Human hemoglobin shares bioactivities ascribed to human tumor necrosis factor-alpha

Hemoglobin mediated cytotoxicity and apoptosis has been evaluated in Tumor necrosis factor-alpha (TNF-alpha) sensitive cell line, U937 and compared with TNF-alpha. Both species of hemoglobin, Hemoglobin A2 and Hemoglobin A0 induced apoptosis and cytotoxicity in U937 cell as measured by flow cytometry and 3-(4,5-dihydro-6-(4-(3,4-dimethoxybenzooyl)-1-piperazinyl)-2(1H)-quinoline (MTT) assay respectively. Different concentration of Hemoglobin A0 (4 ng/mL to 4000 ng/mL) induced apoptosis ranging from 9% to 16% in U937 cells. 4000 ng/mL hemoglobin A0 showed maximal apoptotic cells. TNF-alpha showed 87% apoptotic U937 cells at concentration of 1 pg/mL. HbA0 displayed cytotoxicity in U937 cell line at higher concentration in comparison to TNF-alpha. 4000 ng/mL of hemoglobin A0 showed optimal cytotoxic response in U937 cells. A dose response curve was also observed with varying doses of hemoglobin A0. U937 cells pretreated with serum activated LPS for 1 hr and incubated with different concentration of hemoglobin or human TNF-alpha for 24 h reduced the cytotoxic effect on U937. Dexamethasone treatment of U937 cells helped in protecting the HbA0 and HbA2 mediated cytotoxicity and anti-TNF-alpha antibody neutralized the hemoglobin mediated apoptosis and cytotoxicity. It is therefore apparent that human hemoglobin shares some of the bioactivities previously ascribed to TNF-alpha. Sharing of bioactivities of TNF-alpha by hemoglobin is interesting and suggests that cell free hemoglobin can mimic TNF-alpha functionally.