Role of HMGB1 in apoptosis-mediated sepsis lethality

Abstract

Severe sepsis, a lethal syndrome after infection or injury, is the third leading cause of mortality in the United States. The pathogenesis of severe sepsis is characterized by organ damage and accumulation of apoptotic lymphocytes in the spleen, thymus, and other organs. To examine the potential causal relationships of apoptosis to organ damage, we administered Z-VAD-FMK, a broad-spectrum caspase inhibitor, to mice with sepsis. We found that Z-VAD-FMK-treated septic mice had decreased levels of high mobility group box 1 (HMGB1), a critical cytokine mediator of organ damage in severe sepsis, and suppressed apoptosis in the spleen and thymus. In vitro, apoptotic cells activate macrophages to release HMGB1. Monoclonal antibodies against HMGB1 conferred protection against organ damage but did not prevent the accumulation of apoptotic cells in the spleen. Thus, our data indicate that HMGB1 production is downstream of apoptosis on the final common pathway to organ damage in severe sepsis.

Figure 1.

Caspase inhibitor Z-VAD-FMK reduces LPS-induced cytokine release, HMGB1 translocation, and NF-κB activation in RAW 264.7 cells and reduces sepsis-induced serum cytokine levels in mice. (A) Mouse macrophage-like RAW 264.7 cells were stimulated with 200 ng/ml LPS in the presence of Z-VAD-FMK or control peptide (Z-FA-FMK) at doses indicated for 16 h. Levels of HMGB1, TNF, and IL-6 in conditioned media were measured, and data are expressed as percentages of stimulation by LPS alone. *, P < 0.05 versus LPS + control group; n = 7. (B) RAW 264.7 cells were stimulated with 100 ng/ml LPS in the presence of 1 or 10 μM Z-VAD-FMK or control peptide for 6 h at 37°C. Nuclear extracts were prepared, and EMSA was performed using biotin-labeled nucleotides to measure NF-κB content. *, P < 0.05 versus LPS alone; n = 5–7 experiments. (C) BALB/c mice underwent CLP and received either Z-VAD-FMK or control peptide (0.5 mg/mouse) injected intraperitoneally at 90 min and 12 h after CLP. Mice were killed 24 h after CLP surgery. Serum levels of HMGB1, IL-6, KC, and MIP-2 were measured. *, P < 0.05 versus CLP group; n = 7–11 mice per group. Error bars represent SEM. (D) RAW 264.7 cells were incubated with 100 ng/ml LPS alone or in the presence of 1 μm Z-VAD-FMK or control peptide for 16 h. Cells were then incubated with polyclonal anti-HMGB1 antibodies followed by FITC-labeled anti–rabbit antibodies and viewed by fluorescent confocal microscopy. Note that LPS caused the export of HMGB1 into the cytosol, whereas Z-VAD-FMK (but not control peptide) prevented its cytoplasmic translocation and preserved HMGB1 in the nucleus. Data are representative of four separate experiments.

Figure 2.

Necrotic and apoptotic cells induce HMGB1, TNF, and MIP-2 release in RAW 264.7 cells; Z-VAD-FMK inhibits apoptotic cell-induced HMGB1 release. (A) Normal, apoptotic, or necrotic cells were added to RAW 264.7 cells at the amounts indicated for 16 h at 37°C. HMGB1, TNF, and MIP-2 released in conditioned media were measured. *, P < 0.05 versus normal control group; n = 6–8 independent experiments. (B) Apoptotic cells were added to RAW 264.7 cells at the amounts indicated in the presence of 10 μM Z-VAD-FMK or control peptide for 16 h at 37°C, and the HMGB1 released was measured by Western blotting (9, 10). *, P < 0.05 versus apoptotic cells alone; n = 6–8 independent experiments. Error bars represent SEM.

Figure 3.

Treatment with anti-HMGB1 mAb increases survival and reduces serum levels of cytokines and enzymes in sepsis induced by CLP. (A) BALB/c mice were subjected to CLP surgery (n = 10–22 mice per group). At 24 h after CLP surgery, mice received a single dose of anti-HMGB1 mAb at 0.1, 1, or 10 μg/mouse or control IgG injected intraperitoneally. No significant difference was observed in mice treated with different amounts of IgG (0.1, 1, or 10 μg/mouse; not depicted). Animal survival was monitored for 2 wk. *, P < 0.01 versus the IgG-treated group. (B and C) BALB/c mice had CLP surgery and received anti-HMGB1 mAb or nonimmune IgG administered intraperitoneally at 10 μg per mouse 24 h after CLP and were killed 40 h after surgery. (B) Serum levels of LDH, BUN, alkaline phosphatase (Alk Phos), SGPT, and SGOT were measured using commercially obtained assay kits. (C) Serum levels of IL-6, KC, MIP-1α, and IL-10 were measured by ELISAs, and HMGB1 was measured. Data are means ± SEM (error bars) of 5–13 animals in each group. *, P < 0.05 versus CLP group.

Figure 4.

Anti-HMGB1 mAb reduces serum levels of proinflammatory cytokines and organ damage without altering apoptosis in the spleen of septic mice. BALB/c mice were subjected to CLP surgery and received anti-HMGB1 mAb or nonimmune IgG administered intraperitoneally at 10 μg/mouse 24 h after CLP. Mice were killed 40 h after CLP surgery. In some experiments, between 30 and 72 h after CLP, any mice that looked very ill were killed (another mouse was killed from the control group at the same time). (A) Tissue sections of spleens (top) and livers (bottom) of normal or septic mice were prepared by using the standard formalin-fixed, paraffin-embedded procedure and were mounted on glass slides, and TUNEL assay, caspase 3 (top), and Annexin VI (top) stainings were performed. Arrows indicate the positive-staining cells. (B) Tissues were stained with hematoxylin and eosin. (top left) Normal lung shows thin alveolar septal wall and normal cellularity. (top middle) Lung from a septic mouse showing alveolar septal wall thickening, increase in cellular infiltrates, alveolar congestion, hemorrhage, and edema (arrow). (top right) Lung from a septic mouse treated with anti-HMGB1 mAb showing near normal alveoli with thin septum. (middle left) Normal liver showing a central vein (arrow) surrounded by hepatocytes and sinusoids. (middle) Liver from a septic mouse showing a congested central vein (arrow) and necrotic lesions (arrows) as revealed by the loss of cells and the structure of hepatic acinus. (middle right) Liver from a septic mouse treated with anti-HMGB1 mAb showing a central vein (arrow) and surrounding near normal hepatocytes. (bottom left) Normal kidney showing cortex with the glomerulus (G). (bottom middle) Kidney from a septic mouse showing the cortex with swelling tubule epithelia and congestion. (bottom right) Kidney from a septic mouse treated with anti-HMGB1 mAb showing swelling tubule epithelia and congestion. Data are representative of four to eight animals per group.

Figure 5.

Model of the protective effects of anti-HMGB1 treatment in sepsis lethality. The excessive release of HMGB1 is toxic, and it causes organ damage and death. Sepsis can induce cell apoptosis. Apoptotic cells phagocytosed by macrophages release HMGB1. Caspase inhibitor Z-VAD-FMK inhibits both cell apoptosis and HMGB1 release directly and, thus, improves sepsis survival. Anti-HMGB1 mAbs improve sepsis survival by neutralizing HMGB1 toxicity without altering sepsis-induced apoptosis in the spleen.