[Skip to Content]
Access to paid content on this site is currently suspended due to excessive activity being detected from your IP address 54.205.176.107. Please contact the publisher to request reinstatement.
[Skip to Content Landing]
Download PDF
Figure 1.
Effect of histamine H1-antagonists on histamine-induced phosphoinositide (PI) turnover in human conjunctival epithelial cells. Tritiated ([3H])myoinositol–labeled cells were exposed to drug for 20 minutes before stimulation with histamine for 60 minutes. [3H]inositol phosphates were quantified by ion exchange chromatography followed by liquid scintillation spectrometry.

Effect of histamine H1-antagonists on histamine-induced phosphoinositide (PI) turnover in human conjunctival epithelial cells. Tritiated ([3H])myoinositol–labeled cells were exposed to drug for 20 minutes before stimulation with histamine for 60 minutes. [3H]inositol phosphates were quantified by ion exchange chromatography followed by liquid scintillation spectrometry.

Figure 2.
Effect of histamine H1-antagonists on histamine-induced interleukin 6 secretion from human conjunctival epithelial cells. Cells were incubated with test compound for 30 minutes before stimulation with histamine (30 µmol/L) for 24 hours. Supernatants were analyzed for cytokines by specific enzyme-linked immunosorbent assay.

Effect of histamine H1-antagonists on histamine-induced interleukin 6 secretion from human conjunctival epithelial cells. Cells were incubated with test compound for 30 minutes before stimulation with histamine (30 µmol/L) for 24 hours. Supernatants were analyzed for cytokines by specific enzyme-linked immunosorbent assay.

Figure 3.
Effect of histamine H1-antagonists on histamine-stimulated interleukin 8 secretion from human conjunctival epithelial cells. Assay and data collection procedures are as noted in Figure 2.

Effect of histamine H1-antagonists on histamine-stimulated interleukin 8 secretion from human conjunctival epithelial cells. Assay and data collection procedures are as noted in Figure 2.

