Phagocytic Uptake of Oxidized Heme Polymer Is Highly Cytotoxic to Macrophages

Apoptosis in macrophages is responsible for immune-depression and pathological effects during malaria. Phagocytosis of PRBC causes induction of apoptosis in macrophages through release of cytosolic factors from infected cells. Heme polymer or beta-hematin causes dose-dependent death of macrophages with LC50 of 132 mu g/ml and 182 mu g/ml respectively. The toxicity of hemin or heme polymer was amplified several folds in the presence of non-toxic concentration of methemoglobin. beta-hematin uptake in macrophage through phagocytosis is crucial for enhanced toxicological effects in the presence of methemoglobin. Higher accumulation of beta-hematin is observed in macrophages treated with beta-hematin along with methemoglobin. Light and scanning electron microscopic observations further confirm accumulation of beta-hematin with cellular toxicity. Toxicological potentiation of pro-oxidant molecules toward macrophages depends on generation of H2O2 and independent to release of free iron from pro-oxidant molecules. Methemoglobin oxidizes beta-hematin to form oxidized beta-hematin (beta H*) through single electron transfer mechanism. Pre-treatment of reaction mixture with spin-trap Phenyl-N-t-butyl-nitrone dose-dependently reverses the beta-hematin toxicity, indicates crucial role of beta H* generation with the toxicological potentiation. Acridine orange/ethidium bromide staining and DNA fragmentation analysis indicate that macrophage follows an oxidative stress dependent apoptotic pathway to cause death. In summary, current work highlights mutual co-operation between methemoglobin and different pro-oxidant molecules to enhance toxicity towards macrophages. Hence, methemoglobin peroxidase activity can be probed for subduing cellular toxicity of pro-oxidant molecules and it may in-turn make up for host immune response against the malaria parasite.