Potentiation of lipopolysaccharide-induced chemokine and adhesion molecule expression in corneal fibroblasts by soluble CD14 or LPS-binding protein.
OBJECTIVE
The detection of bacterial lipopolysaccharide (LPS) by human cells is facilitated by LPS-binding protein (LBP) and soluble (s)CD14. The effects of these proteins on chemokine release and adhesion molecule expression in cultured human corneal fibroblasts were examined.


METHODS
The release of chemokines into culture supernatants and the expression of the intercellular adhesion molecule (ICAM)-1 on the cell surface were determined by enzyme-linked immunosorbent assays. The intracellular abundance of chemokine and ICAM-1 mRNAs was quantitated by reverse transcription and real-time polymerase chain reaction analyses. The phosphorylation and degradation of IkappaB-alpha and the subcellular localization of NF-kappaB were examined by immunoblot and immunofluorescence analyses, respectively.


RESULTS
Neither sCD14 nor LBP alone affected the expression of chemokines or ICAM-1 in cultured human corneal fibroblasts. However, sCD14 or LBP enhanced the LPS-induced upregulation of ICAM-1 and the chemokines interleukin-8 and monocyte chemoattractant protein (MCP)-1 in these cells at the protein and mRNA levels. Combined stimulation with LPS and either sCD14 or LBP also induced the phosphorylation and degradation of IkappaB-alpha and the translocation of NF-kappaB from the cytoplasm to the nucleus of corneal fibroblasts.


CONCLUSIONS
LBP and sCD14 may play important roles in the defense of the cornea against bacterial infection, by facilitating the detection of LPS by corneal fibroblasts.
Fukuda
Ken
K
Department of Ocular Pathophysiology, Yamaguchi University School of Medicine, 1-1-1 Minami-Kogushi, Ube City, Yamaguchi 755-8505, Japan. k.fukuda@yamaguchi-u.ac.jp
Kumagai
Naoki
N
Yamamoto
Kazutaka
K
Fujitsu
Youichiro
Y
Chikamoto
Nobuhiko
N
Nishida
Teruo
T
D000209:Acute-Phase Proteins / Q000494:pharmacology*; D000293:Adolescent; D000328:Adult; D000368:Aged; D002352:Carrier Proteins / Q000494:pharmacology*; D002478:Cells, Cultured; D018925:Chemokines / Q000235:genetics* / Q000378:metabolism; D002648:Child; D002675:Child, Preschool; D003315:Cornea / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D004357:Drug Synergism; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D015151:Immunoblotting; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics* / Q000378:metabolism; D018950:Lipopolysaccharide Receptors / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008297:Male; D008562:Membrane Glycoproteins / Q000494:pharmacology*; D008875:Middle Aged; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012995:Solubility; D015854:Up-Regulation; D000099231:Lipopolysaccharide-Binding Protein
D000209:Acute-Phase Proteins; D002352:Carrier Proteins; D018925:Chemokines; D020672:I-kappa B Proteins; D018799:Intercellular Adhesion Molecule-1; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B; D012333:RNA, Messenger; D000099231:Lipopolysaccharide-Binding Protein; C000606204:NFKBIA protein, human