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, Volume 16, Issue 1–2, pp 21–22 | Cite as

Leucocytes activated by C5a des Arg promote endothelial prostacyclin (PGI2) production via release of oxygen speciesin vitro

  • M. Rampart
  • P. J. Jose
  • T. J. Williams
Microcirculation in Inflammation


Prostacyclin (PGI2) production by an intact endothelial cell layer on the surface of isolated rabbit aortae was investigated in the presence of rabbit C5a des Arg and autologous blood leucocytes. Leucocytes themselves did not produce detectable amounts of PGI2, nor did they alter basal endothelial PGI2 formation. In the absence of leucocytes, C5a des Arg produced a dose-dependent stimulation of PGI2 release from endothelial cells. This effect was greatly enhanced when C5a des Arg and leucocytes were incubated together on the endothelium. Experiments with superoxide dismutase and catalase suggest that release of H2O2 may partly explain the enhanced PGI2 stimulatory activity of C5a des Arg in the presence of leucocytes.

Intravascular activation of the complement system in rabbits leads to the appearance of PGI2 in the circulation and trapping of leucocytes, mainly in the lungs [1]. In this, and in other inflammatory models (e.g. the peritoneal cavity of the rabbit, ref. 2), a correlation has been observed between the generation of C5a and the formation of PGI2, suggesting a causal relationship between these two events. Furthermore, C5a des Arg has been shown to be a very potent attractant for leucocytes and to induce adherence of these cells to vascular endothelium, which is considered to be a prominent feature in the development of the inflammatory response [3]. Since endothelial cells are involved in haemostasis and vasoregulation, partly via secretion of PGI2 [4], we investigated whether the interaction of leucocytes with vascular endothelial cells might affect the functional response of the latter cells, especially with respect to their ability to form PGI2.

Leucocytes were isolated from citrated rabbit blood by dextran sedimentation and differential centrifugation, followed by hypotonic lysis of remaining red cells. Leucocytes were suspended (2×106/0.1 ml) in phosphate-buffered (10 mM, pH 7.3). Dulbecco's modified Eagle medium, containing 1% fatty acid-free rabbit serum albumin. During cell preparation, the thoracic part of the aorta of the same animal was isolated and mounted in a two-plate well device, as described previously [5]. The upper plate contains six circular holes (6 mm diameter), which each form an individual incubation chamber, the base of each chamber being formed by the luminal surface of the aorta. This procedure made it possible to test the animal's own leucocytes on its own vascular endothelium within one hour after sacrifice.

Each of the six wells was incubated for 30 min at room temperature (22–25°C) with 0.1 ml medium, with or without leucocytes and different concentrations of purified rabbit C5a des Arg [2]. PGI2 production was estimated by radioimmunoassay of its non-enzymic degradation product 6-oxo-PGF [6].

Leucocytes incubated in a test tube (i.e., in the absence of endothelial cells) produced no detectable amounts of PGI2 (<0.05 pmol/2×106 cells) even in the presence of C5a des Arg (10−7M). Pretreatment of the leucocytes with cytochalasin B (5 μg/ml) for 5 min before addition of C5a des Arg (10−7M) resulted in the release of significant amounts of superoxide anion (20–50 nmol O 2 /30 min) but had no detectable effect on PGI2 formation. These results show that the lack of PGI2 formation by leucocytes was not due to an inability of the cells to respond to the stimulus.

The PGI2 formation by the aortic endothelium under different experimental conditions is summarized in the table. The presence of leucocytes did not affect basal endothelial PGI2 formation in the absence of C5a des Arg (see table, first row). In the absence of leucocytes, C5a des Arg produced a dose-dependent increase in endothelial PGI2 formation. When the same doses of C5a des Arg were incubated with endothelium in the presence of leucocytes a significantly greater stimulation in endothelial PGI2 production was observed (see table). These results show that C5a des Arg, in concentrations as low as 10−8M, can trigger endothelial PGI2 formation, via interaction with leucocytes.

Endothelial PGI2 production in the presence of leucocytes and C5a des Arg was further stimulated by the addition of superoxide dismutase, but was greatly reduced by the addition of catalase (see table). Neither of the two enzymes had any effect on the direct response of endothelium to C5a des Arg, suggesting that H2O2 may be partly responsible for the stimulatory activity of activated leucocytes, as recently reported by other investigators [7].


Prostacyclin Cytochalasin PGI2 Stimulatory Activity Endothelial Cell Layer 
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Copyright information

© Birkhäuser Verlag 1985

Authors and Affiliations

  • M. Rampart
    • 1
  • P. J. Jose
    • 1
  • T. J. Williams
    • 1
  1. 1.Department of Pharmacology, Institute of Basic Medical SciencesRoyal College of Surgeons of EnglandLondonUK

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