A detailed kinetic analysis of three extranuclear end points of apoptosis, phosphatidylserine exposure, ␣-fodrin degradation, and plasma membrane blebbing, was performed and compared with nuclear fragmentation and the activation of the interleukin-1␤-converting enzyme (ICE)-like proteases in Jurkat T lymphocytes stimulated by anti-Fas monoclonal antibody (anti-Fas mAb) and in monocytic U937 cells stimulated by tumor necrosis factor (TNF) and cycloheximide. Phosphatidylserine exposure was quantitated by plasma clotting time, as well as annexin V-fluorescein isothiocyanate binding, and the ICE-like protease activity was examined by the cleavage of a specific fluorogenic peptide substrate AcAsp-Glu-Val-Asp-amino-4-methylcoumarin. VAD-chloromethylketone (VAD-cmk), an inhibitor of ICE-like proteases, effectively inhibited ICE-like activity in both cell types studied, whereas the calpain inhibitor calpeptin was ineffective. VAD-cmk also effectively inhibited all three extranuclear events, as well as nuclear fragmentation, in Jurkat cells stimulated by anti-Fas monoclonal antibody, indicating that ICE-like proteases play an important role in the regulation of this apoptotic system. Calpain inhibitors were ineffective in this system. TNF-induced extranuclear and nuclear changes in U937 cells were inhibited by calpeptin but were not as effectively inhibited by VAD-cmk as in Jurkat cells. This suggests that ICE-like enzymes predominate in anti-Fas monoclonal antibody-stimulated Jurkat cells, whereas proteases affected by calpain inhibitors as well as the ICE-like enzymes are involved in the signaling of apoptotic events in TNF-induced U937 cells. Importantly, the two apoptotic systems seem to be regulated by different proteases.The apoptotic process, beginning with receptor-mediated signaling, leading to cytoplasmic and nuclear changes and culminating in the phagocytosis of dying cells, defines an important biological continuum. Although morphological changes have been well characterized, it is necessary to investigate how extranuclear and nuclear events relate to each other temporally and how they relate to the earlier signaling events. Characteristic morphological changes include plasma membrane blebbing, chromatin condensation and fragmentation into high molecular weight (HMW) 1 (50 -300-kilobase pair) and oligonucleosomal-length (180-base pair) DNA fragments, and the

Heat shock protein 10 (Hsp10) and heat shock protein 60 (Hsp60) were originally described as essential mitochondrial proteins involved in protein folding. However, both proteins have also been shown to have a number of extracellular immunomodulatory activities. Here we show that purified recombinant human Hsp10 incubated with cells in vitro reduced lipopolysaccharide (LPS)-induced nuclear factor-B activation and secretion of several inflammatory mediators from RAW264.7 cells, murine macrophages, and human peripheral blood mononuclear cells. Induction of tolerance by contaminating LPS was formally excluded as being responsible for Hsp10 activity. Treatment of mice with Hsp10 before endotoxin challenge resulted in the reduction of serum tumor necrosis factor-␣ and RANTES (regulated upon activation, normal T cell expressed and secreted) levels and an elevation of serum interleukin-10 levels. Hsp10 treatment also delayed mortality in a murine graft-versus-host disease model, where gut-derived LPS contributes to pathology. We were unable to confirm previous reports that Hsp10 has tumor growth factor properties and suggest that Hsp10 exerts anti-inflammatory activity by inhibiting Toll-like receptor signaling possibly by interacting with extracellular Hsp60.

Steatosis is increasingly recognized as a cofactor influencing the progression of fibrosis in chronic hepatitis C; however, the mechanisms by which it contributes to liver injury remain uncertain. We studied 125 patients with chronic hepatitis C to assess the effect of steatosis on liver cell apoptosis and the expression of Bcl-2, Bcl-x L , Bax, and tumor necrosis factor alpha (TNF-␣) and the relationship between liver cell apoptosis and disease severity. A significant increase in liver cell apoptosis was seen in liver sections with increasing grade of steatosis (r ‫؍‬ 0.42; P < .0001). Hepatic steatosis and previous heavy alcohol consumption were the only two variables independently associated with the apoptotic index. Increasing steatosis was associated with decreased Bcl-2 mRNA levels and an increase in the proapoptotic Bax/Bcl-2 ratio (r ‫؍‬ ؊0.32, P ‫؍‬ .007; and r ‫؍‬ 0.27, P ‫؍‬ .02, respectively). In the absence of steatosis, increased liver cell apoptosis was not associated with stellate cell activation or fibrosis (r ‫؍‬ 0.26, P ‫؍‬ .11; r ‫؍‬ 0.06, P ‫؍‬ .71, respectively). In contrast, in the presence of steatosis, increasing apoptosis was associated with activation of stellate cells and increased stage of fibrosis (r ‫؍‬ 0.35, P ‫؍‬ .047; r ‫؍‬ 0.33, P ‫؍‬ .03, respectively), supporting the premise that the steatotic liver is more vulnerable to liver injury. In patients with hepatitis C virus genotype 3, there was a significant correlation between TNF-␣ mRNA levels and active caspase-3 (r ‫؍‬ 0.54, P ‫؍‬ .007). In conclusion, these observations suggest a mechanism whereby steatosis contributes to the progression of liver injury in chronic hepatitis C. Further investigation will be required to determine the molecular pathways responsible for the proapoptotic effect of steatosis and whether this increase in apoptosis contributes directly to fibrogenesis.

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