Tuberc Respir Dis.  2015 Jul;78(3):218-226. 10.4046/trd.2015.78.3.218.

EphA2 Receptor Signaling Mediates Inflammatory Responses in Lipopolysaccharide-Induced Lung Injury

• Hong JY
• Shin MH
• Chung KS
• Kim EY
• Jung JY
• Kang YA
• Kim YS
• Kim SK
• Chang J
• Park MS

• KMID: 2320646
• DOI: http://doi.org/10.4046/trd.2015.78.3.218

Abstract

BACKGROUND
Eph receptors and ephrin ligands have several functions including angiogenesis, cell migration, axon guidance, fluid homeostasis, oncogenesis, inflammation and injury repair. The EphA2 receptor potentially mediates the regulation of vascular permeability and inflammation in response to lung injury.
METHODS
Mice were divided into 3 experimental groups to study the role of EphA2 signaling in the lipopolysaccharide (LPS)-induced lung injury model i.e., IgG+phosphate-buffered saline (PBS) group (IgG instillation before PBS exposure), IgG+LPS group (IgG instillation before LPS exposure) and EphA2 monoclonal antibody (mAb)+LPS group (EphA2 mAb pretreatment before LPS exposure).
RESULTS
EphA2 and ephrinA1 were upregulated in LPS-induced lung injury. The lung injury score of the EphA2 mAb+LPS group was lower than that of the IgG+LPS group (4.30+/-2.93 vs. 11.45+/-1.20, respectively; p=0.004). Cell counts (EphA2 mAb+LPS: 11.33x10(4)+/-8.84x10(4) vs. IgG+LPS: 208.0x10(4)+/-122.6x10(4); p=0.018) and total protein concentrations (EphA2 mAb+LPS: 0.52+/-0.41 mg/mL vs. IgG+LPS: 1.38+/-1.08 mg/mL; p=0.192) were decreased in EphA2 mAb+LPS group, as compared to the IgG+LPS group. In addition, EphA2 antagonism reduced the expression of phospho-p85, phosphoinositide 3-kinase 110gamma, phospho-Akt, nuclear factor kappaB, and proinflammatory cytokines.
CONCLUSION
This results of the study indicated a role for EphA2-ephrinA1 signaling in the pathogenesis of LPS-induced lung injury. Furthermore, EphA2 antagonism inhibits the phosphoinositide 3-kinase-Akt pathway and attenuates inflammation.

Keyword

Lipopolysaccharides; Lung Injury; EphA2 Protein

MeSH Terms

Animals
Axons
Capillary Permeability
Carcinogenesis
Cell Count
Cell Movement
Cytokines
Homeostasis
Inflammation
Ligands
Lipopolysaccharides
Lung Injury*
Methods
Mice
Receptor, EphA1
Receptor, EphA2*
Receptors, Eph Family
Cytokines
Ligands
Lipopolysaccharides
Receptor, EphA1
Receptor, EphA2
Receptors, Eph Family

Figure

• 

  Figure 1 The expression of EphA2 and ephrinA1 in lung lysates after lipopolysaccharide (LPS) exposure, as shown by Western blots (A) and densitometry (B, C). IgG pretreatment increased the expression of EphA2 and ephrinA1 after LPS exposure; whereas, EphA2 monoclonal antibody pretreatment reduced the expression of EphA2 and ephrinA1 after LPS exposure. Values are presented as mean±SD. PBS: phosphate buffered saline.

• 

  Figure 2 The effect of EphA2 monoclonal antibody pretreatment in lipopolysaccharide (LPS)-induced lung injury. Mice were intranasally treated with IgG or EphA2 antibody. One hour later, they were intranasally treated with phosphate buffered saline (PBS) or 40 µg LPS in PBS. The lungs were removed after 24 hr and stained with hematoxylin and eosin (H&E). (A) IgG+PBS group, (B) IgG+LPS group, (C) EphA2 Ab 2 µg+LPS group, and (D) EphA2 Ab 4 µg+LPS group, n=4 per group. The IgG+LPS group (B) showed obvious neutrophil infiltration and alveolar septal infiltration; however, the EphA2 Ab+LPS groups (C, D) showed attenuated lung injury (H&E stain, ×400).

• 

  Figure 3 The effect of EphA2 monoclonal antibody pretreatment on semiquantitative lung injury scores (A), total bronchoalveolar fluid (BALF) cell count (B) and BALF protein concentration (C). In the lipopolysaccharide (LPS)-induced lung injury model, EphA2 antagonism with EphA2 monoclonal antibody led to improved lung injury scores, decreased BALF cell counts and decreased BALF proteins (p<0.05, except BALF protein), as compared to IgG pretreatment. Values are presented as mean±SD (n=4 per group).

• 

  Figure 4 The expression of phosphoinositide 3-kinase (PI3K) protein (pp85 and 110γ) in lung lysates after lipopolysaccharide (LPS) exposure, as shown by Western blots (A) and densitometry (B, C). Phosphorylated 110γ (catalytic subunit of PI3K) levels were increased in mice with LPS exposure (IgG+LPS), as compared with controls (IgG+phosphate buffered saline [PBS]). Activation of P110γ was reduced in response to EphA2 monoclonal antibody pretreatment (EphA2+LPS) (C). However, the expression of p85 (regulatory subunit of PI3K) protein was not consistently influenced by EphA2 monoclonal antibody (B). Values are presented as mean±SD.

• 

  Figure 5 The expression of Akt, p65 nuclear factor κB (NF-κB), and IκB in lung lysates after lipopolysaccharide (LPS) exposure, as shown by representative Western blots (A) and densitometry (B-D). The amount of phosphorylated Akt was increased in mice with LPS exposure (IgG+LPS), as compared with controls (IgG+PBS). LPS-induced activation of Akt was reduced in response to EphA2 monoclonal antibody pretreatment (EphA2+LPS) (B). The expression of phosphorylated NF-κB protein was similar to that of Akt (C). The expression of phosphorylated IκB after LPS exposure was increased in mice with EphA2 monoclonal antibody pretreatment (D). Values are presented as mean±SD.

• 

  Figure 6 The expression of cytokines (interleukin [IL]-1β, IL-6, KC, macrophage inflammatory protein 2 [MIP-2], and tumor necrosis factor α [TNF-α]) in response to EphA2 monoclonal antibody pretreatment in a lipopolysaccharide (LPS) induced lung injury model. Enzyme linked immunosorbent assay measurement showed differential expression of cytokines (IL-1β, IL-6, KC, MIP-2, and TNF-α) in lung tissue lysates. Cytokine levels were reduced in response to LPS exposure in mice with EphA2 monoclonal antibody pretreatment, as compared to those with IgG pretreatment (n=3 per group). Values are presented as mean±SD.

• 

  Figure 7 The effects of ephrinA1-mediated activation of EphA2 on the phosphoinositide 3-kinase (PI3K)/Akt signaling pathways in lipopolysaccharide (LPS)-induced lung injury. EphA2-ephrinA1 signaling may contribute to the development of LPS-induced lung injury. Exposure to LPS resulted in activation of the EphA2-ephrinA1 and PI3K/Akt/nuclear factor κB (NF-κB) signaling pathway. EphA2 monoclonal antibody-induced inhibition of EphA2 signaling resulted in decreased PI3K/Akt/NF-κB-dependent inflammatory processes. These results suggested crosstalk between EphA2 signaling and PI3K/Akt/NF-κB signaling in LPS-induced lung injury. TNF-α: tumor necrosis factor α; IL: interleukin.

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