Histamine H1 Antagonists: Inhibition of IL-6 and IL-8 Secretion and PI Turnover in Human Conjunctival Epithelial Cells and H1 Receptor Binding Affinities*
Histamine H1 Antagonists: Inhibition of IL-6 and IL-8 Secretion and PI Turnover in Human Conjunctival Epithelial Cells and H1 Receptor Binding Affinities*
1.
Gamache  DADimitrijevich  SDWeimer  LK  et al.  Secretion of proinflammatory cytokines by human conjunctival epithelial cells. Ocul Immunol Inflamm. 1997;5117- 128Article
2.
Churchill  LFriedman  BSchleimer  RProud  D Production of granulocyte-macrophage colony-stimulating factor by cultured human tracheal epithelial cells. Immunology. 1992;75189- 195
3.
Takizawa  HOhtoshi  TKikutani  T  et al.  Histamine activates bronchial epithelial cells to release inflammatory cytokines in vitro. Int Arch Allergy Immunol. 1995;108260- 267Article
4.
Marini  MVittori  EHollemborg  JMattoli  S Expression of the potent inflammatory cytokines, granulocyte-macrophage colony-stimulating factor and interleukin-6 and interleukin-8, in bronchial epithelial cells of patients with asthma. J Allergy Clin Immunol. 1992;891001- 1009Article
5.
Murray  PIHoekzema  Rvan Haren  MACde Hon  FDKijlstra  A Aqueous humor interleukin-6 levels in uveitis. Invest Ophthalmol Vis Sci. 1990;31917- 920
6.
Grossman  RMKrueger  JYourish  D  et al.  Interleukin-6 is expressed in high levels in psoriatic skin and stimulates proliferation of cultured keratinocytes. Proc Natl Acad Sci U S A. 1989;866367- 6371Article
7.
Gosset  PMalaquin  FDelneste  Y  et al.  Interleukin-6 and interleukin-1α production is associated with antigen-induced late nasal response. J Allergy Clin Immunol. 1993;92878- 890Article
8.
Hoekzema  RMurray  PIvan Haren  MAHelle  MKijlstra  A Analysis of interleukin-6 in endotoxin-induced uveitis. Invest Ophthalmol Vis Sci. 1991;288- 95
9.
Albelda  SMSmith  CWWard  PA Adhesion molecules and inflammatory injury. FASEB J. 1994;8504- 512
10.
Leonard  EJYoshimura  TTanaka  SRaffeld  M Neutrophil recruitment by intradermally injected neutrophil attractant/activation protein-1. J Invest Dermatol. 1991;96690- 694Article
11.
Willems  JJoniau  MClinque  Svan Damme  J Human granulocyte chemotactic peptide (IL-8) as a specific neutrophil degranulator: comparison with other monokines. Immunology. 1989;67540- 542
12.
Tanimoto  YTakahashi  KKimura  I Effects of cytokines on human basophil chemotaxis. Clin Exp Allergy. 1992;221020- 1025Article
13.
Collins  PDWeg  VBFaccioli  LHWatson  MLMoqbel  RWilliams  TJ Eosinophil accumulation induced by human interleukin-8 in the guinea-pig in vivo. Immunology. 1993;79312- 318
14.
Trocme  SDAldave  AJ The eye and the eosinophil. Surv Ophthalmol. 1994;39241- 252Article
15.
Moy  JNGleich  GJThomas  LL Noncytotoxic activation of neutrophils by eosinophil granule major basic protein: effect of superoxide anion generation and lysosomal enzyme release. J Immunol. 1990;1452626- 2632
16.
Proud  DSweet  JStein  P  et al.  Inflammatory mediator release on conjunctival provocation of allergic subjects with allergen. J Allergy Clin Immunol. 1990;85896- 905Article
17.
Abelson  MBSoter  NASimon  MADohlman  JAllansmith  MR Histamine in human tears. Am J Ophthalmol. 1977;83417- 418
18.
Sharif  NAXu  SXMagnino  PEPang  I-H Human conjunctival epithelial cells express histamine-1 receptors coupled to phosphoinositide turnover and intracellular calcium mobilization: role in ocular allergic and inflammatory diseases. Exp Eye Res. 1996;63169- 178Article
19.
Weimer  LKGamache  DAYanni  JM Histamine-stimulated cytokine secretion from human conjunctival epithelial cells: inhibition by the histamine H1 antagonist emedastine. Int Arch Allergy Immunol. 1998;115288- 293Article
20.
Sharif  NAXu  SXYanni  JM Emedastine: a potent, high affinity histamine H1-receptor-selective antagonist for ocular use: receptor binding and second messenger studies. J Ocul Pharmacol. 1994;10653- 664Article
21.
Sharif  NAXu  SX Pharmacological characterization of bradykinin receptors coupled to phosphoinositide turnover in SV40-immortalized human trabecular meshwork cells. Exp Eye Res. 1996;63631- 637Article
22.
Yanni  JMMiller  STGamache  DASpellman  JMXu  SSharif  NA Comparative effects of topical ocular anti-allergy drugs on human conjunctival mast cells. Ann Allergy Asthma Immunol. 1997;79541- 545Article
23.
Friedlander  MH Ocular allergy. Middelton  EReed  CEEllis  EFAdkinson  NFYunginger  JWeds.Allergy: Principles and Practice Vol 2 St Louis, Mo CV Mosby Co1988;1469- 1480
24.
Delneste  YLassalle  PJeannin  PJoseph  MTonnel  ABGosset  P Histamine induces IL-6 production by human endothelial cells. Clin Exp Immunol. 1994;98344- 349Article
25.
Jeannin  PDelneste  YGosset  P  et al.  Histamine induces interleukin 8 secretion by endothelial cells. Blood. 1994;842229- 2233
26.
Tonnel  ABGosset  PMolet  STillie-Leblond  IJeannin  PJoseph  M Interactions between endothelial cells and effector cells in allergic inflammation. Ann N Y Acad Sci. 1996;7969- 20Article
27.
Noah  TLParadiso  AMMadden  MCMcKinnon  KPDevlin  RB The response of a human bronchial epithelial cell line to histamine: intracellular calcium changes and extracellular release of inflammatory mediators. Am J Respir Cell Mol Biol. 1991;5484- 492Article
28.
Amsellem  CGormand  FHosni  R  et al.  Antihistamines and production of granulocyte-macrophage colony-stimulating factor and interleukin-8 by human bronchial epithelial cells in vitro: evaluation of the effects of loratadine and cetirizine. Gen Pharmacol. 1996;27269- 272Article
29.
Yanni  JMStephens  DJParnell  DWSpellman  JM Preclinical efficacy of emedastine, a potent, selective histamine H1 antagonist for topical ocular use. J Ocul Pharmacol. 1994;10665- 675Article
30.
Verin  PHSecchi  AEasty  DL  et al.  Efficacy and safety of emedastine eye drops 0.05% compared to levocabastine eye drops 0.05% in allergic conjunctivitis [abstract]. Invest Ophthalmol Vis Sci. 1998;39 ((suppl)) S549
31.
Yanni  JMStephens  DJMiller  ST  et al.  The in vitro and in vivo ocular pharmacology of olopatadine (AL-4943A), an effective anti-allergic/antihistaminic agent. J Ocul Pharmacol Ther. 1996;12389- 400Article
32.
Sharif  NAXu  SXMiller  STGamache  DAYanni  JM Characterization of the ocular antiallergic and antihistaminic effects of olopatadine (AL-4943A), a novel drug for treating ocular allergic disease. J Pharmacol Exp Ther. 1996;2781252- 1261
33.
Ishii  HSasaki  YToshihide  IShigeto  KOhmori  K Pharmacological studies on KW-4679, an antiallergic drug: inhibitory effect on passive cutaneous anaphylaxis (PCA) and bronchoconstriction in rats and guinea pigs. Clin Rep. 1995;297- 23
34.
Miki  IKusano  AOhta  S  et al.  Histamine enhanced the TNF-α–induced expression of E-selectin and ICAM-1 in vascular endothelial cells. Cell Immunol. 1996;171285- 288
35.
Ikemura  TManabe  HSasaki  Y  et al.  KW-4679, an antiallergic drug, inhibits the production of inflammatory lipids in human polymorphonuclear leukocytes and guinea pig eosinophils. Int Arch Allergy Immunol. 1996;11057- 63Article
Laboratory Sciences
May 1999

