Effect of tyrosine kinase inhibitors, imatinib and nilotinib, in murine lipopolysaccharide-induced acute lung injury during neutropenia recovery

Abstract

Introduction: Neutrophil recovery has been implicated in deterioration of oxygenation and exacerbation of preexisting acute lung injury (ALI). The aim of this study was to investigate whether imatinib or nilotinib was effective on lipopolysaccharide (LPS)-induced ALI during neutropenia recovery in mice.

Methods: Mice were rendered neutropenic with cyclophosphamide prior to the intratracheal instillation of LPS. Imatinib or nilotinib was administrated by oral gavage during neutropenia recovery. In order to study the effects of drugs, mice were killed on day 5 and blood, bronchoalveolar lavage (BAL) fluid and lung tissue samples were obtained. The lung wet/dry weight ratio and protein levels in the BAL fluid or lung tissue were determined.

Results: Treatment with imatinib or nilotinib significantly attenuated the LPS-induced pulmonary edema, and this result was supported by the histopathological examination. The concentrations of tumor necrosis factor-α, interleukin (IL)-1β, IL-6 and myeloperoxidase in BAL fluid were significantly inhibited by imatinib or nilotinib in mice of ALI during neutropenia recovery. The mRNA expressions of platelet-derived growth factor receptor-β and c-KIT in imatinib or nilotinib group were significantly lower than LPS group.

Conclusions: Our data indicated that imatinib or nilotinib effectively attenuated LPS-induced ALI during neutropenia recovery. These results provide evidence for the therapeutic potential of imatinib and nilotinib in ALI during neutropenia recovery.

Figures

Figure 1

Effect of cyclophosphamide in mice. All mice received 150 mg/kg cyclophosphamide by intraperitoneal injection on day (D) -5 before they became neutropenic and 100 mg/kg on day -2 before administration of imatinib or nilotinib. Neutrophils were measured in the peripheral blood every day to monitor the cytotoxic effect of cyclophosphamide until mice were sacrificed. WBC, white blood cell.

Figure 2

Effect of imatinib or nilotinib on the histopathological changes in the lung in mice lipopolysaccharide-induced acute lung inury. Representative images of H&E-stained lung sections from four experimental groups: (A) control group; the lung structure is normal, (B) cyclophosphamide (C) + lipopolysaccharide (LPS) group; LPS induced acute lung damage with interstitial edema, hemorrhage, thickening of the alveolar wall and infiltration of inflammatory cells into the interstitium and alveolar spaces, (C) C + LPS + imatinib (I) group, (D) C + LPS + nilotinib (N) group; lung injury was attenuated by treatment with imatinib or nilotinib.

Figure 3

Effect of imatinib or nilotinib on lung edema in mice with lipoplysaccharide-induced acute lung injury. After lipopolysaccharide (LPS) administration, treatment with imatinib or nilotinib markedly decreased (A) lung wet/dry (W/D) weight ratio and (B) albumin. The lower lobe of the right lung was removed and weighed (wet weight), dried at 60°C for 72 hrs, and weighed again (dry weight). The W/D ratio was used as an indicator of lung edema formation. C, cyclophosphamide; I, imatinib; N, nilotinib. Asterisks indicates significant differences to the C + LPS group (*P <0.05, **P <0.01).

Figure 4

Effect of imatinib or nilotinib on the protein level of inflammatory cytokines and myeloperoxidase in mice with lipoplysaccharide-induced acute lung injury. The levels of (A) TNF-α, (B) IL-6, (C) IL-1β and (D) myeloperoxidase (MPO) were assayed in bronchiolar lavage (BAL) fluid and the sensitivities of the assay were 15.6 pg/ml, 3 pg/ml, 7 pg/ml and 0.78 ng/ml, respectively. Intratracheal administration of lipopolysaccharide (LPS) induced increased levels of TNF- α, IL-6, IL-1β and MPO. Compared with the cyclophosphamide (C) + LPS group, the level of inflammatory cytokines and MPO was significantly lower in the imatinib or nilotinib group. I, imatinib; N, nilotinib. Asterisks indicates significant differences to the C + LPS group (*P <0.05, **P <0.01).

Figure 5

Effect of imatinib or nilotinib on activation of p-platelet-derived growth factor receptor-ß in mice with lipoplysaccharide-induced acute lung injury. Lung tissues were homogenized and the extracted protein samples were separated by SDS-PAGE using 8% PAGE gel, followed by transfer to 0.45 μm polyvinylidene fluoride membranes. The membranes were blocked with 5% skimmed milk and exposed overnight at 4°C to specific anti-platelet-derived growth factor receptor-ß (PDGFR-β) and anti-phospho-PDGFR-β antibodies. LPS, lipopolysaccharide; C, cyclophosphamide; I, imatinib; N, nilotinib.

Figure 6

Effect of imatinib or nilotinib treatment on mRNA expression of platelet-derived growth factor receptor in mice with lipoplysaccharide-induced acute lung injury. The mRNA expression of platelet-derived growth factor receptor-ß (PDGFR) was measured in lung tissues. In real-time PCR analysis, to normalize the content of cDNA samples, the comparative threshold (CT) cycle method, consisting of the normalization of the number of target gene copies versus the housekeeping gene β-actin, was used. LPS, lipopolysaccharide; C, cyclophosphamide; I, imatinib; N, nilotinib. Asterisks indicate significant differences from the C + LPS group (**P <0.01).

Figure 7

Comparison of degree of lung injury. (A) Representative images of H&E-stained lung sections from four experimental groups. (B) Number of inflammatory cells in bronchoalveolar lavage (BAL) fluid. (C) Result of myeloperoxidase (MPO) analysis. LPS, lipopolysaccharide; C, cyclophosphamide; I, imatinib; N, nilotinib. *P <0.05; **P <0.01; NS, non-significant.

Figure 8

Effect of post-treatment with imatinib or nilotinib. (A) Representative images of H&E-stained lung sections from six experimental groups. (B) Number of inflammatory cells in bronchoalveolar lavage (BAL) fluid. (C) Albumin level in BAL fluid. (D) Myeloperoxidase (MPO) analysis. LPS, lipopolysaccharide; C, cyclophosphamide; I, imatinib; N, nilotinib. *P <0.05 and **P <0.01 for C + LPS versus C + LPS + I/N (post); #P <0.05 and ##P <0.01 for C + LPS + I/N versus C + LPS + I/N (post). Post, post-induction of LPS.