James L. Stahl

Olopatadine inhibits TNFα release from human conjunctival mast cells

Background Tumor necrosis factor-α (TNFα) release likely plays a crucial role in allergic ocular inflammation via increasing ICAM-1 on epithelial cells and triggering other proinflammatory events. The immediate and prolonged release of TNFα from human conjunctival mast cells in response to allergen challenge is potentially an important target for therapeutic intervention, yet the effect of ocular anti-allergic agents on this process has not been examined. Olopatadine (Patanol) is a clinically effective dual-action ophthalmic anti-allergic agent that has been shown to inhibit mast cell histamine, tryptase, and PGD 2 release in vitro and promote decreased H 1 receptor binding activity in vitro and functional H 1 receptor antagonism in vivo. Objective To investigate the effect of olopatadine on TNFα release from anti-IgE antibody challenged purified human conjunctival mast cells. Methods Human conjunctival mast cells were purified (>95%) from cadaveric tissues using a procedure combining enzymatic digestion and Percoll gradient centrifugation. These cells were incubated with olopatadine for 30 minutes then challenged with anti-IgE antibody for 90 minutes. Supernatants were analyzed for TNFα. Results Purified human conjunctival mast cells responded to anti-IgE antibody challenge with TNFα release in a concentration dependent manner (optimum concentration was 10 μg/mL). Olopatadine pre-incubation resulted in a dose-dependent decrease in anti-IgE antibody mediated TNFα release (IC 50 = 13.1 μM). At a concentration of 3 mM olopatadine reduced TNFα release to the level of unchallenged controls. Conclusion Olopatadine inhibited anti-IgE antibody-mediated release of TNFα from human conjunctival mast cells. This effect could contribute to the long duration of anti-allergic activity reported for the drug.

Ocular allergic disease.

Purpose of reviewThis review will focus on recent advances in our understanding of the pathogenesis of allergic eye diseases. Common findings in acute allergic conjunctivitis (seasonal and perennial) and chronic allergic conjunctivitis (vernal keratoconjunctivitis, atopic keratoconjunctivitis, and giant papillary conjunctivitis) include evidence of mast cell activation and eosinophil attraction and activation. Cytokine levels found in tears, conjunctival impression cytology and biopsy specimens, and serum have been evaluated as markers of disease, and as targets of therapeutic intervention. Recent findingsHuman conjunctival epithelial cells respond to tumor necrosis factor α, interleukin-1β, and interferon-γ individually and in combination. Intracellular adhesion molecule-1 expression is upregulated by interleukin-1β and tumor necrosis factor α. Conjunctival epithelial cells release interleukin-8 in response to interleukin-1β and tumor necrosis factor α but not interferon-γ. Supernatants from activated mast cells cause increased adhesion of eosinophils to conjunctival epithelium. Tear levels of tumor necrosis factor α were elevated in vernal keratoconjunctivitis patients compared with normal controls. T cell lines from chronic allergic eye disease patients showed inconsistent production of cytokines in atopic and vernal keratoconjunctivitis and low levels in giant papillary conjunctivitis. Vernal keratoconjunctivitis patients have differing levels of eosinophil cationic protein in their serum if they were serum specific immunoglobulin E positive compared to serum specific immunoglobulin E negative patients. SummaryRecent findings continue to expand our basic knowledge of mechanisms and differences between seasonal and perennial allergic conjunctivitis and atopic and vernal keratoconjunctivitis. Understanding the complex interactions and cross talk between cells, cytokines and other mediators is relevant for new therapeutic approaches directed at specific disease entities.

Cytokines, chemokines, RANTES, and eotaxin.

Over the past several years, a number of cytokines with chemoattractive properties (chemokines) have been identified. These low molecular weight molecules have been shown to be important leukocyte chemical attractants to sites of inflammation and infection. Chemokines act on leukocytes through selective receptors and are now known to function also in leukocyte maturation, trafficking, and homing of these cells. RANTES and eotaxin (among other chemokines) are important chemoattractants for eosinophils. Since eosinophils seem to play a critical role in the production of allergic inflammation, an understanding of the mechanism of action of these chemokines may lead to new therapies for asthma and other allergic processes.

Olopatadine inhibits anti-immunoglobulin E- stimulated conjunctival mast cell upregulation of ICAM-1 expression on conjunctival epithelial cells

