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J.H. Salmon, S.H. Loter, S. Jones, B.C. Gilger; Neutrophil Chemotaxis to Normal Equine Tears . Invest. Ophthalmol. Vis. Sci. 2004;45(13):627.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Horses are more often affected by infectious keratitis than other domestic species. The purpose of this project is to determine the effect of tear film of normal horses on neutrophil (PMN) chemotaxis and to compare this PMN chemotaxis to that induced by human tear film. Methods: Tears from horses and humans with no ocular abnormalities were collected using cellulose sponges placed into the ventral conjunctival fornix. Tears were removed from the sponges by centrifugation and stored at –80C until tested. PMNs used for the assays were obtained from healthy adult individuals. Chemotaxis was assessed using microtiter plates with 2µm filters (ChemoTx disposable chemotaxis system 101–2, Neuro Probe, Inc.). The percentage of chemotaxis was determined by measuring the fluorescence of calcein labeled PMNs that migrated into each well compared to the fluorescence of the control wells. To determine the mechanism of chemotaxis, tears were incubated with chemotactic inhibitors prior to placement on microtiter plate. Results: Normal equine tears activate chemotaxis to PMNs in a dilution dependent manner. Normal human tears also activate chemotaxis although it appears to be quantitatively less than equine tears over a range of dilutions. Platelet activating factor, our positive control, consistently induced 80% chemotaxis with equine PMNs. Elevation of intracellular cAMP (icAMP) with rolipram, a selective phosphodiesterase 4 inhibitor, did not significantly affect equine PMN chemotaxis when tested alone. Clenbuterol, a beta–2–adrenergic agonist that increases icAMP production, demonstrated a dose dependent inhibition of chemotaxis toward equine tears. Furthermore, combined treatments with the two drugs synergistically inhibited chemotaxis to a greater extent. Conclusions: Normal equine tears more potently activate chemotaxis compared to human tears. Assays using chemotactic inhibitors showed that the combination of rolipram, inhibiting icAMP breakdown, and clenbuterol, increasing icAMP production, showed the greatest chemotactic inhibition. Therefore, levels of cAMP likely play a role in ocular surface immunology. Further study on tear film component differences between humans and horses may determine the substance in tears that induces PMN chemotaxis in horses.
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