Fibronectin inhibits cytokine production induced by CpG DNA in macrophages without direct binding to DNA.
Fibronectin (FN) is known to have four DNA-binding domains although their physiological significance is unknown. Primary murine peritoneal macrophages have been shown to exhibit markedly lower responsiveness to CpG motif-replete plasmid DNA (pDNA), Toll-like receptor-9 (TLR9) ligand, compared with murine macrophage-like cell lines. The present study was conducted to examine whether FN having DNA-binding domains is involved in this phenomenon. The expression of FN was significantly higher in primary macrophages than in a macrophage-like cell line, RAW264.7, suggesting that abundant FN might suppress the responsiveness in the primary macrophages. However, electrophoretic analysis revealed that FN did not bind to pDNA in the presence of a physiological concentration of divalent cations. Surprisingly, marked tumor necrosis factor - (TNF-)α production from murine macrophages upon CpG DNA stimulation was significantly reduced by exogenously added FN in a concentration-dependent manner but not by BSA, laminin or collagen. FN did not affect apparent pDNA uptake by the cells. Moreover, FN reduced TNF-α production induced by polyI:C (TLR3 ligand), and imiquimod (TLR7 ligand), but not by LPS (TLR4 ligand), or a non-CpG pDNA/cationic liposome complex. The confocal microscopic study showed that pDNA was co-localized with FN in the same intracellular compartment in RAW264.7, suggesting that FN inhibits cytokine signal transduction in the endosomal/lysosomal compartment. Taken together, the results of the present study has revealed, for the first time, a novel effect of FN whereby the glycoprotein modulates cytokine signal transduction via CpG-DNA/TLR9 interaction in macrophages without direct binding to DNA through its putative DNA-binding domains.
Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Yoshida-Shimo-Adachi-cho, Sakyo-ku, Kyoto 606-8501, Japan. h.yoshida@nihs.go.jp
Hiroyuki
H
Yoshida
Makiya
M
Nishikawa
Sachiyo
S
Yasuda
Hiroyasu
H
Toyota
Tsuyoshi
T
Kiyota
Yuki
Y
Takahashi
Yoshinobu
Y
Takakura
D000634:Aminoquinolines / Q000494:pharmacology; D000818:Animals; D019138:Antibodies, Blocking / Q000276:immunology / Q000494:pharmacology; D002413:Cations, Divalent / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005353:Fibronectins / Q000378:metabolism* / Q000494:pharmacology; D000077271:Imiquimod; D007150:Immunohistochemistry; D039081:Integrin alpha5beta1 / Q000276:immunology / Q000378:metabolism; D007369:Interferon Inducers / Q000494:pharmacology; D008247:Lysosomes / Q000378:metabolism; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D008813:Mice, Inbred ICR; D018613:Microscopy, Confocal; D009838:Oligodeoxyribonucleotides / Q000378:metabolism* / Q000494:pharmacology; D011070:Poly I-C / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D000634:Aminoquinolines; D019138:Antibodies, Blocking; C408982:CPG-oligonucleotide; D002413:Cations, Divalent; D016207:Cytokines; D005353:Fibronectins; D039081:Integrin alpha5beta1; D007369:Interferon Inducers; D009838:Oligodeoxyribonucleotides; D014409:Tumor Necrosis Factor-alpha; D011070:Poly I-C; D000077271:Imiquimod