Role of heparin in pulmonary cell populations in an in-vitro model of acute lung injury

Abstract

Background: In the early stages of acute respiratory distress syndrome (ARDS), pro-inflammatory mediators inhibit natural anticoagulant factors and initiate an increase in procoagulant activity. Previous studies proved the beneficial effects of heparin in pulmonary coagulopathy, which derive from its anticoagulant and anti-inflammatory activities, although it is uncertain whether heparin works. Understanding the specific effect of unfractioned heparin on cell lung populations would be of interest to increase our knowledge about heparin pathways and to treat ARDS.

Methods: In the current study, the effect of heparin was assessed in primary human alveolar macrophages (hAM), alveolar type II cells (hATII), and fibroblasts (hF) that had been injured with LPS.

Results: Heparin did not produce any changes in the Smad/TGFß pathway, in any of the cell types evaluated. Heparin reduced the expression of pro-inflammatory markers (TNF-α and IL-6) in hAM and deactivated the NF-kß pathway in hATII, diminishing the expression of IRAK1 and MyD88 and their effectors, IL-6, MCP-1 and IL-8.

Conclusions: The current study demonstrated that heparin significantly ameliorated the cells lung injury induced by LPS through the inhibition of pro-inflammatory cytokine expression in macrophages and the NF-kß pathway in alveolar cells. Our results suggested that a local pulmonary administration of heparin through nebulization may be able to reduce inflammation in the lung; however, further studies are needed to confirm this hypothesis.

Keywords: Acute Respiratory Distress Syndrome (ARDS); Alveolar cells; Alveolar macrophages; Anticoagulants; Fibroblasts; Inflammation.

Figures

Fig. 1

Purity of isolated cells. hAM were stained with Diff quick to differentiate macrophages, neutrophils and lymphocytes and also CD68 immunofluorescence. hATII were stained with alkaline phosphatase and also with Surfactant C protein immunofluorescence. hF were stained with Diff Quick and ACTA2 immunofluorescence. Diff quick and alkaline phosphatase images had x400 magnification. Images of Hoechst, CD68, SPC and ACTA2 immunofluorescences had x600 magnification (hAM: human alveolar macrophages; hATII: human alveolar type II cells; hF: human fibroblasts, SPC: surfactant C protein and ACTA2: alpha smooth actin)

Fig. 2

Human alveolar macrophages gene expression. a Expression of TNF-α, IL-6, IL-8, MCP-1, IRAK-1, MyD88, TGF-β, Smad2 and Smad3 evaluated by q-PCR at 7 h after LPS treatment. Data are expressed mean ± SEM (ΔCt correction was applied using GAPDH as a housekeeping gene and units are relative to the expression of control group) (n = 8 samples per group). b Protein expression for TNF-α, IL-6, IL-8, MCP-1 and TGF-β (n = 4 samples per group). Data are expressed mean ± SEM. *p ≤ 0.05 vs control groups; #p ≤ 0.05 vs LPS group c Immunofluorescence for NF-kß, IRAK-1, MyD88 and Smad2/3 and all the treatments are shown. Magnification is 400x. (ND: non-detectable; LPS: Lipopolysaccharide from Escherichia coli 055:B5 and HEP: unfractionated heparin)

Fig. 3

Human alveolar type II cells gene expression. a Expression of TNF-α, IL-6, IL-8, MCP-1, IRAK-1, MyD88, TGF-β, Smad2 and Smad3 evaluated by q-PCR at 24 h after LPS treatment. Data are expressed mean ± SEM (ΔCt correction was applied using GAPDH as a housekeeping gene and units are relative to the expression of control group) (n = 8 samples per group). b Protein expression for TNF-α, IL-6, IL-8, MCP-1 and TGF-β (n = 4 samples per group). Data are expressed mean ± SEM. group c Immunofluorescence for NF-kß, IRAK-1, MyD88 and Smad2/3 and all the treatments are shown. Magnification is 400x.*p ≤ 0.05 vs control groups; #p ≤ 0.05 vs LPS group (LPS: Lipopolysaccharide from Escherichia coli 055:B5 and HEP: unfractionated heparin)

Fig. 4

Human fibroblasts gene expression. a Expression of TNF-α, IL-6, IL-8, MCP-1, IRAK-1, MyD88, TGF-β, Smad2 and Smad3 evaluated by q-PCR at 24 h after LPS treatment. Data are expressed mean ± SEM (ΔCt correction was applied using GAPDH as a housekeeping gene and units are relative to the expression of control group) (n = 8 samples per group). b Protein expression for TNF-α, IL-6, IL-8, MCP-1 and TGF-β (n = 4 samples per group). Data are expressed mean ± SEM group c Immunofluorescence for NF-kß, IRAK-1, MyD88 and Smad2/3 and all the treatments are shown. Magnification is 400x. *p ≤ 0.05 vs control groups; #p ≤ 0.05 vs LPS group (LPS: Lipopolysaccharide from Escherichia coli 055:B5 and HEP: unfractionated heparin)

Fig. 5

hAM, hATII cells and hF apoptosis/necrosis measured by Annexin V and PI staining with flow cytometry. The proportion of live cells (Annexin V-FITC-/PI-), early apoptotic cells (Annexin V-FITC+/PI-), necrotic cells (Annexin V-FITC-/PI+), late apoptotic/necrotic cells (Annexin V-FITC+/PI+). No changes were observed in LPS treated samples or in LPS + Hep treated ones (hAM:human alveolar macrophages; hATII: human alveolar type II cells; hF: human fibroblasts; FITC: Fluorescein; PI: propidium iodide; LPS: Lipopolysaccharide from Escherichia coli 055:B5 and HEP: unfractionated heparin)