ABSTRACT. It was recently reported that intravenous administration of phorbol 12-myristate 13-acetate (PMA) showed a therapeutic effect in myelocytic leukemia patients. However, we previously observed that, in serum-free conditions, polymorphonuclear leucocytes (PMNs) were killed rapidly by exposure to PMA, suggesting the possibility of serious side effects. In this study, we found that PMA-induced necrosis of PMNs was prevented by serum, suggesting the existence of a "necrosis-suppressing factor". Next we tried to identify the serum factor. The hemopexins we purified were found to suppress necrosis of PMNs in a dose-dependent fashion. Hemopexins alone could not suppress necrosis, however, as it required the coexistence of another macromolecule such as albumin. Albumin promoted the suppressive activity of hemopexins in a dose-dependent fashion. These results strongly suggest that serum hemopexins may rescue mature PMNs from necrosis in the PMA-administered leukemia patient as previously reported, resulting in avoidance of serious side effects.

ABSTRACT. Polymorphonuclear leucocytes (PMNs) circulating in mammalian peripheral blood are terminally differentiated cells, and once isolated in serum-free medium, they undergo apoptosis within 1 or 2 days. In this study, we studied the effects of phorbol myristate acetate (PMA)on the viability of porcine PMNs in vitro. PMAis known to suppress apoptosis in many cell types. PMAbut not dioctanoyl glycerol (DOG)induced morphological degeneration and cell death within 3 to 5 hours as assessed by light microscopy observation and the MTTviability assay. This occurred despite the fact that DNAfragmentation associated with "spontaneous apoptosis" was not observed. Morphological degeneration and death were not due to the oxidative damage from superoxides or its metabolites produced by polymorphonuclear leucocytes, because PMAand DOGsimilarly stimulated superoxide production. Several other inactive phorbol derivatives tested did not cause cell death, suggesting that the toxicity of PMAdid not result from non-specific effect of the reagent.Polymorphonuclear leucocytes (PMNs) from mammalian peripheral blood are terminally differentiated cells that spontaneously undergo apoptosis in serum-free culture.It is widely accepted that protein kinase C plays a key role in regulation of apoptosis, cell survival, and differentiation (1). Phorbol esters, which selectively stimulate protein kinase C (PKC), block DNAfragmentation and cell death in thymocytes exposed to Ca2+ ionophore or glucocorticoid hormone (2). In U937 cells, DNA fragmentation induced by C2-ceramide is prevented by phorbol 12-myristate 13-acetate (PMA) (3). With respect to terminal neutrophil differentiation leading to apoptosis, modulation of PKCactivity also may be important. For example, during HL-60 cell differentiation into neutrophils, PKCa is specifically down regulated (4). In addition, treatment of human neutrophils with tumor necrosis factor a (TNF a) results in an increase in concentration of ceramide and its metabolite, shingosine, and it induces apoptosis. This suggests that neutrophil apoptosis in response to TNFa maybe related to inhibition of PKC activity (5). In this study, we examined the effects of PKCactivators on viability of porcine peripheral blood PMNssuspended in Ca2+, Mg2+free phosphate buffer under conditions of serum deprivation. Here we report that PMA, but not dioctanoyl glycerol (DOG), induced degenerative changes in cell morphology and cell death within 3 to 5 hours without DNAfragmentation. MATERIALS AND METHODS Materials4-/3-phorbol 12-myristate 13-acetate (PMA), 1 ,2-dioctanoylsw-glycerol (DOG), 4-/3-phorbol 12-myristate, 4-/3-phorbol 13-acetate, staurosporine, MTT, sodium-N-lauroylsarcosinate, proteinase K, RNaseA and cytochrome c were puchased from Sigma Chemical Co., USA. Phorbol derivatives including PMA and DOG were dissolved in DMSO(500 ptg/ml) and ethanol (10 mM), respectively. Methylcellulose 25cP and agarose were from Wako Pure Chemical Co., Japan. Ficoll-400 was from Pharmacia Biotech, Japan. The 100 bp DNAladder marker wa...

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ABSTRACT. Circulating and nonadherent polymorphonuclear leukocytes (PMNs) become activated to attain adhesive state in an integrin-dependent manner by various stimuli, and perform a variety of microbicidal functions such as phagocytosis and superoxide production. We found that, in the absence of serum, a physiological concentration of hemopexin has a strong inhibitory action on Mg 2+ -dependent adhesion of PMA-activated PMNs to fibrinogen-and serum-coated surfaces. Under these conditions, Ca 2+ had no effect on Mg 2+ -dependent adhesion or the adhesion-inhibitory activity of hemopexin. In contrast, PMNs suspended in serum containing sufficient amounts of hemopexin to inhibit adhesion showed marked adherence, which was inhibited by EGTA. Next, we prepared a small-molecule fraction of serum by ultrafiltration followed by boiling. PMA-activated PMNs was found to adhere in the presence of both hemopexin and the small-molecule fraction, and the adhesion was enhanced by exogenous Ca 2+ . EGTA abolished the effect of the small molecule fraction. The data suggest that serum contains adhesion-promoting factor(s) which allows PMNs to adhere despite the presence of hemopexin and that Ca 2+ is required for adhesion-promoting activity. Further study of hemopexin may provide clues for new therapeutic strategies aimed at interfering with PMN adhesion to control inflammation and tissue injury.

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