Activation-induced expression of caps and release of microparticles have been descr ed in many cell types, but it is unclear how these events are related and what their biolog cal significance is. Here, temporal and concentration studies with human renal MVEC compz red endothelial activators TNF-cx and IFN-y for capping and endothelial microparticle (Ei IP) release. Methods: The fluorescent dye La Jolla Blue (LJB) was used to visualize caps by fluorescence microscopy, using a custom filter block with excitation at 610-670 nm md emission at 690 nm. The release of EMP was detected by fluorescent dye labeled mAb's to CD31, CD51, ICAM-1, and VCAM-1. At timed intervals during incubation of MV EC with the activators, supernatants were removed for EMP assay by flow cytometry. The remaining cells were first labeled with mouse mAb to CD31, followed by GAM gG conjugated with LJB and then detached by trypsin plus EDTA and fixed on slic es. Results: Both TNF-a and IFN-y induced similar appearing caps in a similar time course. Capping increased after 3 hr and plateaued at 9 -12 hr, then declined after 18 hr. At the peak about 40 -50% of MVEC were capped but only 5 -8% of cells without activator lad caps over 24 hr. In contrast, the time course and magnitude of EMP shedding v ere different between TNF-a and IFN-y. Detectable TNF-a-induced EMP shedding ai ose after 6 hr and plateaued at 15 -18 hr; IFN-y-induced shedding was not detected until 21 hr and plateaued at 48 -72 hr. At their peaks, the TNF-a-induced EMP was 3 to 5- 'old greater than that induced by IFN-y. The results were different when other markers vere used: the TNF-a induced EMP labeled with mAb to 1CAM-1 resulted in the higiest number of EMP, followed by CD31 CD51 VCAM-1. The ICAM-1 labeled EMP were 10-fold higher than VCAM-1 labeled EMP. This ranking was not the same in c ells induced by IFN-y. Conclusion: The observation that CD31-containing caps prece led EMP shedding suggests that capping may be a prelude to shedding. Although both activators induced similar capping, but TNF-a was much more efficient in inducing EMP. This implies an additional messenger(s) linking the cause of capping to that of shedo ing, responding more strongly to TNF-a. The underlying mechanism and biological signifie: ince of capping and EMP is under investigation.
|Original language||English (US)|
|Issue number||11 PART I|
|State||Published - Dec 1 2000|
ASJC Scopus subject areas
- Cell Biology