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

Author Affiliations

From Ophthalmic Products Research, Alcon Laboratories Inc, Fort Worth, Tex.

Arch Ophthalmol. 1999;117(5):643-647. doi:10.1001/archopht.117.5.643
Abstract

Objective  To evaluate the effects of topical ocular drugs with histamine H1-antagonist activity on histamine-stimulated phosphatidylinositol turnover and interleukin (IL) 6 and IL-8 secretion from human conjunctival epithelial cells.

Methods  Primary human conjunctival epithelial cell cultures were stimulated with histamine in the presence or absence of test drugs. Phosphatidylinositol turnover was quantified by ion exchange chromatography and cytokine content of supernatants by enzyme-linked immunosorbent assay.

Results  Antazoline hydrochloride, emedastine difumarate, levocabastine hydrochloride, olopatadine hydrochloride, and pheniramine maleate attenuated histamine-stimulated phosphatidylinositol turnover and IL-6 and IL-8 secretion. Emedastine was the most potent in ligand binding, phosphatidylinositol turnover, and IL-6 secretion, with dissociation constant and 50% inhibitory concentrations of 1-3 nmol/L. Olopatadine, antazoline, and pheniramine exhibited similar H1-binding affinities (32-39 nmol/L). However, olopatadine was approximately 10-fold more potent as an inhibitor of cytokine secretion (50% inhibitory concentration, 1.7-5.5 nmol/L) than predicted from binding data, while antazoline and pheniramine were far less potent (20- to 140-fold) in functional assays. Levocabastine (dissociation constant, 52.6 nmol/L) exhibited greater functional activity (50% inhibitory concentration, 8-25 nmol/L) than either antazoline or pheniramine.

Conclusions  Histamine-stimulated phosphatidylinositol turnover and cytokine secretion by human conjunctival epithelial cells are attenuated by compounds with H1-antagonist activity. However, antihistaminic potency alone does not predict anti-inflammatory potential. Olopatadine, emedastine, and levocabastine were notably more potent than pheniramine and antazoline.

Clinical Relevance  Selected topical ocular drugs with antihistaminic activity may offer therapeutic advantages to patients with allergic conjunctivitis by inhibiting proinflammatory cytokine secretion from human conjunctival epithelial cells.

HUMAN CONJUNCTIVAL epithelial cells (HCEs) secrete cytokines after stimulation by various cell-activating agents. Gamache et al1 showed that primary cultures of HCEs secrete tumor necrosis factor α, interleukin (IL) 6, IL-8, and granulocyte-macrophage colony-stimulating factor (GM-CSF) after IL-1α, phorbol myristate acetate, and calcium ionophore A23187 treatment. The authors suggested that this capability of the conjunctival epithelium indicates a possible effector function for the tissue in allergic conjunctivitis. Other investigators have reported that histamine, a major mediator of allergic diseases, induces the production of cytokines by airway epithelium. For instance, GM-CSF release from human tracheal epithelial cells was significantly enhanced by exposure to 1-µmol/L histamine.2 In addition, histamine caused a dose-dependent stimulation of IL-6, IL-8, and GM-CSF release by normal and transformed human bronchial epithelial cells that appeared to occur via histamine H1-receptor activation.3 The proinflammatory properties of these cytokines are well documented. Elevated IL-6 levels have been reported to be associated with a variety of inflammatory conditions, including asthma, psoriasis, uveitis, and allergic rhinitis.47 Intravitreal injection of IL-6 has been shown to produce uveitis in rats and rabbits.8 Interleukin 8 is a potent member of the C-X-C family of chemokines. It promotes integrin expression, neutrophil degranulation, and chemotaxis of basophils and eosinophils.913

