Extracellular nucleotides and interleukin-8 production by ARPE cells: potential role of danger signals in blood-retinal barrier activation.
OBJECTIVE
RPE cell activation is an important feature of autoimmune uveitis. This investigation focused on whether extracellular nucleotides could contribute to this activation, and the effects of ATPgammaS, UTP, and UDP on the production of IL-8 by RPE cells was studied in relation to their expression of functional P2Y receptors.


METHODS
ARPE-19 cells were cultured with ATPgammaS, UTP, UDP, and TNF. IL-8 gene transcription and protein production were measured by semiquantitative RT-PCR and ELISA. Western blot analysis and RT-PCR were used to investigate ERK 1/2 activation and P2Y expression. Changes in intracellular calcium and cAMP concentration were analyzed by spectrofluorometry and radioimmunoassay.


RESULTS
Stimulation of ARPE-19 cells with ATPgammaS, UTP, and UDP induced IL-8 gene transcription and protein secretion. TNFalpha induction of IL-8 secretion was also increased by ATPgammaS, UTP, and UDP. Nucleotide induction of IL-8 production was blocked by PD98059, and all nucleotides stimulated ERK 1/2 phosphorylation. P2Y(2) and P2Y(6) mRNAs were detected in ARPE-19 cells. All tested nucleotides induced a pulse of intracellular calcium.


CONCLUSIONS
ATPgammaS, UTP, and UDP stimulate both basal and TNFalpha-induced IL-8 secretion in RPE cells through an ERK 1/2-dependent pathway. The results suggest that those effects are mediated by P2Y(2) and P2Y(6) receptors.
Relvas
Lia Judice M
LJ
Department of Ophthalmology, CHU Centre Hospitalier, Universitaire St-Pierre and Brugmann, Université Libre de Bruxelles-Campus Erasme, Bruxelles, Belgium.
Bouffioux
Christophe
C
Marcet
Brice
B
Communi
Didier
D
Makhoul
Maya
M
Horckmans
Michael
M
Blero
Daniel
D
Bruyns
Catherine
C
Caspers
Laure
L
Boeynaems
Jean-Marie
JM
Willermain
François
F
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D001813:Blood-Retinal Barrier / Q000502:physiology*; D015153:Blotting, Western; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D005419:Flavonoids / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D010766:Phosphorylation; D012333:RNA, Messenger / Q000378:metabolism; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000187:drug effects* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014530:Uridine Diphosphate / Q000494:pharmacology*; D014544:Uridine Triphosphate / Q000494:pharmacology*
D005419:Flavonoids; D016209:Interleukin-8; D012333:RNA, Messenger; D018048:Receptors, Purinergic P2; D014409:Tumor Necrosis Factor-alpha; C099845:purinoceptor P2Y6; C022571:adenosine 5'-O-(3-thiotriphosphate); D014530:Uridine Diphosphate; D000255:Adenosine Triphosphate; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D014544:Uridine Triphosphate