Recent studies have demonstrated that intravenous administration of a plasmid DNA-cationic liposome complex (lipoplex) induced significant proinflammatory cytokine production in blood and inhibited transgene expression in pulmonary endothelial cells. In this study, we examined the effects of gadolinium chloride (GdCl(3)) pretreatment on the biodistribution and induction of proinflammatory cytokine production and transgene expression after intravenous injection of a lipoplex in mice. GdCl(3) is known to transiently deplete liver Kupffer cells and spleen macrophages after intravenous administration. Intravenous administration of a lipoplex triggers high levels of proinflammatory cytokine production, such as TNF-alpha, IFN-gamma and IL-12 in serum and a large amount of (32)P-labeled lipoplex accumulates in the liver 1 h after intravenous administration. However, pretreatment with GdCl(3) dramatically reduces serum levels of these cytokines and liver accumulation of the lipoplex. RT-PCR analysis showed that mRNA expression of TNF-alpha greatly increases in the liver and spleen after lipoplex injection and that pretreatment with GdCl(3) reduces mRNA expression in these organs. Messenger RNA expression of TNF-alpha in the liver occurs in non-parenchymal cells (sinusoidal endothelial cells and/or Kupffer cells). Inhibition of cytokine production by pretreatment with GdCl(3) leads to recovery of transgene expression in the lung following the second injection of lipoplex, which was reduced following the first injection of lipoplex. Thus, the present study demonstrates that tissue macrophages involving liver Kupffer cells and spleen macrophages are closely involved in TNF-alpha production following i.v. administration of the lipoplex. It is also suggested that avoiding lipoplex uptake and subsequent cytokine production by these cells would be a useful method of maintaining a high level of gene expression in the lung after repeated injections.