Hemopexins Suppress Phorbol Ester-induced Necrosis of Polymorphonuclear Leucocytes.

Abstract

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.

An alternative view of the proposed alternative activities of hemopexin

“…42 The human hemopexin used in these studies exhibited the expected apparent molecular weight and banding pattern (see discussion above) on analysis by SDS-PAGE and contained only a trace of lower molecular weight components that cross-reacted with polyclonal anti-hemopexin antibodies. Both hemopexin and the hemopexin-heme complex were found to suppress necrosis, leading the authors to discount their original premise, 42 which was that hemopexin protects cells by sequestering free heme released into the extracellular milieu by oxidative breakdown, thereby preventing self-damage of PMA-exposed PMNs caused by heme-aggravated oxidative cytotoxicity. As a result, the mechanism by which hemopexin suppresses necrosis in this system remained undefined.…”

“…42 Three species of purified hemopexins (bovine, porcine, and human) substantially suppressed the necrosis of PMA-activated PMNs but only if other macromolecules, presumably required for regulation of osmotic pressure under the conditions required for the culture, were present. 42 The human hemopexin used in these studies exhibited the expected apparent molecular weight and banding pattern (see discussion above) on analysis by SDS-PAGE and contained only a trace of lower molecular weight components that cross-reacted with polyclonal anti-hemopexin antibodies.…”

“…42 Three species of purified hemopexins (bovine, porcine, and human) substantially suppressed the necrosis of PMA-activated PMNs but only if other macromolecules, presumably required for regulation of osmotic pressure under the conditions required for the culture, were present. 42 The human hemopexin used in these studies exhibited the expected apparent molecular weight and banding pattern (see discussion above) on analysis by SDS-PAGE and contained only a trace of lower molecular weight components that cross-reacted with polyclonal anti-hemopexin antibodies. Both hemopexin and the hemopexin-heme complex were found to suppress necrosis, leading the authors to discount their original premise, 42 which was that hemopexin protects cells by sequestering free heme released into the extracellular milieu by oxidative breakdown, thereby preventing self-damage of PMA-exposed PMNs caused by heme-aggravated oxidative cytotoxicity.…”

“…The low pH generally used to elute hemopexin from heme or antibody affinity resins (typically pH 2.0-2.5 14,25,[42][43][44] ) is sufficient to unfold the protein. 30 Although the ability of human hemopexin to bind heme following exposure to low pH largely recovers within several hours after return to neutrality, 45 more than 48 h can be required for the protein to elute similarly to native hemopexin during hydrophobic chromatography.…”

“…However, a receptor for the hemopexin-heme complex on the cell surface of PMNs had previously been described by Okazaki et al 117 Their findings suggest that following receptor mediated uptake of hemopexin-heme by PMNs, hemopexin is recycled to the medium. Thus, the original mechanism proposed by Suzuki et al 42 would accommodate this result because hemopexin may have become available from the hemopexin-heme complex to which the cells were initially exposed. As a result, the necrosis-suppressing activity of hemopexin can be most simply explained as another consequence of the hemesequestering antioxidant capacity of hemopexin.…”