J Korean Med Sci.  2008 Apr;23(2):288-295. 10.3346/jkms.2008.23.2.288.

Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) Augments Acute Lung Injury via Its Neutrophil Priming Effects

• Choi JC
• Jung JW
• Kwak HW
• Song JH
• Jeon EJ
• Shin JW
• Park IW
• Choi BW
• Kim JY

• KMID: 1713463
• DOI: http://doi.org/10.3346/jkms.2008.23.2.288

Abstract

Granulocyte macrophage-colony stimulating factor (GM-CSF) has immuno-stimulatory effects. We hypothesized that GM-CSF plays an important role both in lipopolysaccharide (LPS)- and hemorrhage-induced acute lung injury (ALI). We also postulated that GM-CSF augments LPS-induced inflammation by priming neutrophils. ALI was induced in GM-CSF-/- or control C57BL mice either by LPS injection or by hemorrhage. Lung inflammation (by lung expression for tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-2 (MIP-2), interleukin-1beta (IL-1beta), interleukin- 6 (IL-6), and keratinocyte-derived chemokine) and lung injury (by myeloperoxidase and evans blue dye assay) were evaluated after ALI. Incremental doses of LPS (0, 1, 10, and 100 ng/mL) and GM-CSF (0, 1, 10, and 100 ng/mL) were added to bone marrow neutrophils. The expression of TNF-alpha, MIP-2, and IL-1beta was evaluated with enzyme linked immunosorbent assay. The mRNA expression of three cytokines, and the nuclear translocation of nuclear factor kappa B (NF kappa-B) were evaluated by reverse transcriptase-polymerase chain reaction and electropnoretic mobility shift assay, respectively. GM-CSF -/- mice showed decreased neutrophil infiltration, less leakage, and lower expression of cytokines in the lung after LPS or hemorrhage. GM-CSF augmented LPS-induced protein and mRNA expression of TNF-alpha, MIP-2 and IL-1beta, which was mediated by increased intra-nuclear translocation of NF-kappa B. GM-CSF plays an important role in high-dose LPS and hemorrhage-induced ALI, which appears to be mediated by its priming effect on neutrophils.

Keyword

Granulocyte-Macrophage Colony-Stimulating Factor; Adult, Respiratory Distress Syndrome; Lipopolysaccharides; Hemorrhage; Neutrophils

MeSH Terms

Animals
Bone Marrow Cells/cytology
Chemokine CXCL2/metabolism
Granulocyte-Macrophage Colony-Stimulating Factor/metabolism/*physiology
Interleukin-1beta/metabolism
Lipopolysaccharides/metabolism
Lung/metabolism/pathology
*Lung Injury
Male
Mice
Mice, Inbred BALB C
Mice, Inbred C57BL
Mice, Transgenic
Neutrophils/*cytology/metabolism
Peroxidase/metabolism
Tumor Necrosis Factor-alpha/metabolism

Figure

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  Fig. 1 GM-CSF -/- mice showed decreased infiltration of neutrophils in the lung after LPS injection or hemorrhage. GM-CSF -/- mice and control C57BL mice were treated with low or high doses of LPS (0.5 mg/kg or 5 mg/kg, respectively) injected into the peritoneum. Four hours after the injection of LPS or hemorrhage, mice were sacrificed and lung neutrophil infiltration was evaluated by MPO assay. Each group consisted of five mice. GM-/-, GM-CSF knockout mice; C57, C57BL control mice; Hmr, hemorrhage; LPS 0.5, LPS 0.5 mg/kg i.p.; LPS 5, LPS 5 mg/kg i.p. *, p<0.05.

• 

  Fig. 2 GM-CSF-/- mice were resistant to lung leakage after LPS injection or hemorrhage. GM-CSF -/- mice or control C57BL mice were treated with low or high doses of LPS (0.5 mg/kg and 5 mg/kg, respectively) injected into the peritoneum. Three hours after the injection of LPS or hemorrhage, Evans blue dye (50 mg/kg) was injected into the tail vein. One hour after the injection of dye, mice were sacrificed and the lungs were removed. EBD was extracted from the removed lungs by formamide extraction. Each group consisted of five mice. GM-/-, GM-CSF knockout mice; C57, C57BL control mice; Hmr, hemorrhage; LPS 0.5, LPS 0.5 mg/kg i.p.; LPS 5, LPS 5 mg/kg i.p. *, p<0.05.

• 

  Fig. 3 GM-CSF -/- mice showed decreased levels of TNF-α, MIP-2, and IL-1β in the lung after LPS injection. GM-CSF -/- mice and control C57BL mice were treated with low or high doses of LPS (0.5 mg/kg or 5 mg/kg, respectively) injected into the peritoneum. Four hours after the injection of LPS, mice were sacrificed, and expression of TNF-α, MIP-2 and IL-11β in the lung samples were evaluated by ELISA. Each group consisted of five mice.*, p<0.05.

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  Fig. 4 GM-CSF -/- mice showed decreased levels of KC, IL-6, and IL-1β in the lung after hemorrhage. Four hours after hemorrhage, mice were sacrificed and the expression of KC, IL-6, and IL-1β in the lung samples was evaluated by ELISA. Each group consisted of five mice. *, p<0.05.

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  Fig. 5 GM-CSF augments TNF-α, MIP-2, and IL-1β expression in mouse bone marrow neutrophils. Bone marrow neutrophils of control mice (5×105/well) were treated with GM-CSF (0, 1, 10, and 100 ng/mL) in combination with LPS (0, 1, 10, and 100 ng/mL). Four hours after stimulation with GM-CSF and/or LPS, the supernatant was collected and the expression of TNF-α, MIP-2, and IL-1β was evaluated by ELISA. This experiment was done three times independently and the displayed data are mean values. *, p<0.05

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  Fig. 6 GM-CSF augments LPS-induced mRNA expression of TNF-α, MIP-2, and IL-1β in mouse bone marrow neutrophils. LPS (100 ng/mL) and incremental concentration of GM-CSF (0, 1, 10, and 100 ng/mL) was added to bone marrow neutrophils of control mice (1×106/microtube). One hour later, the microtube was centrifuged for 12,000 g at 4℃ and total RNA was collected. The mRNA expression of TNF-α, MIP-2, and IL-1β was measured by RT-PCR. This experiment was done three times independently and the displayed data are mean values. LPS 100, LPS 100 ng/mL; LPS 100/GM1, LPS 100 ng/mL+GM-CSF 1 ng/mL; LPS 100/GM10, LPS 100 ng/mL+GM-CSF 10 ng/mL; LPS 100/GM100, LPS 100 ng/mL+GM-CSF 100 ng/mL. *, p<0.05.

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  Fig. 7 GM-CSF augments LPS-induced nuclear translocation of NF-κB in mouse bone marrow neutrophils. Isolated neutrophils were allocated into 1.5 mL microtubes (107 cells/tube). LPS (100 ng/mL) and incremental concentrations of GM-CSF (0, 1, 10, and 100 ng/mL) were added to bone marrow neutrophils of control mice. One hour later, the microtube was centrifuged at 12,000 g and 4℃ and the nuclear protein was extracted for EMSA. The upper figure and lower graph are representatives of three independent experiments. GM, GM-CSF.

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