The presence of these polymorphonuclear leukocytes (primarily eosinophils) contributes to the pathogenesis of the late-phase allergic response. Degranulation of the eosinophilic leukocytes leads to the release of major basic protein, eosinophil peroxidase, eosinophil cationic protein, and eosinophil-derived neurotoxin.14 Reports have demonstrated that the eosinophil-derived proinflammatory mediator, major basic protein, has the ability to activate neutrophils as well as stimulating additional histamine release, further amplifying the allergic response.15

Elevated levels of histamine in tears of patients with allergic ocular diseases have been reported. Tears collected after ocular antigen challenge contained significantly more histamine than tears collected after sterile saline exposure.16 The concentration of histamine in tears collected from patients with vernal conjunctivitis was increased more than 3 times compared with the concentration in normal subjects.17 Sharif et al18 demonstrated the presence of functional histamine H1-receptors on HCEs. These receptors were coupled to inositol phosphate generation, which mobilized intracellular calcium. Calcium mobilization peaked within 10 seconds and was sustained for 20 minutes after stimulation with histamine. Pharmacological studies indicated that histamine H1-receptor antagonists potently antagonized these effects. Recently, Weimer et al19 demonstrated that histamine stimulation of HCEs induced the secretion of IL-6, IL-8, and GM-CSF in a concentration- and time-dependent manner. They also presented evidence that the potent H1-antagonist emedastine inhibited cytokine secretion at concentrations consistent with its published affinity for the H1 receptor.20

The current experiments were conducted to compare the inhibitory effects of compounds with H1-antagonist activity currently available for topical ocular use on histamine-stimulated activation of the second messenger system and cytokine release from HCEs.

MATERIALS AND METHODS
CELL CULTURES

Methods detailing the preparation of primary epithelial cell cultures and cytokine release studies with the use of these cells have been described.1 Briefly, cultures of HCEs were initiated from donor tissues obtained by various eye banks within 8 hours post mortem. The tissues were enzymatically digested overnight. Epithelial cells were gently scraped from the tissue surface, dissociated into a single cell suspension, and cultured in Clonetics keratinocyte growth medium (Biowhittaker Corp, Walkersville, Md). Cells were used only through passage 6. Cultures were maintained in a preconfluent state to prevent differentiation. Cells were identified as epithelial by positive keratin staining, as described previously.1

CYTOKINE ASSAYS

Several compounds with histamine H1-antagonist activity were evaluated for their ability to inhibit secretion of cytokines (IL-6 and IL-8) from cultured HCEs in response to histamine stimulation. Cells were plated at 2×104 cells per well and cultured overnight in 5% carbon dioxide at 37°C. The following day, keratinocyte growth medium containing test compound was added directly to wells and the cells were incubated for 30 minutes before 24-hour stimulation with histamine (30 µmol/L). Three culture wells were used for each treatment group. At harvest, cell monolayers were examined microscopically to confirm viability and supernatants were collected, centrifuged at 200g, and stored at −20°C. Samples were analyzed for IL-6 and IL-8 by enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, Minn) as directed by the manufacturer. The sensitivities of each enzyme-linked immunosorbent assay are 0.7 pg/mL for IL-6 and 3.0 pg/mL for IL-8.

HISTAMINE-INDUCED PHOSPHATIDYLINOSITOL TURNOVER

The determination of phosphatidylinositol (PI) turnover induced by stimulation of phospholipase C in HCEs was performed as previously described21 with minor modifications. The HCEs were incubated with tritiated ([3H])myoinositol (0.037 MBq/0.5 mL; 55.5-62.9×1010 Bq/mmol; Amersham Life Science, Inc, Arlington Heights, Ill) in Dulbecco modified Eagle medium (GIBCO, Gaithersburg, Md) for 24 hours in 5% carbon dioxide at 37°C to label the cell membrane lipids. Cells were then exposed to histamine (10 nmol/L to 1 mmol/L) for 60 minutes at 23°C. To determine the potencies of the antagonists, the drugs were added to the cells 20 minutes before the addition of histamine (100 µmol/L). The assay was terminated by the addition of ice-cold 0.1-mol/L formic acid. With ion exchange columns containing 1 mL of AG1-X8 resin in formate form, free [3H]myoinositol was removed from the cell lysates with deionized water; the water-soluble [3H]inositol phosphates were then eluted with 1.2-mol/L ammonium formate. The [3H]inositol phosphates were quantified by liquid scintillation spectrometry.

