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

Andersson U, Wang H, Palmblad K, Aveberger AC, Bloom O, Erlandsson-Harris H, Janson A, Kokkola R, Zhang M, Yang H, Tracey KJ - J. Exp. Med. (2000)

Bottom Line: Cytokine release after HMG-1 stimulation was delayed and biphasic compared with LPS stimulation.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Rheumatology Unit, Karolinska Hospital, 17176 Stockholm, Sweden. ulf@mbox313.swipnet.se

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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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.
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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.

Mentions: To characterize the cell type responding to HMG-1, and the kinetics of newly synthesized TNF in individual cells, we performed two-color immunofluorescent staining of PBMCs using TNF-specific and monocyte-specific (anti–calprotectin-1) antibodies (Fig. 1 C). As shown previously, addition of LPS alone to the PBMC cultures activated TNF synthesis in nearly 40% of monocytes within 1 h as judged by morphology and two-color staining (Fig. 1 D). Stimulation of PBMCs by HMG-1 also caused a significant increase in cell-associated TNF synthesis that was restricted to monocytes; TNF synthesis was not increased in lymphocytes after exposure to HMG-1. The kinetic pattern of HMG-1–induced TNF synthesis differed significantly compared with LPS-induced cells (Fig. 1 D, and Fig. 2). HMG-1–activated TNF synthesis occurred in a biphasic pattern, with an early peak at 3 h after addition of HMG-1, followed by another peak after 8–10 h. The percentage of TNF-producing cells during the early peak after HMG-1 stimulation was significantly smaller compared with LPS stimulation. HMG-1 stimulation activated a significantly higher number of TNF-producing monocytes during the later peak (Fig. 1 D), which was surprising because the release of HMG-1 itself is delayed after endotoxin administration 11. Thus, this late mediator of endotoxin lethality is also a monocyte-activating cytokine that stimulates a delayed release of TNF.


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

Andersson U, Wang H, Palmblad K, Aveberger AC, Bloom O, Erlandsson-Harris H, Janson A, Kokkola R, Zhang M, Yang H, Tracey KJ - J. Exp. Med. (2000)

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.
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Related In: Results  -  Collection

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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.
Mentions: To characterize the cell type responding to HMG-1, and the kinetics of newly synthesized TNF in individual cells, we performed two-color immunofluorescent staining of PBMCs using TNF-specific and monocyte-specific (anti–calprotectin-1) antibodies (Fig. 1 C). As shown previously, addition of LPS alone to the PBMC cultures activated TNF synthesis in nearly 40% of monocytes within 1 h as judged by morphology and two-color staining (Fig. 1 D). Stimulation of PBMCs by HMG-1 also caused a significant increase in cell-associated TNF synthesis that was restricted to monocytes; TNF synthesis was not increased in lymphocytes after exposure to HMG-1. The kinetic pattern of HMG-1–induced TNF synthesis differed significantly compared with LPS-induced cells (Fig. 1 D, and Fig. 2). HMG-1–activated TNF synthesis occurred in a biphasic pattern, with an early peak at 3 h after addition of HMG-1, followed by another peak after 8–10 h. The percentage of TNF-producing cells during the early peak after HMG-1 stimulation was significantly smaller compared with LPS stimulation. HMG-1 stimulation activated a significantly higher number of TNF-producing monocytes during the later peak (Fig. 1 D), which was surprising because the release of HMG-1 itself is delayed after endotoxin administration 11. Thus, this late mediator of endotoxin lethality is also a monocyte-activating cytokine that stimulates a delayed release of TNF.

Bottom Line: Cytokine release after HMG-1 stimulation was delayed and biphasic compared with LPS stimulation.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Rheumatology Unit, Karolinska Hospital, 17176 Stockholm, Sweden. ulf@mbox313.swipnet.se

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.

Show MeSH
Related in: MedlinePlus