active TGF-b in MFBcM as mediator of the apoptotic The induction of apoptosis of rat liver parenchymal effects of MFB was proven by preincubation of the condicells (PC) by transforming growth factor b (TGF-b)-extioned medium with human recombinant latency-associpressing transformed fat-storing cells (FSC), i.e., myofiated peptide, which reversed completely MFBcM broblasts (MFB), was studied under culture conditions induced reduction of the XTT-test and the MFBcM-genand compared with the apoptotic effect of human recomerated increase of oligonucleosomal DNA fragments. binant TGF-b 1 . MFB were obtained by subculture of Partial reversibility was reached by preincubation of FSC. The TGF-b concentration in the conditioned methe medium with recombinant soluble type II TGF-b redium of myofibroblasts (MFBcM) determined with the ceptor. The data let us conclude that transformed FSC, Mink cell proliferation inhibition assay was õ0.25 ng/ i.e., MFB in damaged liver, could participate in the mL/24 h in the native medium, but 1.9 ng/mL/24 h after mechanisms of PC apoptosis by paracrine loops involvtransient acidification. MFBcM added in various diluing TGF-b. (HEPATOLOGY 1996;23:571-581.) tions and for different times to PC monolayers induced progressive cell detachment from the plastic support and increase of lactate dehydrogenase (LDH) activity in Cell death, i.e., irreversible loss of vital cellular structhe medium. The reduction of mitochondrial dehydrogetures and functions, can occur principally in two fundanase activity in PC (XTT or WST-1 test) was an early sign mental ways: as necrosis and apoptosis, respectively. 1,2 of MFBcM-induced functional impairment of PC. Short-Necrosis or accidental cell death is a common feature term exposure of PC with MFBcM for 3 hours was suffiof a large array of liver diseases of various causes. 3,4 cient to induce the deleterious effects on PC, but neither It results in cell swelling and rupture, release of cell native (nonactivated) MFBcM nor conditioned medium contents, and extensive inflammation, which is a preof untransformed FSC (FSCcM), in which TGF-b was not requisite for consecutive fibrotic tissue reactions. Bedetectable, were able to impair function and viability of side necrosis, liver cell death occurs also as apoptosis, PC. Activated MFBcM increased strongly (up to 21-fold) the concentration of oligonucleosomal DNA fragments i.e., by a gene-directed program of cell death characterboth in the adherent and detached fraction of PC. Interized morphologically by nuclear and cytosolic condennucleosomal DNA fragments (DNA ladder) were demonsation of single parenchymal cells (PC) (shrinkage) folstrated by electrophoresis of extracted DNA on agarose lowed by nuclear and later by cytosolic fragmentation gels and by in situ end-labeling of DNA breaks (TUNEL leading to the formation of apoptotic bodies. 5-7 Parallel reaction) only in MFBcM-exposed PC. MFBcM-treated to these morphological events, the chromatin is hy-PC exhibited intense fluorescence after staining with drolyzed internucleosomally. Final apoptotic cell bodies DNA-binding dye Hoechst 33342 and an increased numare phagocytosed by adjacent cells without conspicuous ber of cells with fragmented nuclei. All these criteria inflammatory side reactions. 5 Histologically, apoptosis point to MFBcM-generated apoptosis of cultured PC, has been recognized in diseased liver for a long time, which were found to be very similar to those induced by human recombinant TGF-b 1 .
The exclusive role of since former histopathological terms like ''Councilman bodies,'' ''piecemeal necrosis,'' and ''shrinkage necrosis'' describe a phenomenon now known as apoptotic PC Abbreviations: PC, parenchymal cells; MFB, myofibroblasts; FSC, fat-stordeath. 3 Apoptosis has now been recognized in immunoing cells; TGF, transforming growth factor; LAP, latency-associated peptide; logic liver disorders and viral hepatitis, 8,9 primary bili-DMEM, Dulbecco's modified Eagle medium; FCS, fetal calf serum; MFBcM, ary cirrhosis, 10 and experimental alcohol-induced liver myofibroblast-conditioned medium; PBS, phosphate-buffered saline; LDH, lactate dehydrogenase.