DATA ANALYSIS

The antagonist potency (IC50) was defined as the concentration of the drug required to produce 50% inhibition of the agonist-stimulated functional response. Data derived from the cytokine assays were calculated as mean and SEM values that represent the variability among identically treated culture wells. The dose-dependent effect of pharmacological agents and IC50s were determined by linear regression. Data obtained in the PI turnover assays were analyzed by means of a nonlinear, iterative curve fitting program as previously described.18,20 Data are expressed as mean±SEM from 3 to 5 independent experiments.

TEST COMPOUNDS

Compounds were obtained as follows: antazoline hydrochloride and pheniramine maleate (Sigma-Aldrich Corp, St Louis, Mo); emedastine difumarate (Kanebo Ltd, Osaka, Japan); olopatadine hydrochloride (Kyowa Hakko Kogyo Co Ltd, Tokyo, Japan); and levocabastine hydrochloride (Livostin; Ciba Vision Ophthalmics, Atlanta, Ga). Histamine dihydrochloride was obtained from Research Biochemicals International, Natick, Mass.

RESULTS

Exposure of HCEs to 100-µmol/L histamine maximally 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 1.80±0.28-fold above basal levels, respectively. (Basal levels 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.)

Treatment of HCEs with drugs possessing antihistaminic activity and available for topical ocular administration before histamine exposure resulted in concentration-dependent inhibition of PI turnover, IL-6 secretion, and IL-8 secretion. All 5 compounds tested produced concentration-dependent inhibition of the histamine-stimulated cell functional responses (Figure 1, Figure 2, and Figure 3).

Emedastine was the most potent compound tested (Table 1). The potency of emedastine in intact cells was consistent with its activity determined in receptor binding assays with the use of tissue homogenates. Its IC50 values for histamine-induced PI turnover and IL-6 and IL-8 secretion were 1.54, 2.5, and 4.0 nmol/L, respectively. Levocabastine and olopatadine were also potent inhibitors of these histamine-stimulated responses. Levocabastine inhibited the PI turnover and IL-6 and IL-8 secretion (IC50s, 8.32, 25.1, and 11.9 nmol/L, respectively). Olopatadine was more potent than predicted from its published histamine H1-receptor binding affinity (36 nmol/L),22 with IC50s of 10.03, 5.5, and 1.7 nmol/L for PI turnover and IL-6 and IL-8 secretion, respectively. In fact, olopatadine inhibited histamine-stimulated secretion of IL-8 at a concentration (IC50, 1.7 nmol/L) lower than emedastine's efficacious concentration in the same assay system.

Antazoline and pheniramine, 2 first-generation topical ocular antihistamines, were dramatically less potent inhibitors of these histamine-induced cell-based responses (PI turnover, IL-6 and IL-8 secretion) than predicted from their histamine H1-receptor binding affinities (Table 1). The calculated IC50 values for these compounds on the parameters listed above ranged from 652 to 4200 nmol/L for antazoline and 1216 to 4826 nmol/L for pheniramine.

COMMENT

Histamine is recognized as a primary mediator of allergic disease. Its acute vascular effects lead to erythema and edema, and its pruritogenic effects are responsible for the itch characteristic of allergic conjunctivitis.23 Additional biological effects of histamine have been reported. The most interesting of these relative to its role as a mediator of allergic diseases is its ability to stimulate or up-regulate proinflammatory cytokine synthesis and/or secretion.

Delneste et al24 investigated the effect of histamine on adhesion molecule expression and IL-6 production by human vascular endothelial cells. The authors reported that histamine at concentrations ranging from 10 µmol/L to 1 mmol/L increased IL-6 synthesis from these cells. These authors also reported that IL-8 messenger RNA expression and secretion were enhanced by exposure of endothelial cells to histamine in concentrations greater than 1 µmol/L.25 Similar findings by Tonnel et al26 with the use of human umbilical vein endothelial cells indicated that histamine H1 and H2 receptors play a role in cytokine secretion.

