|
KMID : 0369319930130040494
|
|
Allergy
1993 Volume.13 No. 4 p.494 ~ p.508
|
|
|
The effects on the production of placelet activation factor in the cultured human endothelial cells by interleukin-6 and granulocyte macrophage colony stimulating factor
|
|
|
|
|
Abstract
|
|
|
Platelet activating factor (PAF) is a potent mediator implicated in a diverse range of human pathologies, including asthma, shock, systemic anaphylaxis, ulcerations. Recently it is known that PAF plays an important role in microvascular immune
injury.
Tumor necrosis duce cultured endothelial cell (EC) to synthesize PAF. Kawasaki disease, one of the acute systemic vasculitis in children, has been reported to have increased level of IL-6 with vascular damage. This result denotes that increasing
level
of IL-6 causes production of PAF to result in vascular damage.
Recently, IL-6 and granulocyte macrophage colony stimulationg factor (GM-CSF), regarded as important cytokines in inflammatory reactors, may induce production of PAF by stimulating EC like TNF and IL-1. In turn the PAF may damage the vessels to
have
vasculitis.
The purpose of this study is to focus the probable action of IL-6 and GM-CSF to induce the production of PAF by stimulating EC, and to result in vascular damage and vasculitis. In addition. EC was separated from human umbilical vein in a fulterm
newborn
and cultured for morphological observation.
EC was harvested by 0.2% collagenase treatment of normal term, umbilical cord veins and cultured in a 6 well culture plate. After primary and subculture, morphological observation was done and EC was stained for factor VIII related antigen.
[³H]-acetate was added to this supernatant and EC washed after 2 hours, and then stimulated with IL-6 and GM-CSF. After methanol extraction of cells and supernatants, the lipid material was phased into chlorform. Isolation and purification were
performed by thin layer chromatography(TLC) and the portion of Rf value between 0.15 and 0.22 obtained. Then beta-count was done. Also platelet activating factor [³H] Scintillation proximity assay system kit(Amhersham International plc.
Amhersham,
UK)
was used to measure the concentrations of PAF which were produced from endothelial cells and cultured supernatants stimulated by IL-6 and GM-CSF.
@ES The results are as follows:
@EN 1. The isolated cells were the exact form of EC and stained deeply with factor VIII related antigen.
2. Weibel-Palade bodies were seen in EC by electron microscope and confluent microvilli, rough endoplasmic reticula, well developed Golgi bodies, many fenestrations and lamellated myeloid bodies were also seen.
3. Cultured cells showed various forms of oval shape with processes, microvilli on the cell surfaces by scanning microscope and pseudopod-like processes were also seen.
4. IL-6 and GM-CSF stimulated cultured EC to produce PAF, but the release of PAF into supernatant were not detectable.
5. IL-6 stimulated maximal production of PAF in 30 minutes(3.0±0.4ng). As the concentration of IL-6 increased, the production of PAF increased. But there were no increments above the concentration of 10ng/ml of IL-6.
6. GM-CSF stimulated maximal production of PAF in 2 hours(3.2±0.3). As the concentration of CM-CSF increased, the production of PAF increased. But there were no increments above the concentration of 3nM of GM-CSF.
7. The stimulating effects of PAF in EC by IL-6 and GM-CSF were inhibited by each antibodies, actinomycin D and cycloheximide but not by aspirin and dexamethasone.
8. When EC were stimulated with IL-6 and CM-CSF concomitantly for 30 min, and 2hrs., there were no synergistic effects but only the effects by IL-6 alone were observed(3.5±0.5ng, 0.85±0.4ng respectively). When the stimuli were done with IL-6
first
for 30 min, and then with GM-CSF for 2hrs., the stimulating effect of production of PAF were not observed(0.9±0.3ng). But when the stimuli were done with GM-CSF first for 2hrs, and then with IL-6 for 30 min., the stimulating effects with GM-CSF
were
observed(3.2±0.4ng).
These results suggest that IL-6 and GM-CSF stimulate EC to increase the production of PAF and the effects of IL-6 and GM-CSF to inflammatory reactions might be through PAF rather than their direct effects.
|
|
|
KEYWORD
|
|
|
|
|
|
FullTexts / Linksout information
|
|
|
|
|
|
Listed journal information
|
|
|
|