BACKGROUND Olopatadine is a clinically effective dual-action (antihistamine/mast cell stabilizer) ophthalmic antiallergic agent. We have previously demonstrated that olopatadine inhibits tumor necrosis factor alpha (TNF-alpha) release from purified human conjunctival mast cells and that supernates from stimulated mast cells upregulate intercellular adhesion molecule 1 (ICAM-1) expression on epithelial cells via TNF-alpha. OBJECTIVE To investigate the effect of olopatadine on the TNF-alpha-mediated mast cell upregulation of ICAM-1 expression on conjunctival epithelial cells. METHODS Human conjunctival mast cells and epithelial cells were purified (>95%) from cadaveric tissue. Conjunctival mast cells were preincubated with three doses (30, 300, or 3,000 microM) of olopatadine or buffer alone for 30 minutes followed by 90-minute challenge with anti-immunoglobulin E (10 microg/mL). The resulting supernates were incubated with conjunctival epithelial cell monolayers for 24 hours along with the following treatments: rTNF-alpha, mast cell supernate + anti-TNF-alpha, recombinant (r)TNF-alpha + anti-TNF-alpha, the three doses of olopatadine, olopatadine supernates, olopatadine supernates + rTNF-alpha. ICAM-1 expression was measured using flow cytometry. RESULTS Anti-IgE-stimulated human conjunctival mast cell supernates upregulated human conjunctival epithelial cell ICAM-1 expression to the same extent as rTNF-alpha. ICAM-1 upregulation could be completely blocked with anti-TNF-alpha. Preincubation of conjunctival mast cells with olopatadine significantly blocked the ability of supernates to upregulate ICAM-1 on conjunctival epithelial cells. ICAM-1 expression could be restored by adding rTNF-alpha to the olopatadine-preincubated mast cell supernates. CONCLUSIONS Olopatadine is able to significantly decrease the anti-immunoglobulin E mast cell supernate-mediated upregulation of ICAM-1 on human conjunctival epithelial cells in vitro. This seems to be mediated through an effect on a TNF-alpha-specific mechanism.

Toll-like receptor 2 expression on human conjunctival epithelial cells: a pathway for Staphylococcus aureus involvement in chronic ocular proinflammatory responses

BACKGROUND Staphylococcus aureus colonization is common in atopic keratoconjunctivitis, potentially activating epithelial cells via toll-like receptor 2 (TLR-2) and the receptor for platelet-activating factor (PAFR). OBJECTIVES To examine human conjunctival epithelial cells for the expression of TLR-2 in vitro and in vivo and to evaluate the role of TLR-2 in S aureus-mediated activation of these cells. METHODS Conjunctival epithelial cells isolated from cadaveric tissues were stimulated with interferon gamma (IFN-gamma) or a commercial S aureus cell wall extract (Staphylococcus aureus-CWE) (with or without anti-TLR-2 blocking antibody or PAFR antagonist) and were analyzed for tumor necrosis factor alpha (TNF-alpha) and interleukin 8 (IL-8) release; surface expression of TLR-2, intercellular adhesion molecule-1, HLA, and CD14; and TLR-2 messenger RNA expression. Ocular surface cells collected via impression cytology were examined for TLR-2 expression via flow cytometry. RESULTS Expression of TLR-2 was up-regulated on conjunctival epithelial cells by IFN-gamma and Staphylococcus aureus-CWE. Expression of TLR-2 messenger RNA was increased by IFN-gamma. Staphylococcus aureus-CWE up-regulated intercellular adhesion molecule 1, HLA, and CD14 expression and increased TNF-alpha and IL-8 release in a dose-dependent manner. Anti-TLR-2 significantly inhibited TNF-alpha release, whereas PAFR antagonist significantly inhibited IL-8 release. Toll-like receptor 2 was expressed on conjunctival epithelial cells from 4 of 5 patients with atopic keratoconjunctivitis, 3 of 5 with seasonal allergies, and 0 of 3 without allergies. CONCLUSIONS Conjunctival epithelial cells express TLR-2 and may play an active role in the chronic ocular inflammatory response to S aureus through pathways that involve TLR-2 and PAFR.

Epithelial cells are a major cellular source of the chemokine eotaxin in the guinea pig lung.

Eotaxin is the major eosinophil chemoattractant found in bronchoalveolar lavage (BAL) fluid from sensitized guinea pigs after antigen challenge. In this study we have performed immunostaining for eotaxin in airways obtained from challenged animals and examined purified guinea pig lung cells (epithelial cells > 98% purity, mast cells > 90% purity) for eotaxin mRNA and protein. In the airways of antigen (ovalbumin) challenged animals, significant amounts of epithelial cell eotaxin immunostaining were observed. Northern analysis of total RNA obtained from unchallenged, freshly isolated airway epithelial cells contained high levels of eotaxin mRNA. Semi-pure and high purity lung mast cell preparations (challenged or unchallenged) did not express eotaxin mRNA. Western analysis of supernatant fluids obtained from incubated airway epithelial cells demonstrated detectable amounts of eotaxin protein, with the majority of the protein being cell-associated. Thus, airway epithelial cells are identified as a major cellular source of eotaxin in the guinea pig pulmonary system.

