Nebulized Heparin Attenuates Pulmonary Coagulopathy and Inflammation through Alveolar Macrophages in a Rat Model of Acute Lung Injury

Abstract

Objective Alveolar macrophages play a key role in the development and resolution of acute respiratory distress syndrome (ARDS), modulating the inflammatory response and the coagulation cascade in lungs. Anti-coagulants may be helpful in the treatment of ARDS. This study investigated the effects of nebulized heparin on the role of alveolar macrophages in limiting lung coagulation and inflammatory response in an animal model of acute lung injury (ALI). Methods Rats were randomized to four experimental groups. In three groups, ALI was induced by intratracheal instillation of lipopolysaccharide (LPS) and heparin was nebulized at constant oxygen flow: the LPS/Hep group received nebulized heparin 4 and 8 hours after injury; the Hep/LPS/Hep group received nebulized heparin 30 minutes before and 4 and 8 hours after LPS-induced injury; the LPS/Sal group received nebulized saline 4 and 8 hours after injury. The control group received only saline. Animals were exsanguinated 24 hours after LPS instillation. Lung tissue, bronchoalveolar lavage fluid (BALF) and alveolar macrophages isolated from BALF were analysed. Results LPS increased protein concentration, oedema and neutrophils in BALF as well as procoagulant and proinflammatory mediators in lung tissue and alveolar macrophages. In lung tissue, nebulized heparin attenuated ALI through decreasing procoagulant (tissue factor, thrombin-anti-thrombin complexes, fibrin degradation products) and proinflammatory (interleukin 6, tumour necrosis factor alpha) pathways. In alveolar macrophages, nebulized heparin reduced expression of procoagulant genes and the effectors of transforming growth factor beta (Smad 2, Smad 3) and nuclear factor kappa B (p-selectin, CCL-2). Pre-treatment resulted in more pronounced attenuation. Conclusion Nebulized heparin reduced pulmonary coagulopathy and inflammation without producing systemic bleeding, partly by modulating alveolar macrophages.

Fig.1

Experimental design. ( a ) Treatment group and ( b ) pre-treatment group.

Fig. 2

Bronchoalveolar lavage analysis and wet/dry weight ratio . Absolute ( a ) neutrophil (PMN) and ( b ) alveolar macrophage (AM) cell counts in the bronchoalveolar lavage fluid of rats 24 hours after the induction of the injury. ( c ) Wet/dry weight ratio and ( d ) protein concentration in the bronchoalveolar lavage fluid. Data are presented as mean ± SEM. ANOVA followed by the post hoc Fisher's PLSD test were used. * p  < 0.05; ** p  < 0.001; *** p  < 0.0001.

Fig. 3

Lung tissue analysis . ( a–d ) Representative images of haematoxylin and eosin staining lung tissue sections and ( e ) histological score in animals 24 hours after induction of the injury. Original magnification ×200. Data are presented as mean ± SEM. ANOVA followed by the post hoc Fisher's PLSD test were used. * p  < 0.05; ** p  < 0.001; *** p  < 0.0001.

Fig. 4

Coagulant effectors and cytokines . ( a ) Tissue factor (TF), ( b ) thrombin–anti-thrombin complexes (TATc), ( c ) fibrin degradation products (FDPs), ( c ) plasminogen activator inhibitor type-1 activity (PAI-1), ( e ) IL-6, ( f ) TNF-α, ( g ) GRO-κC and ( h ) IL-10 concentrations were measured in lung homogenate of animals 24 hours after induction of the injury. Data are presented as mean ± SEM. ANOVA followed by the post hoc Fisher's PLSD test were used. * p  < 0.05; ** p  < 0.001; *** p  < 0.0001.

Fig. 5

Activation of alveolar macrophages. Gene expression of proinflammatory (M1): ( a ) TNF-α, ( b ) iNOS and alternative (M2), ( c ) IL-10, ( d ) arginase-1 mediators in alveolar macrophages isolated from BALF of animals 24 hours after induction of the injury. Data are presented as mean ± SEM. ANOVA followed by the post hoc Fisher's PLSD test were used. * p  < 0.05; ** p  < 0.001; *** p  < 0.0001.

Fig. 6

Inflammatory and coagulation pathways of alveolar macrophages . Gene expression of TGF-β pathway: ( a ) TGF-β, ( b ) Smad 2 and ( c ) Smad 3, NF-κ≡ pathway, ( d ) IRAK-1, ( e ) p-selectin and ( f ) CCL-2 and coagulation pathway, ( g ) TF, ( h ) PAI-1 and ( i ) plasminogen in alveolar macrophages isolated from BALF of animals 24 hours after induction of the injury. Data are presented as mean ± SEM. ANOVA followed by the post hoc Fisher's PLSD test were used. * p  < 0.05; ** p  < 0.001; *** p  < 0.0001.