Yonsei Med J.  2011 Jan;52(1):65-73. 10.3349/ymj.2011.52.1.65.

Granulocyte Colony Stimulating Factor Attenuates Hyperoxia-Induced Lung Injury by Down-Modulating Inflammatory Responses in Neonatal Rats

• Jeon GW
• Sung DK
• Jung YJ
• Koo SH
• Choi SH
• Chang YS
• Sin JB
• Park WS

• KMID: 1106439
• DOI: http://doi.org/10.3349/ymj.2011.52.1.65

Abstract

PURPOSE
Granulocyte colony stimulating factor (G-CSF) has been known to increase neutrophil production and have anti-inflammatory properties, but the effect of G-CSF on pulmonary system is in controversy. We investigated whether G-CSF treatment could attenuate hyperoxia-induced lung injury, and whether this protective effect is mediated by the down-modulation of inflammatory responses in a neonatal rat model.
MATERIALS AND METHODS
Newborn Sprague-Dawley rats (Orient Co., Seoul, Korea) were subjected to 14 days of hyperoxia (90% oxygen) beginning within 10 h after birth. G-CSF (20 microg/kg) was administered intraperitoneally on the fourth, fifth, and sixth postnatal days.
RESULTS
This treatment significantly improved hyperoxia-induced reduction in body weight gain and lung pathology such as increased mean linear intercept, mean alveolar volume, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling positive cells. Hyperoxia-induced activation of nicotinamide adenine dinucleotide phosphate oxidase, which is responsible for superoxide anion production, as evidenced by upregulation and membrane translocation of p67phox was significantly attenuated after G-CSF treatment, as were inflammatory responses such as increased myeloperoxidase activity and mRNA expression of transforming growth factor-beta. However, the attenuation of other proinflammatory cytokines such as tumor necrosis factor-alpha and interleukin-6 was not significant.
CONCLUSION
In sum, G-CSF treatment significantly attenuated hyperoxia-induced lung injury by down-modulating the inflammatory responses in neonatal rats.

Keyword

Granulocyte colony stimulating factor; bronchopulmonary dysplasia; inflammation; animals; infant; newborn

MeSH Terms

Animals
Animals, Newborn
Blotting, Western
Female
Granulocyte Colony-Stimulating Factor/*therapeutic use
Hyperoxia/*complications
In Situ Nick-End Labeling
Interleukin-6/genetics
Lung/*drug effects/*metabolism
Lung Injury/*drug therapy/etiology/genetics/metabolism
NADPH Oxidase/metabolism
Peroxidase/metabolism
Pregnancy
Random Allocation
Rats
Rats, Sprague-Dawley
Reverse Transcriptase Polymerase Chain Reaction
Transforming Growth Factor beta/genetics
Tumor Necrosis Factor-alpha/genetics
Weight Gain/drug effects

Figure

• 

  Fig. 1 Daily body weight gain in each experimental group. Data; mean ± SEM. NC, normoxia control group (n = 12); NG, normoxia with G-CSF treatment group (n = 12); HC, hyperoxia control group (n = 14); HG, hyperoxia with G-CSF treatment group (n = 14). *p < 0.05 compared to NC. †p < 0.05 compared to HC.

• 

  Fig. 2 Representative optical photomicrographs of P 14 rat lungs stained with hematoxylin and eosin. Scale bar = 100 µm, NC, normoxia control group; NG, normoxia with G-CSF treatment group; HC, hyperoxia control group; HG, hyperoxia with G-CSF treatment group.

• 

  Fig. 3 Comparisons of alveolarization measured by the mean linear intercept (A) and mean alveolar volume (B). Data; mean ± SEM. NC, normoxia control group (n = 6); NG, normoxia with G-CSF treatment group (n = 6); HC, hyperoxia control group (n = 8); HG, hyperoxia with G-CSF treatment group (n = 8). *p < 0.05 compared to NC. †p < 0.05 compared to HC.

• 

  Fig. 4 TUNEL positive cells (A) and number of TUNEL positive cells (B) in P14 rat lungs. TUNEL positive cells labeled with FITC (green, arrow) and the nuclei labeled with DAPI (blue) (Scale bar, 25 µm) (A). Data; mean ± SEM. NC, normoxia control group (n = 6); NG, normoxia with G-CSF treatment group (n = 6); HC, hyperoxia control group (n = 8); HG, hyperoxia with G-CSF treatment group (n = 8). *p < 0.05 compared to NC. †p < 0.05 compared to HC. TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling; FITC, fluorescein isothiocyanate; DAPI, 4, 6-diamidino-2-phenylindole; G-CSF, granulocyte colony stimulating factor.

• 

  Fig. 5 White blood cell count in blood (A) and myeolperoxidase activity in P 14 rat lungs (B). Data; mean ± SEM. NC, normoxia control group (n = 6); NG, normoxia with G-CSF treatment group (n = 6); HC, hyperoxia control group (n = 6); HG, hyperoxia with G-CSF treatment group (n = 6). *p < 0.05 compared to NC. †p < 0.05 compared to HC. MPO, myeloperoxidase.

• 

  Fig. 6 Representative RT-PCR blots and densitometric histograms for TNF-α, IL-6, and TGF-β. NC, normoxia control group (n = 6); NG, normoxia with G-CSF treatment group (n = 6); HC, hyperoxia control group (n = 6); HG, hyperoxia with G-CSF treatment group (n = 6). *p < 0.05 compared to NC. †p < 0.05 compared to HC. RT-PCR, reverse transcriptase polymerase chain reaction; TNF-α, tumor necrosis factor-α; IL-6, interleukin-6; TGF-β, transforming growth factor-β; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

• 

  Fig. 7 Western blot analysis of p67phox in the cytosol and membrane fractions of P 14 rat lung. NC, normoxia control group (n = 6); NG, normoxia with G-CSF treatment group (n = 6); HC, hyperoxia control group (n = 6); HG, hyperoxia with G-CSF treatment group (n = 6). *p < 0.05 compared to NC. †p < 0.05 compared to HC.

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