6881 - Alex Tonks.pdf (12.42 MB)
Download file

The role of phospholipids in the modulation of the monocytic oxidative burst

Download (12.42 MB)
thesis
posted on 20.06.2022, 17:00 authored by Alex Tonks

Alveolar macrophages play a central role in the pulmonary inflammatory response by generating reactive oxygen intermediates (ROIs). This study investigates how pulmonary surfactant (composed of approximately 90% phospholipids and l0% surfactant specific proteins) modulates the production of ROI's in monocytes and macrophages.
 

The human monocyte cell line MonoMac-6 (MM6), peripheral blood monocytes, the murine macrophage RAW 264.7 and the rat alveolar macrophage line NR8383 were primed with lipopolysaccharide (LPS) and stimulated to produce ROIs with opsonised zymosan (OpZ). The surfactants Curosurf®, Survanta® and Exosurf Neonatal™ were all shown to significantly inhibit ROI production (P<0.01) measured by luminol-enhanced chemiluminescence (LCL). Preincubation of MM6 cells with 1,2- dipalmitoylphosphatidylcholine (DPPC), the major surfactant phospholipid, also significantly inhibited ROI production (p<0.001). In contrast, 1-palmitoyl-2- arachidonoyl phosphatidylcholine (PAPC) increased ROI production (P<0.001). DPPC modulation was independent of LPS 'priming' and did not affect cell viability. In addition, DPPC (but not PAPC) significantly inhibited the LPS-stimulated release of TNF-α in MM6 cells (P<0.05). However, DPPC failed to modulate nitric oxide production, measured by the Griess assay, demonstrating the selective nature of the effect.
 

Experiments with a cell-free system showed that DPPC had no direct effect on NADPH oxidase assembly and activation. Flow cytometry analysis indicated that the suppressive effects of DPPC on ROI production were not attributed to CDl4, complement, or Fcy receptor down-regulation. Moreover, the binding of radiolabelled LPS was not decreased in MM6 cells preincubated with DPPC.
 

Transmission electron microscopy demonstrated that DPPC was taken up by MM6 cells and altered the monocyte membrane ultrastructure. Western blotting demonstrated that the mitogen activated protein kinases were not modulated by DPPC. However, the activity of protein kinase C (PKC) measured by 32P radioassay, was shown to be significantly inhibited by DPPC (P<0.01). Taken together these findings indicate that surfactant lipids, particularly DPPC, suppress monocyte NADPH oxidase activation by down-regulation of PKC.

History

Year

2001

School

School of Art and Design

Usage metrics

Exports