The Role of IFN-α and Nitric Oxide in the Release of HMGB1 by RAW 264.7 Cells Stimulated with Polyinosinic-Polycytidylic Acid or Lipopolysaccharide

Abstract

High mobility group protein 1 (HMGB1) is a nonhistone nuclear protein with a dual function. Inside the cell, HMGB1 binds to DNA and modulates a variety of processes, including transcription. Outside the cell, HMGB1 displays cytokine activity and can promote inflammation, serving as a mediator in models of shock and arthritis. In in vitro studies, proinflammatory molecules such as LPS, lipoteichoic acid, dsRNA, TNF-α, and IFN-γ can induce HMGB1 release from macrophages. To define further the release process, we investigated the role of the downstream mediators, NO and IFN-α, in the release of HMGB1 from RAW 264.7 macrophage cells stimulated with LPS or polyinosinic-polycytidylic acid (poly(I:C)). In these experiments, 1400W, an inhibitor of NO production by the inducible NO synthase, reduced HMGB1 release stimulated by LPS, but not poly(I:C), whereas neutralizing IFN-α prevented HMGB1 release induced by poly(I:C), but not LPS. The addition of an NO donor and rIFN-α to RAW 264.7 cells caused HMGB1 release. Furthermore, inhibition of JNK activation attenuated HMGB1 release induced by either LPS or poly(I:C). Analysis of bone marrow-derived macrophages stimulated by LPS or poly(I:C) showed patterns of HMGB1 release similar to those of RAW 264.7 cells. Together, these experiments indicate that, although both LPS and poly(I:C) induce HMGB1 release from RAW 264.7 cells and murine macrophages, the response is differentially dependent on NO and IFN-α.

The Relationship between Apoptosis and High-Mobility Group Protein 1 Release from Murine Macrophages Stimulated with Lipopolysaccharide or Polyinosinic-Polycytidylic Acid

“…In a previous study, we showed that NO and IFN-␣ differentially mediated the release of HMGB1 by LPS and poly(I:C) in a process that may depend on JNK activation (16). In these experiments, we showed that inhibiting iNOS or neutralizing IFN-␣ by an Ab reduced HMGB1 release.…”

“…In previous experiments, we showed that inhibiting JNK activation reduced extracellular release of HMGB1 induced by poly(I:C) or LPS (16). To evaluate the role of JNK in apoptosis induced by LPS or poly(I:C), we analyzed the effects of JNK inhibitor on caspase activities and LDH levels in RAW 264.7 cells stimulated with LPS or poly(I:C).…”

Bacterial Endotoxin Induces the Release of High Mobility Group Box 1 via the IFN-β Signaling Pathway

“…The level of HMGB1 was determined by Western blotting using anti-HMGB1 Abs, and quantified by densitometric scanning of the exposed x-ray film. Relative densitometric value of HMGB1 of each sample shown as arbitrary units in the figures was calculated by arbitrarily assigning that of control (a LPS (or Escherichia coli)-treated sample) as 1 (16).…”

Acteoside Improves Survival in Cecal Ligation an Puncture-Induced Septic Mice via Blocking of High Mobility Group Box 1 Release

“…B et al, 2004). Acteoside-induced HO-1 expression in Raw264.7 cells was significantly inhibited by Nrf2 siRNA, indicating the involvement of p38 MARK/Nrf2 signaling; however, others have produced compelling evidence for the lack of involvement of NFkB and p38 in HMGB1 release in LPS-treated macrophages, while emphasizing the role of Janus kinase 2/signal transducer and activator of transcription-1 and iNOS/ NO signaling in HMGB1 release (Jiang and Pisetsky, 2006;Oh et al, 2009). Thus, further study is needed to identify the pathways involved in the inhibition of HMGB1 downstream of HO-1.…”