Gadolinium Chloride-Induced Hepatocyte Proliferation Is Prevented by Antibodies to Tumor Necrosis Factor α

Abstract Gadolinium chloride (GdCl 3 ) destroys large Kupffer cells and has been used extensively in mechanistic studies in a number of disease and toxicity processes; however, it cannot be used to study hepatocyte turnover since it increases cell proliferation itself. The mechanism by which GdCl 3 activates cell turnover in liver is unknown, but several possibilities exist. Here it was demonstrated that a direct mitogenic action on hepatocytes is unlikely since GdCl 3 did not stimulate the growth of primary rat hepatocyte in vitro . Therefore, it was hypothesized that GdCl 3 acts indirectly through mitogenic cytokines of nonparenchymal cell origin. Antibodies to tumor necrosis factor α (TNFα) were used to evaluate if TNFα is causally responsible for GdCl 3 -induced cell proliferation. GdCl 3 treatment of rats in vivo increased hepatocyte replication 5-fold in 24 h and 3-fold in 48 h. Pretreatment with specific anti-TNFα antibodies completely prevented these effects. However, when antibody treatment was delayed until 24 h after GdCl 3 , increased cell proliferation was not prevented, suggesting that TNFα production during the first 24 h after treatment is responsible for activation of a signaling cascade involving other mitogens that sustain hepatocyte replication at 48 h. Twenty-four hours after treatment with GdCl 3 , TNFα mRNA transcripts were increased 2-fold over control, an effect that was prevented by pretreatment with anti-TNFα antibody. NFκB, which is known to be involved in TNFα transcription, was activated by GdCl 3 about 4.5-fold over control 8 h after treatment in vivo , an increase not observed when antibodies to TNFα were present. When GdCl 3 was added to macrophages in culture, TNFα was nearly doubled 4 h after treatment. Additionally, conditioned media harvested from macrophages treated with GdCl 3 for 2 to 8 h stimulated the growth of HepG2 cells in culture about 2-fold, while antibodies to TNFα completely prevented this effect. Taken together, these data are consistent with the hypothesis that TNFα released from Kupffer cells at early time points prior to their destruction is causally responsible for triggering a cascade of events responsible for GdCl 3 -induced cell proliferation.