Histamine has also been reported to stimulate cytokine secretion from epithelial cells. Secretion of IL-6, IL-8, and GM-CSF by bronchial epithelial cells has been demonstrated.3 Noah et al27 suggested that a correlation exists between calcium influx and IL-6 secretion in a bronchial epithelial cell line in response to stimulation with histamine (100 µmol/L). However, using the antihistamines loratadine and cetirizine hydrochloride, Ansellem et al28 failed to demonstrate an inhibitory effect on cytokine secretion from bronchial epithelial cells. These investigators therefore suggested that histamine does not play a role in cytokine production. Further experimentation has shown that human tracheal epithelial cells do produce GM-CSF after exposure to histamine.2 Recently, Weimer et al19 demonstrated that histamine induces a concentration- and time-dependent secretion of IL-6, IL-8, and GM-CSF from HCEs. These authors presented evidence that the histamine-stimulated cytokine response was the result of histamine H1-receptor activation in these cells. These findings are supported by data showing that the effects of histamine on PI turnover and intracellular Ca2+ concentration in HCEs were not significantly blocked by H2 and H3 antagonists but were dramatically reduced by H1 antagonists.18

Our data confirm histamine's ability to stimulate PI turnover and cytokine secretion from HCEs. The concentrations of the agonist used in the present studies (30-100 µmol/L) are consistent with previous cytokine-stimulating concentrations of the biogenic amine.2,3,19,24,25

Current data demonstrate that compounds capable of antagonizing histamine H1 receptors prevent histamine-stimulated IL-6 and IL-8 secretion. These data confirm emedastine's ability to prevent cytokine secretion19 and characterize the effects of other compounds that possess antihistaminic activity. First-generation topical ocular antihistamines antazoline and pheniramine have reported affinities for the histamine H1 receptor of 38.4 and 33.9 nmol/L, respectively.20 The binding paradigms used to generate these values used cell membranes as the receptor source. In the present studies, which used living, whole cells as the test systems, the 2 compounds were surprisingly less potent. This was true not only for cytokine secretion but also for histamine-stimulated PI turnover. These physiological effects are linked via increased intracellular Ca2+ concentrations, which are known to facilitate secretory events. Supporting data obtained with calcium ionophore A23187 exposure of HCEs have shown a stimulation of cytokine secretion.1 The IC50 values for antazoline and pheniramine ranged from 652 to 4200 nmol/L and from 1216 to 4826 nmol/L, respectively. These data suggest that the first-generation antihistamines have limited ability to interact with intact cells of the human conjunctiva. These findings may explain the limited clinical utility of these early antihistamines when used as single-entity products without vasoconstrictors.

Second-generation topical ocular antihistamines, levocabastine and emedastine, inhibited histamine-stimulated PI turnover and cytokine secretion. The potency of these molecules in these assays was consistent with their affinities for the H1 receptor (52.6 nmol/L for levocabastine and 1.22 nmol/L for emedastine).20 Levocabastine's IC50 values ranged from 8.3 to 25.1 nmol/L. Emedastine has been reported to be the most potent antihistamine available for topical ocular use.20,29 The drug's histamine H1-receptor affinity of 1.22 nmol/L is reflected in the data obtained in the present experiments. Emedastine's potency in preventing cytokine secretion by HCE may partially explain the advantages noted with this compound during clinical comparative trials with levocabastine.30

Interesting results were obtained with olopatadine. Olopatadine is marketed as Patanol for topical ocular use. The compound is a human conjunctival mast cell degranulation inhibitor and antihistamine.22,31,32 Published reports demonstrate the compound's antiallergic activity in vivo.33 Results presented herein indicate that the compound is more potent as an inhibitor of cytokine secretion than would have been predicted from its H1-receptor affinity (36 nmol/L22). Olopatadine potently inhibited secretion of IL-6 and IL-8 from HCEs. The IC50 values for the drug were 5.5 and 1.7 nmol/L, respectively. These IC50 values were also approximately 2-fold and 10-fold lower, respectively, than those predicted from the functional second messenger data. Olopatadine was reported to be more potent as an inhibitor of histamine-enhanced tumor necrosis factor α–stimulated adhesion molecule expression than predicted from the drug's receptor binding affinity.34 One possible explanation for this increased efficacy is a nonspecific antisecretory effect. However, this does not appear to be the case. A report by Ikemura et al35 presented data showing that olopatadine (1-100 µmol/L) did not inhibit the release of β-glucuronidase from human polymorphonuclear neutrophils after stimulation with the calcium ionophore A23187.

