The C-terminal acidic tail is responsible for the inhibitory effects of HMGB1 on efferocytosis

Abstract

HMGB1 was described originally as a nuclear protein involved in DNA binding and transcriptional regulation. However, HMGB1 also has an extracellular role as a potent mediator of inflammation and can diminish the uptake of apoptotic cells by phagocytes, a process called efferocytosis. To explore the mechanism responsible for the ability of HMGB1 to inhibit efferocytosis, we examined the role of the C-terminal acidic tail, a region of HMGB1 that has been shown to participate in specific intramolecular interactions. Deletion of the C-terminal tail abrogated the ability of HMGB1 to decrease murine macrophage ingestion of apoptotic neutrophils and to diminish phagocytosis-induced activation of Erk and Rac-1 in macrophages. We found that RAGE plays a major role in efferocytosis, and deletion of the C-terminal tail of HMGB1 prevented binding of HMGB1 to RAGE but not to other macrophage receptors involved in efferocytosis, such as the α(V)β(3) integrin. Whereas HMGB1 decreased ingestion of apoptotic neutrophils significantly by alveolar macrophages under in vivo conditions in the lungs of mice, this effect was lost when the C-terminal acidic tail was absent from HMGB1. These results demonstrate that the HMGB1 C-terminal tail is responsible for the inhibitory effects of HMGB1 on phagocytosis of apoptotic neutrophils under in vitro and in vivo conditions.

Figure 1.. The C-terminal acidic tail is required for HMGB1 to inhibit efferocytosis.

(A) Schematic diagram showing constructs that express 3× Flag-fused human HMGB1 and ΔC-HMGB1. The amino acid number at the end of each domain is shown. (B) Effects of full-length HMGB1 and ΔC-HMGB1 on phagocytosis of apoptotic neutrophils by peritoneal macrophages.. Phagocytosis assays were performed as described in Materials and Methods. Three additional independent experiments provided similar results; ***P<0.001.

Figure 2.. Absence of the C-terminal acidic tail abolishes the inhibitory effect of HMGB1 on efferocytosis-induced Erk phosphorylation and Rac-1 activation.

(A) Peritoneal macrophages were washed with serum-free medium and incubated in serum-free media for 1 h followed by exposure for 1 h to 1 μg/ml BSA (control), full-length HMGB1, or ΔC-HMGB1 in serum-free media. The macrophages were washed and incubated with RPMI-1640 medium with 1% FBS, with or without apoptotic neutrophils. After 15 min, the cells were washed, and cell extracts from the macrophages were prepared, Western blotting was performed to determine the levels of phosphorylated (p-Erk) and total Erk. A representative gel as well as mean ± sd of phosphorylated Erk/total Erk ratios for each condition using optical band intensity measurements from 3 independent experiments are shown. (B) Phagocytosis assays were performed for 15 min as described above. The macrophages were then washed 3 times and cell lysates prepared to determine Rac-1 activation. A representative gel as well as mean ± sd of activated/total Rac-1 ratios for each condition using optical band intensity measurements from 3 independent experiments are shown; *P < 0.05 versus control.

Figure 3.. The C terminus is required for binding of HMGB1 to RAGE but not to the α_Vβ₃ integrin.

HMGB1-Flag or ΔC-HMGB1-Flag (50 ng) was incubated with 100 ng soluble α_Vβ₃ (A) or soluble RAGE (sRAGE)-Fc chimeric protein (B). α_Vβ₃- or RAGE-bound HMGB1 or ΔC-HMGB1 was pulled down using agarose beads conjugated with anti-Flag antibody. The precipitated proteins were eluted and resolved by 12% SDS-PAGE, and Western blotting was performed using anti-β₃ antibody (A) or anti-RAGE antibody. (B) Blots were stripped and reprobed with anti-Flag antibody to demonstrate the amount of Flag-tagged HMGB1 or ΔC-HMGB1 that was pulled down by the beads. A second independent experiment provided similar findings. IP, Immunoprecipitation; IB, immunoblotting.

Figure 4.. RAGE plays a major role in modulating phagocytosis of apoptotic neutrophils by macrophages.

(A) Peritoneal macrophages from RAGE^+/+ and RAGE^−/− mice were pretreated with BSA or HMGB1 protein (1 μg/ml) for 1 h followed by incubation with apoptotic neutrophils for 90 min and the phagocytic index then determined. Data shown are representative of 2 independent experiments; ***P < 0.001; *P < 0.05. (B) RAGE^+/+ and RAGE^−/− macrophages were incubated with apoptotic neutrophils for 15 min and then washed. Western blotting was performed to determine the levels of phosphorylated and total Erk. A representative experiment is shown. A second independent experiment provided similar results.

Figure 5.. Removal of the C-terminal acidic tail abrogates the inhibitory effect of HMGB1 on phagocytosis in vivo.

Apoptotic neutrophils (10⁷) with HMGB1, ΔC-HMGB1, or mouse albumin (2 μg) were administered intratracheally in 50 μl PBS into anesthesized mice. BAL was performed with 1 ml cold PBS containing 5 mM EDTA 90 min later. Cytospin slides were prepared using 200 μl BALF, and phagocytosis of apoptotic neutrophils by alveolar macrophages was assessed. The phagocytosis index is represented as the percentage of alveolar macrophages containing at least 1 ingested neutrophil; n = 3 mice in each group; ***P < 0.001. A second independent experiment provided similar results.