Lipopolysaccharide pre-conditioning is protective in lung ischemia-reperfusion injury

Abstract

Background: The availability of suitable lung donors has remained a significant barrier to lung transplantation. The clinical relevance of an isolated positive Gram stain in potential donor lungs, which occurs in >80%, is unclear. Low doses of lipopolysaccharide (LPS) have been protective in several models of ischemia-reperfusion injury through a pre-conditioning response. We sought to demonstrate that low-dose LPS is protective against subsequent lung ischemia-reperfusion injury.

Methods: Pathogen-free Long-Evans rats were pre-treated with vehicle or LPS 24 hours before 90 minutes of ischemia and up to 4 hours of reperfusion. Lungs were assessed for vascular permeability, myeloperoxidase content, bronchoalveolar lavage inflammatory cell and cytokine/chemokine content, as well as nuclear translocation of nuclear factor kappaB (NFkappaB) and activator protein-1 (AP-1), and interleukin-1 receptor-associated kinase-1 (IRAK-1) and stress-activated protein kinase (SAPK) activation.

Results: Compared with positive controls, LPS pre-treatment resulted in reductions in vascular permeability (70%, p < 0.001), myeloperoxidase content (93%, p < 0.001), bronchoalveolar lavage inflammatory cells (91%, p < 0.001), and inflammatory cytokine/chemokine content (cytokine-induced neutrophil chemoattractant, 99%, p = 0.003; interleukin-1beta, 72%, p < 0.0001; tumor necrosis factor-alpha, 76%, p < 0.0001), NFkappaB (86%, p < 0.001) and AP-1 (97%, p < 0.001) nuclear translocation, and IRAK-1 (87%, p < 0.001) and SAPK (80%, p < 0.001) phosphorylation.

Conclusions: Lipopolysaccharide pre-treatment reduced lung injury and inflammatory mediator production after subsequent exposure to ischemia-reperfusion. Understanding the clinical significance of lipopolysaccharide in donor lungs has the potential to expand and clarify donor inclusion criteria.

Figure 1

Lipopolysaccharide (LPS) pre-treatment dose response of the lung permeability index is shown after ischemia-reperfusion. Pre-treatment with LPS before ischemia-reperfusion yielded protection from lung injury across the spectrum of LPS doses studied compared with positive controls (PC), but lung permeability remained elevated above that seen in negative controls (NC). A LPS pre-treatment dose of 15 ng was the lowest dose associated with the greatest protection from lung injury.

Figure 2

Western blot analysis is shown for phosphorylated interleukin-1 receptor-associated kinase-1 (IRAK-1). Positive controls exhibited significantly more IRAK-1 phosphorylation (lane 2) than unmanipulated negative controls (lane 1) or lipopolysaccharide (LPS) controls (lane 5). LPS-treated animals demonstrated significantly reduced IRAK-1 phosphorylation (lanes 3, 4) compared with positive controls (p. 0.001) after undergoing 90 minutes of ischemia, followed by 15 minutes of reperfusion.

Figure 3

Relative optical densitometry of Western blot analysis is shown for phosphorylated interleukin-1 receptor-associated kinase-1 (IRAK-1). IRAK-1 phosphorylation was reduced by 87% in lipopolysaccharide-treated animals compared with positive controls as measured by relative optical densitometry.

Figure 4

Western Blot analysis for phosphorylated c-Jun N-terminal kinase (JNK) and p38. Positive controls exhibit significantly more p38 and JNK phosphorylation (lane 3) than unmanipulated negative controls (lane 1) or lipopolysaccharide (LPS) controls (lane 2). LPS-treated animals demonstrate significantly reduced JNK phosphorylation and p38 phosphorylation (lane 4) compared with positive controls (p < 0.001, p < 0.001, respectively) after undergoing 90 minutes of ischemia, followed by 15 minutes of reperfusion.

Figure 5

Relative optical densitometry of Western blot analysis is shown for phosphorylated c-Jun N-terminal kinase (JNK) and p38. JNK and p38 phosphorylation were both reduced by 80% in lipopolysaccharide-treated animals compared with positive controls as measured by relative optical densitometry.

Figure 6

Results of electromobility shift assay are shown for nuclear factor κB (NFκB). Positive controls demonstrated marked nuclear translocation of NFκB (lanes 3, 4, and 5) compared with negative controls (lane 1) and lipopolysaccharide (LPS) controls (lane 2). LPS-treated animals demonstrate significantly reduced nuclear translocation of NFκB (lanes 6, 7, 8, and 9) compared with positive controls (p < 0.001) after undergoing 90 minutes of ischemia, followed by 15 minutes of reperfusion.

Figure 7

Electromobility shift assay is shown for activator protein-1 (AP-1). Positive controls demonstrated marked nuclear translocation of AP-1 (lanes 3, 4, and 5) compared with negative controls (lane 1) and lipopolysaccharide (LPS) controls (lane 2). LPS-treated animals demonstrated significantly reduced nuclear translocation of AP-1 (lanes 6, 7, and 8) compared with positive controls (p < 0.001) after undergoing 90 minutes of ischemia, followed by 15 minutes of reperfusion.

Figure 8

Relative optical densitometry of electromobility shift assay showed that nuclear translocation of factor κB (NFκB) and activator protein-1 (AP-1) were reduced by 86% and 97%, respectively, in lipopolysaccharide-treated rats compared with positive controls.