Lori K. Weimer

Secretion of proinflammatory cytokines by human conjunctival epithelial cells.

The production of cytokines by human conjunctival epithelial cells following stimulation was investigated. Primary cultures of human conjunctival epithelial cells were characterized by morphology and keratin expression. Cultured epithelial cells were treated with varying concentrations of lipopolysaccharide, interleukin (IL)-1 beta, calcium ionophore A23187, or phorbol myristate acetate, and cytokine secretion was determined over specified intervals. Culture supernatants and cell lysates were analyzed by ELISA for IL-1 beta, IL-3, IL-4, IL-5, IL-6, IL-8, IL-11, IL-1 receptor antagonist (IL-1ra), tumor necrosis factor-alpha (TNF-alpha), and granulocyte-macrophage colony stimulating factor (GM-CSF). With the exception of IL-1ra, unstimulated conjunctival epithelial cells produced cytokines at relatively low or undetectable levels. IL-1ra was detected in both culture supernatants and cell lysates under basal conditions. In response to stimuli, conjunctival epithelial cells secreted the proinflammatory cytokines TNF-alpha, IL-6, IL-8, and GM-CSF in a dose- and time-dependent fashion. After stimulation, the intracellular levels of IL-1ra increased in these cells but the supernatant-associated levels remained unchanged. None of the other cytokines evaluated (IL-1 beta, IL-3, IL-4, IL-5, and IL-11) were detected in supernatants or lysates of resting or stimulated cells. These findings suggest that conjunctival epithelial cells may contribute to the pathogenesis of human ocular diseases by production of proinflammatory cytokines. Further evaluation of these cells as targets of therapy is warranted.

Histamine-stimulated cytokine secretion from human conjunctival epithelial cells: inhibition by the histamine H1 antagonist emedastine.

The present studies demonstrate that histamine induces the secretion of IL-6, IL-8 and GM-CSF from human conjunctival epithelial cells in a dose- and time-dependent manner. The histamine antagonists emedastine (H1), ranitidine (H2) and thioperamide (H3) were evaluated for their ability to inhibit secretion of these cytokines. Emedastine potently inhibited histamine-induced IL-6, IL-8 and GM-CSF secretion with mean IC50 values of 2.23, 3.42 and 1.50 nM, respectively. Ranitidine and thioperamide failed to inhibit cytokine secretion over a wide dose range. These data suggest that mast cell derived histamine may stimulate inflammatory cytokine production in allergic conjunctivitis via activation of epithelial cell H1 receptors. The histamine H1 antagonist emedastine potently inhibits this response.

Effect of Lodoxamide on in vitro and in vivo Conjunctival Immediate Hypersensitivity Responses in Rats

The antiallergic compound, lodoxamide, was evaluated for its abilities to attenuate a local allergic reaction in rat conjunctiva in vivo and to inhibit rat conjunctival mast cell mediator release in vitro. Topically applied lodoxamide (0.01, 0.10 and 1.0%, w/v) dose-dependently reduced the allergic response (23, 43 and 72%, respectively) in vivo. Lodoxamide was more effective than cromolyn sodium, N-acetyl aspartyl glutamic acid (Naaxia) and levocabastine, and 25 (7-200) times more potent than nedocromil sodium in direct comparisons. Addition of lodoxamide (10 micrograms/ml) to sensitized conjunctival tissue in vitro immediately prior to antigen challenge significantly reduced the amount of histamine released by the tissue. These data suggest that lodoxamides in vivo anti-allergic activity in the conjunctiva is associated with its ability to prevent allergic mediator release from mast cells contained in this same tissue.

