Reduction Of Histone H1 Cytotoxicity By Activated Protein C and Its Exosite Variants

In vivo and in vitro data in murine and baboon sepsis models have shown pathogenic effects of extracellular histones H3 and H4 in the circulation. The plasma serine protease, activated protein C (APC), is protective in these models by proteolysis of histones H3 and H4, and APC remarkably reduces mortality in these models. Histones H3 and H4 are also pathogenic in lung, liver, and kidney injury models. The function of extracellular histone H1, the linker histone between nucleosomes, has not been well investigated. Here we test the hypothesis that H1 can exert cytotoxic activity on lung epithelial cells and endothelial cells, and that H1 can affect endothelial cell barrier. Survival of lung epithelial (NCI H460) and endothelial (EA.hy926) cells was measured with an LDH (lactate dehydrogenase) release cytotoxicity assay in which LDH release follows loss of cell membrane integrity. Histone H1 (calf thymus-derived lysine-rich histone fraction) exhibited significant cytotoxicity on both lung epithelial and endothelial cells. Compared to other histone preparations (e.g., recombinant H2A, H2B, H3, and H4), H1 showed similar cytotoxicity on endothelial cells, whereas, on epithelial cells, H1 was more cytotoxic than other histones. Pre-incubation of all histones with plasma-derived human APC blunted their cytotoxic effects. Immunoblot analysis demonstrated site-specific, limited proteolysis of H1 and of other histones by APC, suggesting that APC protected the cells by cleaving H1 and other histones. When TransEndothelial Resistance (TER) measurements were performed on endothelial cells with the iCelligence system, the results showed that H1 histones exerted complex effects on cells, including a significant reduction in transcellular impedance observed at 10-20 hr post-treatment, implying disruption of the endothelium by H1. Studies showed that APC protected endothelial cells from this H1-induced decrease in TER. For example, the normalized cell index value for endothelial cells in TER assays was reduced by 10%, 16%, and 30% following treatment with 100, 200, and 400 µg/mL histone H1, respectively, and APC potently blunted this H1-induced reduction of the normalized cell index in TER assays. These experiments show that histone H1 has potent cytotoxic effects on endothelial and epithelial cells, as previously observed by others for histones H3 and H4, and that APC can prevent these effects. Future work should identify cell surface receptors and signaling mechanisms for H1 cytotoxic effects that may involve toll-like receptors. Our findings also suggest that histone H1 could be targeted for therapeutic purposes. Identification of the surface residues of APC that interact with H1 or other histones may help provide APC mutants defective in histone cleavage that could be used for in vivo proof of concept studies. Exosites around the active site of APC can directly interact with its substrates and influence APC activity by determining APC’s affinity for substrates. A recombinant APC variant with Lys191-193 mutated to Ala (3K3A-APC) appeared to have enhanced cytoprotective effects in H1-mediated cytotoxicity assays whereas a variant of APC with residues Glu330 and Glu333 mutated to Ala (E330A/E333A-APC) showed reduced protection against H1-mediated cytotoxicity. Immunoblot analysis of reaction mixtures showed reduced cleavage of H1 by the E330A/E333A-APC mutant compared to normal APC, suggesting that residues Glu330 and Glu333 contribute to affinity for highly positively charged histones like H1. In summary, data show that histone H1 exerts cytotoxic effects on endothelial and epithelial cells and that a negatively charged exosite on APC, which includes Glu330 and Glu333, contributes to APC’s ability to proteolyze H1 and reduce its cytotoxicity.