Enhanced activity of lysosomal proteases in activated rat hepatic stellate cells is associated with a concomitant increase in the number of the mannose-6-phosphate/insulin-like growth factor II receptor.

Activated hepatic stellate cells (HSCs) play a central role during hepatic tissue repair through their influence on extracellular matrix remodelling. We have determined whether the activity levels of cathepsin B and D are affected by in vitro activation of rat HSCs, and whether the enzymes were released from the cells. Furthermore, given the important role of the mannose-6-phosphate/insulin-like growth factor II receptor (M6P/IGF-IIR) in the intracellular transport of lysosomal enzymes, we have examined whether changes in the activity of these proteases were associated with parallel changes in the level of the M6P/IGF-IIR. The activity of cathepsin B and D increased ∼4 times between 2 and 8 days of HSC culture. This result was supported by analysing mRNA expression by RT-PCR. The cells released the enzymes into the culture medium, amounting to ∼10% of the cell-associated activity over 24 h. The release of enzymes was not affected by reducing medium pH from 7.4 to 6.2, indicating that the enzymes were transported to the medium independently of the M6P/IGF-II-R. The released cathepsin B was mostly in the inactive proenzyme form. HSC activation led to a particularly large increase in M6P/IGF-IIR expression. A large proportion of the receptors was located on the cell surface and was found to be very suitable for measuring endocytosis of (125) I-IGF-II. The results show that the endocytic activity increased in parallel with the increase in surface receptors and activity of lysosomal enzymes. Degradation of the ligand was reduced by inhibitors of lysosomal proteases and therefore took place in lysosomes.

Calprotectin (S100A8/S100A9) and myeloperoxidase: co-regulators of formation of reactive oxygen species.

INFLAMMATORY MEDIATORS TRIGGER POLYMORPHONUCLEAR NEUTROPHILS (PMN) TO PRODUCE REACTIVE OXYGEN SPECIES (ROS: O(2) (-), H(2)O(2), ∙OH). Mediated by myeloperoxidase in PMN, HOCl is formed, detectable in a chemiluminescence (CL) assay. We have shown that the abundant cytosolic PMN protein calprotectin (S100A8/A9) similarly elicits CL in response to H(2)O(2) in a cell-free system. Myeloperoxidase and calprotectin worked synergistically. Calprotectin-induced CL increased, whereas myeloperoxidase-triggered CL decreased with pH > 7.5. Myeloperoxidase needed NaCl for CL, calprotectin did not. 4-hydroxybenzoic acid, binding ∙OH, almost abrogated calprotectin CL, but moderately increased myeloperoxidase activity. The combination of native calprotectin, or recombinant S100A8/A9 proteins, with NaOCl markedly enhanced CL. NaOCl may be the synergistic link between myeloperoxidase and calprotectin. Surprisingly- and unexplained- at higher concentration of S100A9 the stimulation vanished, suggesting a switch from pro-oxidant to anti-oxidant function. We propose that the ∙OH is predominant in ROS production by calprotectin, a function not described before.

Hepatic clearance of Sonazoid perfluorobutane microbubbles by Kupffer cells does not reduce the ability of liver to phagocytose or degrade albumin microspheres.

This study has been performed to examine which cells are responsible for the hepatic clearance of the new ultrasound contrast agent Sonazoid and to study whether uptake of these gas microbubbles disturbs the function of the cells involved. Sonazoid was injected into rats and perfused fixed livers were studied by electron microscopy, which revealed that the Sonazoid microbubbles were exclusively internalised in Kupffer cells, i.e. by the macrophages located in the liver sinusoids, and not by parenchymal, stellate or endothelial cells. This is the first demonstration of intact phagocytosed gas microbubbles within Kupffer cells. Uptake of the Sonazoid perfluorobutane microbubbles by the Kupffer cells following injection of a dose corresponding to 20x the anticipated clinical dose for liver imaging did not result in measurable changes in the uptake and degradation of radioactively labelled albumin microspheres previously shown to be a useful indicator marker for Kupffer cell phagocytosis.