Protective Mechanisms Of Inter-Alpha Inhibitor Protein On Extracellular Histone Toxicity

Sepsis is almost invariably associated with hemostatic abnormalities ranging from isolated thrombocytopenia to acute disseminated intravascular coagulation (DIC). Recent evidence indicates that extracellular histones are important mediators and therapeutic targets in sepsis, inflammation and thrombosis. Baboons challenged with E. coli release large amounts of histones into systemic blood circulation. Conversely, infusion of histones into mice and baboons directly induces an inflammatory response, fibrin and platelet deposition in the tissues, leading to organ failure and death. In vitro, histones cytotoxicity is significantly decreased in the presence of plasma or serum. Here, we demonstrate that Inter-alpha Inhibitor Protein (IAIP), an abundant plasma protein, effectively neutralizes the cytotoxic effects of histones and decreases platelet aggregation in vitro and in vivo. IAIP consists of three polypeptides: two heavy chains (HC) and one light chain called bikunin. Bikunin, which has anti-proteolytic activity, carries a chondroitin sulfate (CS) chain to which the heavy chains are covalently linked. The heavy chains can be transferred from IAIP to hyaluronan (HA) and become covalently linked to form HC-HA complex. The mechanism of histone inhibition by IAIP is unclear. Since histones are cationic molecules and can be electrostatically attracted to the negatively charged glycosaminoglycans, we hypothesize that the inhibitory activity of IAIP against histone toxicity could be at least in part mediated by HA and CS. The major aim of this study is to investigate the interaction of IAIP and its components (bikunin, CS, and HA) with histones and to validate the protective effect of IAIP against histone induced thrombocytopenia, prolonged bleeding time, and organ injury.

We first determined the effect of the different components of IAIP on histone-induced toxicity, by incubating HL60 cells with histones +/- IAIP, CS, high molecular weight HA (HMW-HA) or bikunin. Cell damage was measured by flow cytometry for propidium iodide (PI) staining. We also tested the effects of IAIP, CS, HMW-HA, and CS on histone-induced platelet aggregation. Interestingly, IAIP, HMW-HA and CS, but not bikunin prevented histone-induced toxicity and platelet aggregation in a concentration dependent manner. Next, we determined if HA or CS are partly responsible for the protective effect of IAIP by pre-incubating IAIP with hyaluronidase or chondoritinase. Hyaluronidase, but not chondoritinase, slightly decreased the protective effects of IAIP on histone-induced platelet aggregation and cytotoxicity, suggesting that HA has a partial role. In addition, using BIACORE 3000, we proved that both IAIP and HMW HA bind to recombinant histone H4 in a dose dependent manner.

To validate the protective effect against histone toxicity in vivo, we injected C57BL/6 mice with intravenous calf thymus histones (50 mg/kg) with/without IAIP (50 mg/kg) or HMW HA (90 mg/kg). Platelets were measured before and 30 minutes after histone challenge. As expected, mice injected with only histones developed severe thrombocytopenia (up to ∼ 90%) and prolonged bleeding time (> 10 minutes). Co-injecting mice with histones and IAIP or HMW HA significantly reduced histone-induced thrombocytopenia, with platelet consumption of 82% in the histone-only group versus 37% in the histone + IAIP group and 23% in the histone + HMW-HA group. Both treatments also reduced tail bleeding time from 10 minutes for histone only group to 2.4 minutes in histone + IAIP group and 2.2 minutes for histone + HMW-HA. In addition, mice co-injected with histones and IAIP or HMW HA had significantly reduced proinflammatory cytokine levels (IL-1β, IL-6, TNF-α, KC, MCP-1) and lung injury markers, such as decreased neutrophil infiltration, decreased fibrin deposition and microvascular thrombosis, as noted by immunofluorescence and electron microscopy.

In conclusion, our data demonstrate that IAIP and HMW-HA could protect against histone-induced cytotoxicity, platelet activation and microthrombosis, prevent thrombocytopenia and prolonged bleeding time, and protect against systemic inflammation and organ injury. It is likely that IAIP protective effects are at least partially mediated by the HA associated with this protein. Our data suggest that IAIP and HMW-HA could be developed as novel therapies for multiple organ failure in patients with sepsis and trauma.