The results presented herein confirm histamine's ability to stimulate PI turnover and cytokine secretion from HCEs. First-generation antihistamines pheniramine and antazoline are dramatically less potent in the whole cell assays used compared with their receptor affinities, possibly providing some insight into their limited clinical utility. The current generation of topical ocular antihistamines, emedastine and levocabastine, exhibit potencies consistent with their receptor affinities (deviation approximately 2-fold). Importantly, olopatadine is 10 times more potent as an inhibitor of histamine-stimulated cytokine secretion than predicted from its histamine H1-receptor affinity. These data suggest that olopatadine may offer additional therapeutic benefits that complement the mast cell stabilization and antihistaminic activities observed in the clinic.

Back to top
Article Information

Accepted for publication November 9, 1998.

Reprints: John M. Yanni, PhD, Allergy/Inflammation Research, Alcon Laboratories Inc, 6201 South Freeway, Fort Worth, TX 76134-2099 (e-mail: John.Yanni@AlconLabs.com).

References
1.
Gamache  DADimitrijevich  SDWeimer  LK  et al.  Secretion of proinflammatory cytokines by human conjunctival epithelial cells. Ocul Immunol Inflamm. 1997;5117- 128Article
2.
Churchill  LFriedman  BSchleimer  RProud  D Production of granulocyte-macrophage colony-stimulating factor by cultured human tracheal epithelial cells. Immunology. 1992;75189- 195
3.
Takizawa  HOhtoshi  TKikutani  T  et al.  Histamine activates bronchial epithelial cells to release inflammatory cytokines in vitro. Int Arch Allergy Immunol. 1995;108260- 267Article
4.
Marini  MVittori  EHollemborg  JMattoli  S Expression of the potent inflammatory cytokines, granulocyte-macrophage colony-stimulating factor and interleukin-6 and interleukin-8, in bronchial epithelial cells of patients with asthma. J Allergy Clin Immunol. 1992;891001- 1009Article
5.
Murray  PIHoekzema  Rvan Haren  MACde Hon  FDKijlstra  A Aqueous humor interleukin-6 levels in uveitis. Invest Ophthalmol Vis Sci. 1990;31917- 920
6.
Grossman  RMKrueger  JYourish  D  et al.  Interleukin-6 is expressed in high levels in psoriatic skin and stimulates proliferation of cultured keratinocytes. Proc Natl Acad Sci U S A. 1989;866367- 6371Article
7.
Gosset  PMalaquin  FDelneste  Y  et al.  Interleukin-6 and interleukin-1α production is associated with antigen-induced late nasal response. J Allergy Clin Immunol. 1993;92878- 890Article
8.
Hoekzema  RMurray  PIvan Haren  MAHelle  MKijlstra  A Analysis of interleukin-6 in endotoxin-induced uveitis. Invest Ophthalmol Vis Sci. 1991;288- 95
9.
Albelda  SMSmith  CWWard  PA Adhesion molecules and inflammatory injury. FASEB J. 1994;8504- 512
10.
Leonard  EJYoshimura  TTanaka  SRaffeld  M Neutrophil recruitment by intradermally injected neutrophil attractant/activation protein-1. J Invest Dermatol. 1991;96690- 694Article
11.
Willems  JJoniau  MClinque  Svan Damme  J Human granulocyte chemotactic peptide (IL-8) as a specific neutrophil degranulator: comparison with other monokines. Immunology. 1989;67540- 542
12.
Tanimoto  YTakahashi  KKimura  I Effects of cytokines on human basophil chemotaxis. Clin Exp Allergy. 1992;221020- 1025Article
13.
Collins  PDWeg  VBFaccioli  LHWatson  MLMoqbel  RWilliams  TJ Eosinophil accumulation induced by human interleukin-8 in the guinea-pig in vivo. Immunology. 1993;79312- 318
14.
Trocme  SDAldave  AJ The eye and the eosinophil. Surv Ophthalmol. 1994;39241- 252Article
15.
Moy  JNGleich  GJThomas  LL Noncytotoxic activation of neutrophils by eosinophil granule major basic protein: effect of superoxide anion generation and lysosomal enzyme release. J Immunol. 1990;1452626- 2632
16.
Proud  DSweet  JStein  P  et al.  Inflammatory mediator release on conjunctival provocation of allergic subjects with allergen. J Allergy Clin Immunol. 1990;85896- 905Article
17.
