AsialoGM1-mediated IL-8 release by human corneal epithelial cells requires coexpression of TLR5.
OBJECTIVE
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 Ca(2+) 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 Ca(2+) 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 Ca(2+) influx through L-type voltage-dependent Ca(2+) channels. This led to P2Y receptor stimulation along with membrane depolarization, resulting from increases in ATP release into the medium. Intracellular Ca(2+) 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 Ca(2+) influx through L-type voltage-dependent Ca(2+) channels, as well as stimulation of the ERK pathway.
Du
Jojo W
JW
Department of Biological Sciences, College of Optometry, State University of New York, 33 West 42nd Street, New York City, NY 10036, USA.
Zhang
Fan
F
Capó-Aponte
José E
JE
Tachado
Souvenir D
SD
Zhang
Jing
J
Yu
Fu-Shin X
FS
Sack
Robert A
RA
Koziel
Henry
H
Reinach
Peter S
PS
D000255:Adenosine Triphosphate / Q000378:metabolism; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000378:metabolism; D002460:Cell Line; D019573:Epithelium, Corneal / Q000378:metabolism*; D005434:Flow Cytometry; D005677:G(M1) Ganglioside / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D016209:Interleukin-8 / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000502:physiology; D008856:Microscopy, Fluorescence; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D010766:Phosphorylation; D018048:Receptors, Purinergic P2 / Q000378:metabolism; D051198:Toll-Like Receptor 5 / Q000378:metabolism*
D020746:Calcium Channels, L-Type; D016209:Interleukin-8; D018048:Receptors, Purinergic P2; C495346:TLR5 protein, human; D051198:Toll-Like Receptor 5; D005677:G(M1) Ganglioside; C018835:asialo GM1 ganglioside; D000255:Adenosine Triphosphate; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D002118:Calcium