High mobility group 1 protein (HMG-1) stimulates proinflammatory cytokine synthesis in human monocytes - PubMed (original) (raw)

High mobility group 1 protein (HMG-1) stimulates proinflammatory cytokine synthesis in human monocytes

U Andersson et al. J Exp Med. 2000.

Abstract

Lipopolysaccharide (LPS) is lethal to animals because it activates cytokine release, causing septic shock and tissue injury. Early proinflammatory cytokines (e.g., tumor necrosis factor [TNF] and interleukin [IL]-1) released within the first few hours of endotoxemia stimulate mediator cascades that persist for days and can lead to death. High mobility group 1 protein (HMG-1), a ubiquitous DNA-binding protein, was recently identified as a "late" mediator of endotoxin lethality. Anti-HMG-1 antibodies neutralized the delayed increase in serum HMG-1, and protected against endotoxin lethality, even when passive immunization was delayed until after the early cytokine response. Here we examined whether HMG-1 might stimulate cytokine synthesis in human peripheral blood mononuclear cell cultures. Addition of purified recombinant HMG-1 to human monocyte cultures significantly stimulated the release of TNF, IL-1alpha, IL-1beta, IL-1RA, IL-6, IL-8, macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta; but not IL-10 or IL-12. HMG-1 concentrations that activated monocytes were within the pathological range previously observed in endotoxemic animals, and in serum obtained from septic patients. HMG-1 failed to stimulate cytokine release in lymphocytes, indicating that cellular stimulation was specific. Cytokine release after HMG-1 stimulation was delayed and biphasic compared with LPS stimulation. Computer-assisted image analysis demonstrated that peak intensity of HMG-1-induced cellular TNF staining was comparable to that observed after maximal stimulation with LPS. Administration of HMG-1 to Balb/c mice significantly increased serum TNF levels in vivo. Together, these results indicate that, like other cytokine mediators of endotoxin lethality (e.g., TNF and IL-1), extracellular HMG-1 is a regulator of monocyte proinflammatory cytokine synthesis.

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Figures

Figure 1

Figure 1

(A) HMG-1 stimulates release of TNF from cultured human PBMCs. Ficoll-separated human PBMCs were stimulated with purified rHMG-1 (0.001–1 μg/ml) in the presence of polymyxin B (10 μg/ml), and the supernatant was assayed for TNF by ELISA 4 h after stimulation. Data represent mean ± SEM of two separate experiments (in triplicate). (B) HMG-1 induces increased expression of TNF mRNA in human PBMCs. Human PBMCs were cultured in OPTI-MEM I medium with rHMG-1 (1 μg/ml) and polymyxin B (10 μg/ml). Cells were harvested at the indicated time points and assayed for TNF mRNA and GAPDH mRNA levels in controls by RNase Protection assay. Data are representative of two independent experiments. (C) Phenotypic characterization of TNF-producing PBMCs stimulated with HMG-1. Typical localization of intracellular TNF accumulation demonstrated by indirect immunofluorescence (A, Oregon Green) 8 h after HMG-1 stimulation. The same cells were stained with rhodamine for a monocytic marker (B, calprotectin 1). (D) Intracellular TNF expression after HMG-1 stimulation of PBMCs. Human PBMCs were cultured alone or with HMG-1 (1 μg/ml) in the presence of polymyxin B or LPS (100 ng/ml). The cells were harvested at the indicated time points, fixed, and stained for TNF using indirect immunofluorescence. Only monocytes contributed to the TNF formation as judged by morphology and two-color staining. The results are expressed as frequency of TNF-producing cells in the monocyte population. Data represent mean ± SEM of three separate experiments (in duplicate). *P < 0.05 vs. LPS stimulation.

Figure 1

Figure 1

(A) HMG-1 stimulates release of TNF from cultured human PBMCs. Ficoll-separated human PBMCs were stimulated with purified rHMG-1 (0.001–1 μg/ml) in the presence of polymyxin B (10 μg/ml), and the supernatant was assayed for TNF by ELISA 4 h after stimulation. Data represent mean ± SEM of two separate experiments (in triplicate). (B) HMG-1 induces increased expression of TNF mRNA in human PBMCs. Human PBMCs were cultured in OPTI-MEM I medium with rHMG-1 (1 μg/ml) and polymyxin B (10 μg/ml). Cells were harvested at the indicated time points and assayed for TNF mRNA and GAPDH mRNA levels in controls by RNase Protection assay. Data are representative of two independent experiments. (C) Phenotypic characterization of TNF-producing PBMCs stimulated with HMG-1. Typical localization of intracellular TNF accumulation demonstrated by indirect immunofluorescence (A, Oregon Green) 8 h after HMG-1 stimulation. The same cells were stained with rhodamine for a monocytic marker (B, calprotectin 1). (D) Intracellular TNF expression after HMG-1 stimulation of PBMCs. Human PBMCs were cultured alone or with HMG-1 (1 μg/ml) in the presence of polymyxin B or LPS (100 ng/ml). The cells were harvested at the indicated time points, fixed, and stained for TNF using indirect immunofluorescence. Only monocytes contributed to the TNF formation as judged by morphology and two-color staining. The results are expressed as frequency of TNF-producing cells in the monocyte population. Data represent mean ± SEM of three separate experiments (in duplicate). *P < 0.05 vs. LPS stimulation.