Corneal Protection by the Ocular Mucin Secretagogue 15(S)-HETE in a Rabbit Model of Desiccation-induced Corneal Defect

The mucin secretagogue 15(S)-HETE was found to stimulate glycoprotein secretion in human ocular tissue at submicromolar concentrations in the present studies. Therefore, the ability of topically applied 15(S)-HETE to preserve corneal integrity was investigated in a rabbit model of desiccation-induced corneal defect. Desiccation-induced corneal injury was elicited in anesthetized rabbits by maintaining one eye open with a speculum. Corneal staining and corneal thickness changes were determined immediately following desiccation. 15(S)-HETE dose-dependently reduced corneal damage (ED50 = 120 nM) during a two-hour desiccation. Corneal staining was unchanged relative to control using a 1 microM dose of 15(S)-HETE. Through four hours of desiccation, 15(S)-HETE (500 nM) decreased corneal staining by 71% and completely prevented corneal thinning. 15(S)-HETE (1 microM) was significantly more efficacious than an artificial tear product over the 4-hour desiccation period. There was no evidence of tachyphylaxis following repeated topical ocular dosing of 15(S)-HETE. These studies demonstrate that 15(S)-HETE stimulates ocular mucin secretion in vitro and effectively protects the cornea in a rabbit model of desiccation-induced injury. The results suggest that the ocular mucin secretagogue 15(S)-HETE may have therapeutic utility in dry eye patients, alleviating corneal injury and restoring corneal integrity.

Preservation of Corneal Integrity by the Mucin Secretagogue 15(S)-HETE in a Rabbit Model of Desiccation-Induced Dry Eye

Mucins are high molecular weight hydrophilic glycoproteins that form a gel with aqueous lacrimal secretions spanning approximately 30 µm of the 35–40 µm thick tear film.1 This protective mucinous gel hydrates the cornea and provides a smooth refractive surface. Dry eye conditions are associated with reduced tear film mucin as well as alterations in mucin distribution and composition.2 The ability of a mucin-containing ophthalmic solution to accelerate healing of injured rabbit corneas illustrates the critical role of tear film mucin.3 Mucins are produced by conjunctival goblet cells4 and epithelial cells of the conjunctiva and cornea.5 This suggests the entire ocular surface epithelium is capable of producing mucins. Topical application of an ocular mucin secretagogue could provide therapeutic benefit to the injured cornea in the dry eye condition.

Characterization of the In Vitro Anti-inflammatory Activity of AL-5898 and Related Benzopyranyl Esters and Amides

Selected ester- (AL-5898 and AL-8417) and amide-linked benzopyran analogues (AL-7538 and AL-12615) were evaluated in vitro for their ability to inhibit key enzymes/processes of the inflammatory response. AL-7538 and AL-12615 exhibited weak intrinsic cyclooxygenase inhibitory activity (IC50 = 13 μM, 37 μM). In contrast, 5-HETE and LTB4 synthesis in A23187-stimulated neutrophils was effectively inhibited by both ester and amide analogs (IC50 = 2–3 μM). While there was some indication for differing sensitivities among benzopyran esters and amides in the suppression of cytokine synthesis in stimulated U-937 cells, there appeared to be no great discrimination when assessing their effect on U-937 cell adhesion to IL-1β activated HMVEC-L cells. Inhibition of cell adhesion was concentration-dependent, with IC50 values ranging between 18 μM and 30 μM for AL-5898. Concentration-dependent inhibition of inflammatory cytokine production (i.e., IL-1β, TNF-α, GM-CSF and IL-6) was also apparent in LPS-stimulated, cultured PBMC as well as in PMA/A23187 activated U-937 cells monitoring the synthesis of IL-1β, IL-8, TNF-α, and MCP-1. Notably, the hydrolysis products of the benzopyranyl ester, AL-5692 and (S)-6-methoxy-α-methyl-2-naphthaleneacetic acid, were devoid of pharmacological activity when assessed for inhibition of monocyte adhesion or IL-1β synthesis. Collectively, our data demonstrate the unique in vitro polypharmacology of a novel series of benzopyran analogs that suppress pivotal enzymes and processes in the inflammatory response.