Abelson  MBSoter  NASimon  MADohlman  JAllansmith  MR Histamine in human tears. Am J Ophthalmol. 1977;83417- 418
18.
Sharif  NAXu  SXMagnino  PEPang  I-H Human conjunctival epithelial cells express histamine-1 receptors coupled to phosphoinositide turnover and intracellular calcium mobilization: role in ocular allergic and inflammatory diseases. Exp Eye Res. 1996;63169- 178Article
19.
Weimer  LKGamache  DAYanni  JM Histamine-stimulated cytokine secretion from human conjunctival epithelial cells: inhibition by the histamine H1 antagonist emedastine. Int Arch Allergy Immunol. 1998;115288- 293Article
20.
Sharif  NAXu  SXYanni  JM Emedastine: a potent, high affinity histamine H1-receptor-selective antagonist for ocular use: receptor binding and second messenger studies. J Ocul Pharmacol. 1994;10653- 664Article
21.
Sharif  NAXu  SX Pharmacological characterization of bradykinin receptors coupled to phosphoinositide turnover in SV40-immortalized human trabecular meshwork cells. Exp Eye Res. 1996;63631- 637Article
22.
Yanni  JMMiller  STGamache  DASpellman  JMXu  SSharif  NA Comparative effects of topical ocular anti-allergy drugs on human conjunctival mast cells. Ann Allergy Asthma Immunol. 1997;79541- 545Article
23.
Friedlander  MH Ocular allergy. Middelton  EReed  CEEllis  EFAdkinson  NFYunginger  JWeds.Allergy: Principles and Practice Vol 2 St Louis, Mo CV Mosby Co1988;1469- 1480
24.
Delneste  YLassalle  PJeannin  PJoseph  MTonnel  ABGosset  P Histamine induces IL-6 production by human endothelial cells. Clin Exp Immunol. 1994;98344- 349Article
25.
Jeannin  PDelneste  YGosset  P  et al.  Histamine induces interleukin 8 secretion by endothelial cells. Blood. 1994;842229- 2233
26.
Tonnel  ABGosset  PMolet  STillie-Leblond  IJeannin  PJoseph  M Interactions between endothelial cells and effector cells in allergic inflammation. Ann N Y Acad Sci. 1996;7969- 20Article
27.
Noah  TLParadiso  AMMadden  MCMcKinnon  KPDevlin  RB The response of a human bronchial epithelial cell line to histamine: intracellular calcium changes and extracellular release of inflammatory mediators. Am J Respir Cell Mol Biol. 1991;5484- 492Article
28.
Amsellem  CGormand  FHosni  R  et al.  Antihistamines and production of granulocyte-macrophage colony-stimulating factor and interleukin-8 by human bronchial epithelial cells in vitro: evaluation of the effects of loratadine and cetirizine. Gen Pharmacol. 1996;27269- 272Article
29.
Yanni  JMStephens  DJParnell  DWSpellman  JM Preclinical efficacy of emedastine, a potent, selective histamine H1 antagonist for topical ocular use. J Ocul Pharmacol. 1994;10665- 675Article
30.
Verin  PHSecchi  AEasty  DL  et al.  Efficacy and safety of emedastine eye drops 0.05% compared to levocabastine eye drops 0.05% in allergic conjunctivitis [abstract]. Invest Ophthalmol Vis Sci. 1998;39 ((suppl)) S549
31.
Yanni  JMStephens  DJMiller  ST  et al.  The in vitro and in vivo ocular pharmacology of olopatadine (AL-4943A), an effective anti-allergic/antihistaminic agent. J Ocul Pharmacol Ther. 1996;12389- 400Article
32.
Sharif  NAXu  SXMiller  STGamache  DAYanni  JM Characterization of the ocular antiallergic and antihistaminic effects of olopatadine (AL-4943A), a novel drug for treating ocular allergic disease. J Pharmacol Exp Ther. 1996;2781252- 1261
33.
Ishii  HSasaki  YToshihide  IShigeto  KOhmori  K Pharmacological studies on KW-4679, an antiallergic drug: inhibitory effect on passive cutaneous anaphylaxis (PCA) and bronchoconstriction in rats and guinea pigs. Clin Rep. 1995;297- 23
34.
Miki  IKusano  AOhta  S  et al.  Histamine enhanced the TNF-α–induced expression of E-selectin and ICAM-1 in vascular endothelial cells. Cell Immunol. 1996;171285- 288
35.
Ikemura  TManabe  HSasaki  Y  et al.  KW-4679, an antiallergic drug, inhibits the production of inflammatory lipids in human polymorphonuclear leukocytes and guinea pig eosinophils. Int Arch Allergy Immunol. 1996;11057- 63Article
×