Figure 1

Figure 1

(A) HMG-1 stimulates release of TNF from cultured human PBMCs. Ficoll-separated human PBMCs were stimulated with purified rHMG-1 (0.001–1 μg/ml) in the presence of polymyxin B (10 μg/ml), and the supernatant was assayed for TNF by ELISA 4 h after stimulation. Data represent mean ± SEM of two separate experiments (in triplicate). (B) HMG-1 induces increased expression of TNF mRNA in human PBMCs. Human PBMCs were cultured in OPTI-MEM I medium with rHMG-1 (1 μg/ml) and polymyxin B (10 μg/ml). Cells were harvested at the indicated time points and assayed for TNF mRNA and GAPDH mRNA levels in controls by RNase Protection assay. Data are representative of two independent experiments. (C) Phenotypic characterization of TNF-producing PBMCs stimulated with HMG-1. Typical localization of intracellular TNF accumulation demonstrated by indirect immunofluorescence (A, Oregon Green) 8 h after HMG-1 stimulation. The same cells were stained with rhodamine for a monocytic marker (B, calprotectin 1). (D) Intracellular TNF expression after HMG-1 stimulation of PBMCs. Human PBMCs were cultured alone or with HMG-1 (1 μg/ml) in the presence of polymyxin B or LPS (100 ng/ml). The cells were harvested at the indicated time points, fixed, and stained for TNF using indirect immunofluorescence. Only monocytes contributed to the TNF formation as judged by morphology and two-color staining. The results are expressed as frequency of TNF-producing cells in the monocyte population. Data represent mean ± SEM of three separate experiments (in duplicate). *P < 0.05 vs. LPS stimulation.

Figure 1

Figure 1

(A) HMG-1 stimulates release of TNF from cultured human PBMCs. Ficoll-separated human PBMCs were stimulated with purified rHMG-1 (0.001–1 μg/ml) in the presence of polymyxin B (10 μg/ml), and the supernatant was assayed for TNF by ELISA 4 h after stimulation. Data represent mean ± SEM of two separate experiments (in triplicate). (B) HMG-1 induces increased expression of TNF mRNA in human PBMCs. Human PBMCs were cultured in OPTI-MEM I medium with rHMG-1 (1 μg/ml) and polymyxin B (10 μg/ml). Cells were harvested at the indicated time points and assayed for TNF mRNA and GAPDH mRNA levels in controls by RNase Protection assay. Data are representative of two independent experiments. (C) Phenotypic characterization of TNF-producing PBMCs stimulated with HMG-1. Typical localization of intracellular TNF accumulation demonstrated by indirect immunofluorescence (A, Oregon Green) 8 h after HMG-1 stimulation. The same cells were stained with rhodamine for a monocytic marker (B, calprotectin 1). (D) Intracellular TNF expression after HMG-1 stimulation of PBMCs. Human PBMCs were cultured alone or with HMG-1 (1 μg/ml) in the presence of polymyxin B or LPS (100 ng/ml). The cells were harvested at the indicated time points, fixed, and stained for TNF using indirect immunofluorescence. Only monocytes contributed to the TNF formation as judged by morphology and two-color staining. The results are expressed as frequency of TNF-producing cells in the monocyte population. Data represent mean ± SEM of three separate experiments (in duplicate). *P < 0.05 vs. LPS stimulation.

Figure 2

Figure 2

TNF expression in HMG-1–stimulated human PBMCs. Human PBMCs were cultured alone or with either rHMG-1 (1 μg/ml) or LPS (100 ng/ml) (1 or 8 h), then stained for intracellular TNF. TNF-producing monocytes are revealed with an intracellular, round brown dot, representing an accumulation of TNF in the Golgi organelle of producer cells. The number of TNF-expressing monocytes in the LPS-stimulated cultures was increased significantly within 1 h compared with HMG-1–stimulated or unstimulated cultures. In contrast, HMG-1 stimulation significantly increased TNF expression at 8 h. No TNF was detected in unstimulated cells. Note pronounced monocyte aggregation after 8 h of culture.

Figure 3

Figure 3

Administration of HMG-1 in vivo stimulates increased serum TNF in mice. Balb/c mice received an intraperitoneal injection of rHMG-1 in the doses indicated; blood was obtained after 6 h and serum was analyzed for TNF by ELISA (means ± SEM, n = 3).

Figure 4

Figure 4

HMG-1 stimulates the expression of cytokines and chemokines in human PBMCs. Human PBMCs were cultured with either rHMG-1 (1 μg/ml) plus polymyxin B (10 μg/ml) or LPS alone (100 ng/ml). Cells were harvested in longitudinal studies, fixed, and stained using indirect immunofluorescence. Monocytes were the only cells contributing to the synthesis of the studied mediators, confirmed by two-color staining. *P < 0.05 vs. LPS.

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