IL-3 and TNFα increase Thymic Stromal Lymphopoietin Receptor (TSLPR) expression on eosinophils and enhance TSLP-stimulated degranulation

BackgroundThymic stromal lymphopoietin (TSLP) and eosinophils are prominent components of allergic inflammation. Therefore, we sought to determine whether TSLP could activate eosinophils, focusing on measuring the regulation of TSLPR expression on eosinophils and degranulation in response to TSLP, as well as other eosinophil activation responses.MethodsEosinophil mRNA expression of TSLPR and IL-7Rα was examined by real-time quantitative PCR of human eosinophils treated with TNFα and IL-5 family cytokines, and TSLPR surface expression on eosinophils was analyzed by flow cytometry. Eosinophils were stimulated with TSLP (with and without pre-activation with TNFα and IL-3) and evaluated for release of eosinophil derived neurotoxin (EDN), phosphorylation of STAT5, and survival by trypan blue exclusion. A blocking antibody for TSLPR was used to confirm the specificity of TSLP mediated signaling on eosinophil degranulation.ResultsEosinophil expression of cell surface TSLPR and TSLPR mRNA was upregulated by stimulation with TNFα and IL-3. TSLP stimulation resulted in release of EDN, phosphorylation of STAT5 as well as promotion of viability and survival. TSLP-stimulated eosinophil degranulation was inhibited by a functional blocking antibody to TSLPR. Pre-activation of eosinophils with TNFα and IL-3 promoted eosinophil degranulation at lower concentrations of TSLP stimulation.ConclusionsThis study demonstrates that eosinophils are activated by TSLP and that eosinophil degranulation in response to TSLP may be enhanced on exposure to cytokines present in allergic inflammation, indicating that the eosinophil has the capacity to participate in TSLP-driven allergic responses.

Zinc, copper and iron utilisation by chicks fed various concentrations of zinc

1. Zinc, iron and copper interactions were examined in chicks (New Hampshire X Single Comb White Leghorn) grown individually in stainless steel cages and fed on purified diets with three concentrations of zinc: control (37 mg Zn/kg), moderate (100 mg Zn/kg) and excess (2000 mg Zn/kg) in three studies. 2. Chicks given either moderate or excess zinc excreted more than twice as much 65Zn from a test diet as those fed on the control diet. Thus chicks given the moderate amount of zinc had concentrations of zinc in tissues similar to those of controls but chicks given excess zinc accumulated zinc in their tissues. 3. The specific activity of 65Zn in the tissues, especially in the bursa of Fabricius, of chicks given excess zinc was not reduced as much as would be predicted. Thus, ingestion of excess zinc appeared to reduce tissue turnover of 65Zn, especially in the bursa. However, humoral immune responses, as measured by antibody titres to sheep red blood cells, were not affected. 4. Chicks given excess zinc or pair-fed on the control diet retained less 59Fe from a test diet than chicks given control or moderate concentrations of zinc ad libitum. The iron concentrations in tibiae of chicks given excess zinc were depressed.

The promotion of eosinophil degranulation and adhesion to conjunctival epithelial cells by IgE-activated conjunctival mast cells

BACKGROUND Allergen-mediated mast cell activation is a key feature of ocular allergic diseases. Evidence of eosinophil-derived mediators in tears and conjunctival biopsy specimens has been associated with chronic ocular allergic inflammation. OBJECTIVE To examine the role of conjunctival mast cell mediators in eosinophil adhesion to conjunctival epithelial cells and eosinophil degranulation. METHODS Conjunctival cells were obtained by enzymatic digestion of cadaveric conjunctival tissues. Eosinophils were obtained from peripheral blood samples using negative magnetic bead selection. The effect of IgE-activated mast cell supernates on eosinophil degranulation and adherence to epithelial cells was compared with supernates obtained from mast cells pretreated with a degranulation inhibitor (olopatadine). Eosinophil adhesion was measured by eosinophil peroxidase assay, and eosinophil degranulation was measured by eosinophil-derived neurotoxin radioimmunoassay. RESULTS IgE-activated conjunctival mast cell supernates stimulated adhesion of eosinophils to epithelial cells (20.4% +/- 6.3% vs 3.1% +/- 1.0%; P = .048). Degranulation was not required for this process (no effect of olopatadine). IgE-activated mast cell supernates stimulated eosinophil-derived neurotoxin release (108.89 +/- 8.27 ng/10(6) cells vs 79.45 +/- 5.21 ng/10(6) cells for controls, P = .02), which was significantly inhibited by pretreatment of mast cells with a degranulation inhibitor (79.22 +/- 4.33 ng/10(6) cells vs 61.09 +/- 5.39 ng/10(6) cells for olopatadine pretreated and untreated, respectively, P = .02). CONCLUSIONS Mediators released from conjunctival mast cells promote eosinophil adhesion to conjunctival epithelial cells and eosinophil degranulation. Degranulation inhibition studies suggest that different mast cell mediators are involved in regulation of these events.

Zinc, iron, and copper contents of eggs from hens fed varying levels of zinc

Abstract We wanted to determine whether eggs which contained more zinc could be produced by fortifying the diets of hens with zinc. In two studies, hens fed very high levels of zinc (1762 or 1861 μg Zn/g diet) for 4 to 40 weeks produced eggs that contained 57–90% more zinc than eggs produced by hens fed control diets (28 or 26 μg Zn/g diet). Hens fed high levels of zinc (218 or 257 μg Zn/g diet) produced eggs that contained as much as 25% more zinc than eggs produced by hens fed control diets. Ingestion of excess zinc did not have consistent effects on the iron or copper content of eggs or on the zinc content of eggshells.