Tuberc Respir Dis.  2010 Nov;69(5):337-347.

The Role of Transglutaminase-2 in Fibroproliferation after Lipopolysaccharide-induced Acute Lung Injury

• Kim JH

Abstract

BACKGROUND
Transglutaminase-2 (TG-2) has been reported to play an important role in the process of fibrosis. However, TG-2 studies on fibroproliferation of acute lung injury (ALI) are absent. The purpose of this study was to investigate the role of TG-2 in the fibroproliferation of lipopolysaccharide (LPS)-induced ALI.
METHODS
The male C57BL/6 mice of 5 weeks age were divided into 3 groups; control group (n=30) in which 50 microL of saline was given intratracheally (IT), LPS group (n=30) in which LPS 0.5 mg/kg/50 microL of saline was given IT, and LPS+Cyst group treated with intraperitoneal 200 mg/kg of cystamine, competitive inhibitor of TG-2, after induction of ALI by LPS. TG-2 activity and nuclear factor (NF)-kappaB were measured in lung tissue homogenate. Tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, myeloperoxidase (MPO), and transforming growth factor (TGF)-beta1 were measured using bronchoalveolar lavage fluids. Histopathologic ALI score and Mallory's phosphotunistic acid hematoxylin (PTAH) for collagen and fibronectin deposition were performed.
RESULTS
The TG-2 activities in the LPS group were significantly higher than the control and LPS+Cyst groups (p<0.05). The TNF-alpha and IL-1beta concentrations and NF-kappaB activity were lower in the LPS+Cyst group than the LPS group (p<0.05). The LPS+Cyst group showed lower MPO, ALI score, TGF-beta1 concentration, and Mallory's PTAH stain than the LPS group, but the differences were not significant (p>0.05).
CONCLUSION
Inhibition of TG-2 activity in the LPS-induced ALI prevented early inflammatory parameters, but had limited effects on late ALI and fibroproliferative parameters.

Keyword

Acute Lung Injury; Lipopolysaccharides; Inflammation; Fibrosis

MeSH Terms

Acute Lung Injury
Animals
Bronchoalveolar Lavage Fluid
Collagen
Cystamine
Fibronectins
Fibrosis
Hematoxylin
Humans
Inflammation
Interleukin-6
Interleukins
Lipopolysaccharides
Lung
Male
Mice
NF-kappa B
Peroxidase
Transforming Growth Factor beta1
Transforming Growth Factors
Tumor Necrosis Factor-alpha
Collagen
Cystamine
Fibronectins
Hematoxylin
Interleukin-6
Interleukins
Lipopolysaccharides
NF-kappa B
Peroxidase
Transforming Growth Factor beta1
Transforming Growth Factors
Tumor Necrosis Factor-alpha

Figure

• 

  Figure 1 (A) The time course of transglutaminase (TG)-2 activity. Thirty six hours after intratracheal instillation of lipopolysaccharide (LPS, 0.5 mg/kg in 50 µL of saline), TG-2 activity showed significantly high activity compared with the control, 3, 6, 12, and 24 hours (*p<0.05). At 48 hours and 72 hours, TG-2 activities decreased, but not significant compared with the activity at 36 hours (†p>0.05). (B) The response of TG-2 activity for LPS doses. Instillation of LPS 1.0 mg/kg in 50 µL of saline showed an increased trend of TG-2 activity, but not significant compared with the activity of LPS 0.5 mg/kg (‡p=0.248). (C) The dose response of TG-2 activity for cystamine. At the cystamine dose of 200 mg/kg, TG-2 activity decreased significantly compared with the LPS group (§p=0.009), which was not different with the control group (∥p=0.806).

• 

  Figure 2 The time course of acute lung injury (ALI) parameters. The concentrations of tumor necrosis factor (TNF)-α (A), interleukin (IL)-1β (B), and IL-6 (C) in bronchoalveolar lavage fluid (BALF) and nuclear factor (NF)-κB activity (G) in lung tissue homogenates showed peaks at 6 hours after lipopolysaccharide (LPS) administration. Transforming growth factor (TGF)-β1 (D) and myeloperoxidase (MPO) activity (E) were highest at 36 hours, and ALI score (F) showed peak at 48 hours (*p<0.05, compared with the control and other time points).

• 

  Figure 3 Overall study design. Control: control group; LPS: lipopolysaccharide group; LPS+Cyst: LPS+cystamine group; BAL: bronchoalveolar lavage; TNF-α: tumor necrosis factor-α; IL-1β: interleukin-1β; IL-6, interleunkin-6; BALF: bronchoalveolar lavage fluid; NF-κB: nuclear factor-κB; TGF-β1: transforming growth factor-β1; Mallory's PTAH: Mallory's phosphotunstic acid hematoxylin; ALI: acute lung injury.

• 

  Figure 4 Transglutaminase (TG)-2 activity (A), tumor necrosis factor (TNF)-α (B), and interleukin (IL)-1β (C) were significantly decreased in the lipopolysaccharide (LPS)+Cyst group compared with the LPS group (*p<0.05). IL-6 (D) concentration was higher than the LPS group, but not significant (†p>0.05).

• 

  Figure 5 Nuclear factor (NF)-κB activity (A) was significantly decrease in the lipopolysaccharide (LPS)+Cyst group compared with the LPS group (*p=0.028). Myeloperoxidase (MPO) activity (B) and transforming growth factor (TGF)-β1 concentration (C) in the LPS+Cyst group was lower than the LPS group, but not significant (†p>0.05).

• 

  Figure 6 Histopathologic examination shows significantly higher levels of intra-alveolar exudates, inflammatory infiltration, hemorrhage, and interstitial edema in the lipopolysaccharide (LPS) group (B), compared with the control group (A). The LPS+Cyst group (C) showed similar findings with the LPS group. Acute lung injury (ALI) score (D) was not different between the LPS and LPS+Cyst groups (*p=0.171) (H&E stain, ×100).

• 

  Figure 7 Mallory's phosphotunstic acid hematoxylin (PTAH) stain (×400). The percent area of positivity (brown color ▴) was significantly larger in the LPS group (B) than the control group (A). Between the LPS and LPS+Cyst (C) groups, the area was similar (D, *p=0.423).

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