Constitutive and inflammatory mediator-regulated fractalkine expression in human ocular tissues and cultured cells.
OBJECTIVE
Fractalkine (FKN) is a dual-adhesion molecule-chemokine that plays a role in inflammation but has not been explored in the eye. In the current study, constitutive expression of FKN was identified in human iris and retina, and its regulation by various cytokines in endothelial cells (ECs) and stromal cells from human iris, retina, and choroid was investigated.


METHODS
Human iris and retina explants were evaluated for FKN mRNA and protein expression using RT-PCR and immunohistochemistry, respectively. Cultured ocular ECs and stromal cells were stimulated with various inflammatory mediators (endotoxin; TNFalpha; interferon-gamma; interleukin (IL)-1alpha, -4, -10, -13, -17, and -18; and/or CD40 ligand, or combinations thereof), with FKN mRNA being subsequently evaluated by cDNA array and/or RT-PCR and FKN protein by enzyme-linked immunoculture assay (ELICA) and/or by Western blot analysis.


RESULTS
Iris and retina explants constitutively expressed FKN protein in microvascular ECs and also in several stromal cell types. Iris and retina both express FKN mRNA. TNFalpha upregulated FKN in iris explants. All ocular microvascular ECs and stromal cultures expressed low FKN mRNA and/or protein levels, which were variably upregulated by endotoxin, TNFalpha, interferon-gamma, IL-1alpha, and/or CD40 ligand, but not by IL-18. In ECs, the Th2 cytokines IL-4 and -13, but not IL-10, reduced TNFalpha-induced FKN protein. IL-17, usually considered proinflammatory, reduced TNFalpha-induced FKN protein in ocular ECs.


CONCLUSIONS
FKN is expressed in various ocular tissues and cells. Inflammatory mediator modulation of ocular FKN expression suggests that this adhesive chemokine may play important roles in regulating leukocyte efflux in inflammatory eye diseases, such as anterior uveitis and retinochoroiditis.
Silverman
Matthew D
MD
Department of Ophthalmology, Casey Eye Institute, Oregon Health & Sciences University, Portland, Oregon 97201, USA.
Zamora
David O
DO
Pan
Yuzhen
Y
Texeira
Paul V
PV
Baek
Seung-Hee
SH
Planck
Stephen R
SR
Rosenbaum
James T
JT
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D054428:Chemokine CX3CL1; D020523:Chemokines, CX3C / Q000235:genetics* / Q000378:metabolism; D002829:Choroid / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation; D006801:Humans; D007124:Immunoenzyme Techniques; D018836:Inflammation Mediators / Q000494:pharmacology*; D007498:Iris / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D012333:RNA, Messenger / Q000378:metabolism; D012160:Retina / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017154:Stromal Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D015854:Up-Regulation
C515538:CX3CL1 protein, human; D054428:Chemokine CX3CL1; D020523:Chemokines, CX3C; C515539:Cx3cl1 protein, mouse; D018836:Inflammation Mediators; D008565:Membrane Proteins; D012333:RNA, Messenger