Identification of CD163 as an antiinflammatory receptor for HMGB1-haptoglobin complexes
Huan Yang, Haichao Wang, Yaakov A. Levine, Manoj K. Gunasekaran, Yongjun Wang, Meghan Addorisio, Shu Zhu, Wei Li, Jianhua Li, Dominique P.V. de Kleijn, Peder S. Olofsson, H. Shaw Warren, Mingzhu He, Yousef Al-Abed, Jesse Roth, Daniel J. Antoine, Sangeeta S. Chavan, Ulf Andersson, Kevin J. Tracey
Huan Yang, Haichao Wang, Yaakov A. Levine, Manoj K. Gunasekaran, Yongjun Wang, Meghan Addorisio, Shu Zhu, Wei Li, Jianhua Li, Dominique P.V. de Kleijn, Peder S. Olofsson, H. Shaw Warren, Mingzhu He, Yousef Al-Abed, Jesse Roth, Daniel J. Antoine, Sangeeta S. Chavan, Ulf Andersson, Kevin J. Tracey
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Research Article Hepatology Immunology

Identification of CD163 as an antiinflammatory receptor for HMGB1-haptoglobin complexes

Abstract

Secreted by activated cells or passively released by damaged cells, extracellular HMGB1 is a prototypical damage-associated molecular pattern (DAMP) inflammatory mediator. During the course of developing extracorporeal approaches to treating injury and infection, we inadvertently discovered that haptoglobin, the acute phase protein that binds extracellular hemoglobin and targets cellular uptake through CD163, also binds HMGB1. Haptoglobin-HMGB1 complexes elicit the production of antiinflammatory enzymes (heme oxygenase-1) and cytokines (e.g., IL-10) in WT but not in CD163-deficient macrophages. Genetic disruption of haptoglobin or CD163 expression significantly enhances mortality rates in standardized models of intra-abdominal sepsis in mice. Administration of haptoglobin to WT and to haptoglobin gene-deficient animals confers significant protection. These findings reveal a mechanism for haptoglobin modulation of the inflammatory action of HMGB1, with significant implications for developing experimental strategies targeting HMGB1-dependent inflammatory diseases.

Authors

Huan Yang, Haichao Wang, Yaakov A. Levine, Manoj K. Gunasekaran, Yongjun Wang, Meghan Addorisio, Shu Zhu, Wei Li, Jianhua Li, Dominique P.V. de Kleijn, Peder S. Olofsson, H. Shaw Warren, Mingzhu He, Yousef Al-Abed, Jesse Roth, Daniel J. Antoine, Sangeeta S. Chavan, Ulf Andersson, Kevin J. Tracey

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Figure 5

HMGB1 and haptoglobin complexes signal through CD163 to induce HO-1 and IL-10 expression in macrophages.

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HMGB1 and haptoglobin complexes signal through CD163 to induce HO-1 and ...
(A) Surface plasmon resonance analysis of HMGB1 and haptoglobin (Hp) binding to CD163. Recombinant human CD163 was coated on the sensor chip; the mixture of HMGB1 and Hp (1:1 molar ratio) at concentrations of 32, 65, 125, or 250 nM flowed over the chip to measure the binding affinity to CD163 (Kd is approximately 130 nM). Data are representative of 3 experiments. (B) Hp enhances HMGB1-induced HO-1 expression and IL-10 release. Human monocytic THP-1 cells were treated with dexamethasone (2.5 × 10–7 M) for 2 days to induce CD163 expression and then stimulated with HMGB1 alone (5 μg/ml) or plus haptoglobin (15 μg/ml) at 37°C for 16 hours. After incubation, extracellular levels of IL-10 were measured by ELISA. Intracellular levels of HO-1 and β-actin were measured by Western blot. Data are expressed as folds of unstimulated group after normalization to β-actin (n =3 experiments). **P < 0.01, ***P < 0.001 vs. HMGB1 alone (t test). (C) Knockdown of CD163 impairs Hp and HMGB1-induced IL-10 and HO-1 expression. Primary human macrophages were transduced with specific shRNA lentiviral particles targeting CD163 or vector alone (control). At 72 hours after transduction, cells were stimulated with HMGB1 (1 μg/ml) with or without Hp (3 μg/ml) for 16 hours. The expression of CD163, HO-1, and β-actin in cell lysate was measured by Western blot. Left: representative blots. Right: Data for HO-1 expression are presented as folds of unstimulated group after normalization to β-actin. Supernatants were collected to measure IL-10 release (n = 3 experiments). ***P < 0.001 vs. control (t test).