bronchial epithelial cells has been potent antihistamine available for topical ocular use.20,29 3 27 demonstrated. Noah et al suggested that a correlation The drug’s histamine H1-receptor affinity of 1.22 nmol/L exists between calcium influx and IL-6 secretion in a bron- is reflected in the data obtained in the present experi- chial epithelial cell line in response to stimulation with ments. Emedastine’s potency in preventing cytokine histamine (100 µmol/L). However, using the antihista- secretion by HCE may partially explain the advantages mines loratadine and cetirizine hydrochloride, Ansel- noted with this compound during clinical comparative lem et al28 failed to demonstrate an inhibitory effect on trials with levocabastine.30 cytokine secretion from bronchial epithelial cells. These Interesting results were obtained with olopatadine. investigators therefore suggested that histamine does not Olopatadine is marketed as Patanol for topical ocular use. play a role in cytokine production. Further experimen- The compound is a human conjunctival mast cell de- tation has shown that human tracheal epithelial cells do granulation inhibitor and antihistamine.22,31,32 Pub- produce GM-CSF after exposure to histamine.2 Re- lished reports demonstrate the compound’s antiallergic cently, Weimer et al19 demonstrated that histamine in- activity in vivo.33 Results presented herein indicate that duces a concentration- and time-dependent secretion of the compound is more potent as an inhibitor of cyto- IL-6, IL-8, and GM-CSF from HCEs. These authors pre- kine secretion than would have been predicted from its

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©1999 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 22 10. Leonard EJ, Yoshimura T, Tanaka S, Raffeld M. Neutrophil recruitment by intra- H1-receptor affinity (36 nmol/L ). Olopatadine potently inhibited secretion of IL-6 and IL-8 from HCEs. dermally injected neutrophil attractant/activation protein-1. J Invest Dermatol. 1991;96:690-694. The IC50 values for the drug were 5.5 and 1.7 nmol/L, 11. Willems J, Joniau M, Clinque S, van Damme J. Human granulocyte chemotactic respectively. These IC50 values were also approximately peptide (IL-8) as a specific neutrophil degranulator: comparison with other mono- 2-fold and 10-fold lower, respectively, than those pre- kines. Immunology. 1989;67:540-542. dicted from the functional second messenger data. 12. Tanimoto Y, Takahashi K, Kimura I. Effects of cytokines on human basophil che- Olopatadine was reported to be more potent as an motaxis. Clin Exp Allergy. 1992;22:1020-1025. 13. Collins PD, Weg VB, Faccioli LH, Watson ML, Moqbel R, Williams TJ. Eosinophil inhibitor of histamine-enhanced tumor necrosis factor accumulation induced by human interleukin-8 in the guinea-pig in vivo. Immu- ␣–stimulated adhesion molecule expression than pre- nology. 1993;79:312-318. dicted from the drug’s receptor binding affinity.34 One 14. Trocme SD, Aldave AJ. The eye and the eosinophil. Surv Ophthalmol. 1994;39: possible explanation for this increased efficacy is a non- 241-252. specific antisecretory effect. However, this does not ap- 15. Moy JN, Gleich GJ, Thomas LL. Noncytotoxic activation of neutrophils by eo- 35 sinophil granule major basic protein: effect of superoxide anion generation and pear to be the case. A report by Ikemura et al pre- lysosomal enzyme release. J Immunol. 1990;145:2626-2632. sented data showing that olopatadine (1-100 µmol/L) did 16. Proud D, Sweet J, Stein P, et al. Inflammatory mediator release on conjunctival provo- not inhibit the release of ␤-glucuronidase from human cation of allergic subjects with allergen. J Allergy Clin Immunol. 1990;85:896-905. polymorphonuclear neutrophils after stimulation with the 17. Abelson MB, Soter NA, Simon MA, Dohlman J, Allansmith MR. Histamine in hu- calcium ionophore A . man tears. Am J Ophthalmol. 1977;83:417-418. 23187 18. Sharif NA, Xu SX, Magnino PE, Pang I-H. Human conjunctival epithelial cells ex- The results presented herein confirm histamine’s abil- press histamine-1 receptors coupled to phosphoinositide turnover and intracel- ity to stimulate PI turnover and cytokine secretion from lular calcium mobilization: role in ocular allergic and inflammatory diseases. Exp HCEs. First-generation antihistamines pheniramine and Eye Res. 1996;63:169-178. antazoline are dramatically less potent in the whole cell 19. Weimer LK, Gamache DA, Yanni JM. 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