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AsialoGM1-Mediated IL-8 Release by Human Corneal Epithelial Cells Requires Coexpression of TLR5

¹From the Department of Biological Sciences, College of Optometry, State University of New York, New York City, New York; the ²Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts; and the ³Department of Ophthalmology, Wayne State University of Medicine, Detroit, Michigan.

PURPOSE. In this study, it was determined that human corneal epithelial cells (HCECs) express asialoganglioside ganliotetraosylceramide (asialoGM1) and toll-like receptor (TLR)-5, and their interaction induces interleukin (IL)-8 release through Ca²⁺ transient activation and mitogen-activated protein kinase (MAPK) stimulation.

METHODS. Expression of asialoGM1 and TLR5 was detected in SV40 HCECs by Western blot and flow cytometry analyses and their association by coimmunoprecipitation. Single-cell fluorescence imaging was used to measure intracellular free Ca²⁺ transients in fura-2–loaded cells. The enzyme-linked immunosorbent assay (ELISA) was used to quantify IL-8 production in both cultured and primary HCECs.

RESULTS. The HCECs expressed both asialoGM1 and TLR5 receptors. Ligation of asialoGM1 resulted in protein–protein interaction with TLR5, followed by transient increases in Ca²⁺ influx through L-type voltage-dependent Ca²⁺ channels. This led to P2Y receptor stimulation along with membrane depolarization, resulting from increases in ATP release into the medium. Intracellular Ca²⁺ transients led to time-dependent extracellular signal-regulated kinase (ERK) MAPK pathway stimulation, followed by a 9.5-fold increase in IL-8 release. Similarly, in primary HCECs, asialoGM1 receptor stimulation resulted in an 8.1-fold increase. With a TLR5 neutralizing antibody, no asialoGM1-induced increases in IL-8 release occurred, and this response was not suppressed in the presence of a TLR2 neutralizing antibody.

CONCLUSIONS. IL-8 release by HCECs is mediated through ligand-induced asialoGM1 protein–protein interactions with TLR5. This response is dependent on ATP efflux into the medium, followed by P2Y receptor stimulation. Such activation, in turn, results in increases in Ca²⁺ influx through L-type voltage-dependent Ca²⁺ channels, as well as stimulation of the ERK pathway.

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