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.

Comparison of the type of liposome involving cytokine production induced by non-CpG Lipoplex in macrophages.

To improve the transfection efficiency of plasmid DNA (pDNA) into cells, various types of cationic liposome have been used to prepare pDNA/cationic liposome complexes (lipoplexes). It is well-known that lipoplexes induce a large amount of proinflammatory cytokines because unmethylated CpG dinucleotides (CpG motifs) abundantly present in pDNA are recognized by Toll-like receptor-9 (TLR9) expressed in immune cells such as macrophages and dendritic cells. This nonspecific cytokine production is problematic in nonviral gene therapy. Moreover, recent studies have demonstrated that lipoplexes induce not only proinflammatory cytokines but also another type of cytokine, type I interferons (IFNs), irrespective of the frequency of CpG motifs in DNA and the expression of TLR9. To gain more insight into the CpG motif- and TLR9-independent induction of type I IFNs and proinflammatory cytokines by lipoplex, macrophage activation was evaluated in vitro using various cationic liposomes complexed with pDNA containing no CpG motifs. The production of IFN-beta, TNF-alpha and IL-6 by lipoplex was confirmed to be induced independently of the interaction between CpG DNA and TLR9 in macrophages from TLR9-knockout mice. Then, the release of the cytokines, the mRNA expression of Z-DNA binding protein-1 (Zbp1), a cytosolic double-stranded DNA sensor, and the cellular uptake of pDNA were examined in a macrophage-like cell line, RAW264.7. The level of cytokine production and the increase in the Zbp1 mRNA varied depending on the type of cationic liposome used. A good correlation was observed between the cytokine level and the Zbp1 mRNA. A confocal microscopic study using fluorescently labeled pDNA complexes showed that the complexes that released a lot of cytokines showed an enhanced distribution of pDNA-derived fluorescence into the cytosol. These results suggest that different intracellular trafficking derived from the type of liposomes determines the recognition of pDNA by ZBP1 after uptake of lipoplexes by the macrophages, followed by the release of type I IFNs and inflammatory cytokines. The present study demonstrates that cationic liposomes should be selected based on these findings for optimization of DNA-based therapies using lipoplexes.

TLR9-dependent systemic interferon-beta production by intravenous injection of plasmid DNA/cationic liposome complex in mice.

BACKGROUND: The type I interferon (IFN) response to DNA/cationic liposome complex, or lipoplex, has been reported in cultured cells, but little is known about the response in vivo. Studies of the pro-inflammatory cytokine response to lipoplex have shown the importance of the unmethylated CpG dinucleotide (CpG motif) and its receptor, Toll-like receptor (TLR)-9. METHODS: CpG- and non-CpG lipoplex consisting of CpG- or non-CpG plasmid DNA, respectively, and N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride/cholesterol liposomes were intravenously injected into mice. IFN-beta and interleukin (IL)-6 in the serum and organs were measured by the enzyme-linked immunosorbent assay. The involvement of TLR9, phagocytic cells and the spleen in the responses was evaluated using TLR9(-/-), clodronate liposome-treated-, and splenectomized mice, respectively. Accumulation of blood cells in the lung was evaluated histologically. RESULTS: CpG lipoplex induced a large increase in the levels of IFN-beta and IL-6 in the serum, liver, spleen, lung and kidney, whereas non-CpG lipoplex scarcely had any effect. Neither formulation led to significant cytokine production in TLR9(-/-) mice. Clodronate liposome-treated mice showed a large reduction in both IFN-beta and IL-6 levels. Splenectomized mice receiving CpG lipoplex also showed a significantly low production of IL-6 but a similar level of IFN-beta production to that of unsplenectomized mice. A large number of monocytes were found in the capillary vessels around the pulmonary alveoli of mice receiving lipoplex. CONCLUSIONS: These findings indicate that, in contrast to the production of IL-6 from splenic macrophages, IFN-beta is produced from phagocytic cells other than splenic macrophages after the injection of CpG lipoplex through the TLR9-dependent pathway.

Cellular activation by plasmid DNA in various macrophages in primary culture.

Macrophages are an important group of cells responsible for the inflammatory response to unmethylated CpG dinucleotide (CpG motif) in plasmid DNA (pDNA) via Toll-like receptor 9 (TLR9). This finding is primarily based on in vitro studies. Previous in vivo studies also have suggested that tissue macrophages are involved in inflammatory cytokine release in the circulation following intravenous administration of pDNA to mice. However, the relationship between the in vitro and in vivo studies has not been sufficiently clarified. To gain insight into which types of cells are responsible for the production of cytokines upon interaction with pDNA, peritoneal macrophages, splenic macrophages, hepatic nonparenchymal cells (NPCs) including Kupffer cells and mesangial cells were isolated from mice. All types of primary cultured cells, except for mesangial cells, express TLR9 at varying levels. Splenic macrophages and hepatic NPCs were activated to produce tumor necrosis factor-alpha (TNF-alpha) by naked pDNA, whereas peritoneal macrophages and mesangial cells were not. pDNA complexed with N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethyl-ammonium chloride/cholesterol liposome induced TNF-alpha in the splenic macrophages but not in the other cell types. These results indicate that splenic macrophages and hepatic NPCs are closely involved in TNF-alpha production in response to pDNA.

Determination of p-glycoprotein ATPase activity using luciferase.

We investigated whether P-glycoprotein (P-gp) ATPase activity of Caco-2 cell membranes could be estimated by measuring consumption of ATP using luciferin-luciferase reaction, and whether the results would be useful for assessment of the interactions between P-gp and drugs. The vanadate-sensitive ATPase activity of Caco-2 cell membranes was measured rapidly with high sensitivity using luciferin-luciferase reaction. Cyclosporin A, verapamil, digoxin and quinidine stimulated the ATPase activity concentration-dependently with Km values of 5.3, 0.9, 1.2 and 4.1 microM, respectively. These values except for digoxin were comparable with previous reports. The ATPase activity and P-gp mRNA expression in Caco-2 cells were induced by all-trans-retinoic acid, digoxin and levothyroxine, but not dexamethasone or rifampicin. This method was useful to assess interactions with P-gp and drugs, and was used to elucidate the mechanisms of interaction of levothyroxine and digoxin.