The Fas system has been extensively investigated as a model of apoptosis and the caspase cascade has been shown to be a characteristic mechanism of signaling of apoptosis. We have identi®ed and puri®ed a kinase that was activated after the stimulation of Fas on human thymoma-derived HPB ± ALL cells. Partial amino acid sequencing of the puri®ed kinase revealed it to be MST/ Krs, member of the yeast STE20 family of protein kinases. MST/Krs was activated by proteolytic cleavage and proteolytic activation was blocked by the caspase inhibitor, Z-VAD-FK. A mutant MST with Asp?Asn replacement at a putative caspase cleavage site was resistant to either the proteolytic cleavage or the activation of the kinase activity. These ®ndings suggest that proteolytic activation is one activation mechanism of MST and plays a role in apoptosis.

M2 macrophages have been subdivided into subtypes such as IL-4-induced M2a and IL-10-induced M2c in vitro. Although it was reported that IL-10 stimulation leads to an increase in IL-4Rα, the effect of IL-4 and IL-10 in combination with macrophage subtype differentiation remains unclear. Thus, we sought to clarify whether IL-10 enhanced the M2 phenotype induced by IL-4. In this study, we showed that IL-10 enhanced IL-4Rα expression in M-CSF-induced bone marrow-derived macrophages (BMDMs). Global gene expression analysis of M2 macrophages induced by IL-4, IL-10 or IL-4 + IL-10 showed that IL-10 enhanced gene expression of M2a markers induced by IL-4 in M-CSF-induced BMDMs. Moreover, IL-4 and IL-10 synergistically induced CCL24 (Eotaxin-2) production. Enhanced CCL24 expression was also observed in GM-CSF-induced BMDMs and zymosan-elicited, thioglycolate-elicited and naive peritoneal macrophages. CCL24 is a CCR3 agonist and an eosinophil chemoattractant. In vitro, IL-4 + IL-10-stimulated macrophages produced a large amount of CCL24 and increased eosinophil migration, which was inhibited by anti-CCL24 antibody. We also showed that IL-4 + IL-10-stimulated (but not IL-4 or IL-10 alone) macrophages transferred into the peritoneum of C57BL/6J mice increased eosinophil infiltration into the peritoneal cavity. These results demonstrate that IL-4 + IL-10-simulated macrophages have enhanced M2a macrophage-related gene expression, CCL24 production and eosinophil infiltration-inducing activity, thereby suggesting their contribution to eosinophil-related diseases.

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We purified a protein of 32 kDa from human thymoma HPB-ALL cells that was co-purified with a catalytic fragment of MST (mammalian STE-20-like), a kinase of the STE20 family, which is proteolytically activated by caspase in apoptosis (

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AIP (apoptosis-inducing protein) is a protein purified and cloned from Chub mackerel infected with the larval nematode, Anisakis simplex, which induces apoptosis in various mammalian cells including human tumor cell lines. AIP has shown structural and functional homology to Lamino acid oxidase (LAO) which oxidizes several L-amino acids including L-lysine and AIP-induced apoptosis has been suggested to be mediated by H 2 O 2 generated by LAO activity of AIP. In this study, we confirmed that recombinant AIP generated enough H 2 O 2 in culture medium to induce rapid apoptosis in cells and this apoptosis was clearly inhibited by co-cultivation with antioxidants such as catalase and Nacetyl-cysteine. Surprisingly, however, we found that AIP still could induce H 2 O 2 -independent apoptosis more slowly than H 2 O 2 -dependent one in HL-60 cells even in the presence of antioxidants. In addition, the HL-60-derived cell line HP100-1, which is a H 2 O 2 -resistant variant, underwent apoptosis on treatment with AIP with a similar delayed time course. The latter apoptosis was completely blocked by addition of L-lysine to the culture medium, which is the best substrate of AIP as LAO, indicating that decreased concentration of L-lysine in the culture medium by AIPtreatment induced apoptosis. We also showed that the both apoptosis by AIP were associated with the release of cytochrome c from mitochondria and activation of caspase-9, and overexpressed Bcl-2 could inhibit both of the AIPinduced apoptosis. These results indicate that AIP induces apoptosis in cells by two distinct mechanisms; one rapid and mediated by H 2 O 2 , the other delayed and mediated by deprivation of L-lysine, both of which utilize caspase-9/ cytochrome c system. Cell Death and Differentiation (2001) 8, 298 ± 307.

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