Hepatomegaly is a dose-dependent response to TLR4 agonists. After enlarging for a few days, the liver returns to its previous size. This transient phenomenon has attracted little interest and its physiology has evidently not been studied. Here we explored the basis for the much longer-lasting hepatomegaly that occurs in mice that cannot inactivate LPS because they lack the LPS-deacylating enzyme, AOAH. These animals exhibit wildtype acute cytokine responses to intravenous doses of LPS (), yet they develop impressive hepatic enlargement that lasts for many weeks. Importantly, TLR4 activation by a non-LPS agonist, the monoclonal antibody UT12, induced hepatomegaly of similar degree and duration in wildtype and Aoah−/−
animals (Fig. S1
), indicating that the AOAH-dependent phenotype is also LPS-dependent.
For these studies we used E. coli
Ra (O14) LPS. A complete “rough-form” LPS, it offered several advantages over smooth (long polysaccharide-containing) LPS preparations: a more uniform size and structure, CD14-and LBP-independent activation of TLR4 (27
), and an aggregation state that promotes rapid uptake from the blood, largely by Kupffer cells. We found previously that Kupffer cells produce AOAH (6
). Whereas Kupffer cell depletion reduced hepatic LPS deacylation by approximately 90%, LPS uptake by the liver fell only 60%, indicating that other hepatic cells can also remove LPS from the blood (6
). Here we found that some FITC-LPS remained associated with Kupffer cells for at least 7 days, long after the liver has returned to baseline size in wildtype animals; although dispersion of the FITC-LPS may have limited its detection, there were no evident differences in LPS location in the livers of wildtype and Aoah−/−
mice. LPS induced Aoah−/−
Kupffer cells to become larger and more phagocytic than Aoah+/+
Kupffer cells for at least one week. Kupffer cell depletion reduced LPS-induced hepatomegaly in Aoah−/−
mice by ~40%, suggesting that molecules produced by Kupffer cells contribute importantly to the prolonged hepatomegaly phenotype.
The most striking morphological feature of the phenotype was found in the sinusoids, where there were aggregates of cells (KCs, PMN, platelets, erythrocytes) with occasional hemophagocytosis. Scanning electron microscopy suggested subtle changes in the appearance of sinusoidal endothelial cell fenestrae (), and intravital flow studies (D. Rockey, not shown) found slower, less consistent flow through the sinusoids of LPS-treated Aoah−/−
livers. Leukocyte recruitment or trapping in the liver peaked during the second week after LPS exposure, then returned to baseline by 21 days (). Importantly, spleen size increased transiently then returned to baseline whereas liver size remained large (6
), suggesting that portal hypertension does not develop during prolonged hepatomegaly. Hepatomegaly persisted for at least 3 weeks, with no evident increase in hepatocyte proliferation (Fig. S2
) or accumulation of triglyceride (6
). Serum transaminase and alkaline phosphatase levels also did not increase (6
We used several interventions to neutralize potential mediators in vivo
). We prevented coagulation using low-molecular weight heparin (28
), blocked prostanoid synthesis using ibuprofen, and, to look for a role for the sympathetic nervous system (29
), we provided adrenoreceptor stimulation (epinephrine and norepinephrine) or antagonism (metroprolol and prazosin) using indwelling osmotic pumps. None of these interventions prevented LPS-induced prolonged hepatomegaly in Aoah−/−
animals. Damping nitric oxide/nitrite synthesis using L-NAME or providing nitrite in the drinking water (26
) also had no effect on the phenotype.
Acute plasma TNF levels were somewhat lower in LPS-infused Aoah−/−
mice than in the wildtype controls and we were unable to detect TNF in plasma or liver lysates more than 2 hours after LPS injection. Liver TNF mRNA levels remained elevated for at least one week, however, and a role for TNF was found when pre-treatment with the TNF-binding protein, PEGsTNF-R1, reduced prolonged hepatomegaly by 27% (Table S2
). IL-1β was similarly implicated using an IL-1 receptor antagonist (Anakinra). Increases in several anti-inflammatory mediators, most notably IL-10, were unable to check the response.
In summary, AOAH is required to prevent prolonged hepatomegaly after intravenous challenge with low doses of LPS. LPS is largely taken up from the bloodstream by Kupffer cells, which retain at least some of it for a week or more. In animals that lack AOAH, the Kupffer cells enlarge and phagocytose blood cells; many leukocyte cell types are either recruited to the liver or retarded there as blood flow slows through congested sinusoids. Dexamethasone pre-treatment largely prevents LPS-induced prolonged hepatomegaly, neutralizing TNF or IL-1β has a partial inhibitory effect, and blocking the IL-10 receptor greatly enhances the phenotype. Future studies will address the ability of persistently-active (fully acylated) LPS to stimulate Kupffer cells for prolonged periods in vivo.
AOAH’s key role in recovery from endotoxin exposure in mice should encourage efforts to identify the enzyme’s role in human liver physiology and disease. The enzyme may be particularly important in those conditions, such as alcoholic liver disease (8
), in which gut-derived endotoxin is thought to play a contributory role.