Prednisolone inhibits cytokine-induced adhesive and cytotoxic interactions between endothelial cells and neutrophils in vitro

Abstract

We assessed whether prednisolone influenced the ability of human polymorphonuclear neutrophils (PMN) to adhere to and cause lysis of human umbilical vein endothelial cells (HUVEC) in vitro (as measured by the release of 51Cr). Pretreatment of the endothelium with IL-1beta or tumour necrosis factor-alpha (TNF-alpha) caused prominent endothelial E-selectin expression and endothelial hyperadhesiveness for neutrophils, as well as PMN-mediated cytotoxicity. All these processes were dose-dependently reduced when prednisolone was added to the assay system. This protective effect remained when HUVEC alone were pretreated with the drug prior to washing and cytokine activation. Likewise, when HUVEC cytotoxicity was induced by the nitric oxide (NO) donor S-nitroso-acetyl-penicillamine (SNAP), prednisolone reduced cell injury significantly. In contrast, prednisolone did not interfere with signalling systems between TNF-alpha-stimulated HUVEC and quiescent PMN such as IL-8 generation and release of cytosolic Ca2 + in the PMN. Thus, in this in vitro model of vasculitis, prednisolone dose-dependently reduced cytokine-induced E-selectin expression and HUVEC hyperadhesiveness for neutrophils, as well as reducing neutrophil-dependent cytotoxicity against HUVEC via NO-dependent steps.

Fig. 1

Effect of prednisolone on human umbilical vein endothelial cell (HUVEC) expression of E-selectin, assessed by flow cytometry. HUVEC were activated with IL-1β (a,b) or tumour necrosis factor-alpha (TNF-α) (c,d) for 3 h. Subsequently, dispersed HUVEC were labelled with fluorescent antibody to E-selectin. (a) HUVEC activated with IL-1β or medium (control). (c) HUVEC activated with TNF-α or medium (control). (b,d) HUVEC were treated with prednisolone, at concentrations indicated, for 30 min prior to and during subsequent activation with IL-1β 5 U/ml (b) or TNF-α 10 ng/ml (d). The Figure depicts one representative experiment out of three with similar results.

Fig. 2

Effects of prednisolone on adhesion of polymorphonuclear neutrophils (PMN) to human umbilical vein endothelial cell (HUVEC) monolayers. (a) HUVEC were activated with IL-1β or tumour necrosis factor-alpha (TNF-α) at indicated concentrations for 3 h. Subsequently, HUVEC were washed three times, then PMN were added and allowed to adhere for 10 min. The results are given as the relative increase of adherent PMN compared with no stimulation, i.e. medium alone. (b) Spontaneous adhesion. HUVEC were treated with prednisolone or medium alone for 3 h and 30 min. PMN were added, and thus exposed to prednisolone (if added) for 10 min. (c) Activated adhesion. HUVEC were treated with prednisolone at indicated concentrations, or medium alone for 30 min prior to and during subsequent activation with IL-1β (5 U/ml) or TNF-α (10 ng/ml) for 3 h. Subsequently, HUVEC were washed three times, prednisolone or medium was added again. Finally, PMN were added and allowed to adhere for 10 min. The results of (b) and (c) are given as the change of adherence when compared with cells not treated with prednisolone. Mean and s.e.m. values for the number of separate experiments given at the bottom of the bars, run in triplicates. *P < 0·05; **P < 0·01; ***P < 0·001 compared with medium-treated controls.

Fig. 3

Effect of prednisolone on cytokine-induced cytotoxicity. (a) Human umbilical vein endothelial cells (HUVEC) were exposed to either 10 U/ml of IL-1β (□) for 4 h or to 100 ng/ml of tumour necrosis factor-alpha (TNF-α; hatched bar) for 24 h, rinsed, and then unstimulated polymorphonuclear neutrophils (PMN) together with prednisolone were added (the complete system). (b) HUVEC were treated for 30 min with prednisolone, rinsed and then exposed to IL-1β (□) for 4 h or to TNF-α (hatched bar) for 24 h; subsequently unstimulated PMN were added. Mean and s.e.m. values for five experiments, run in duplicates. *P < 0·05; **P < 0·01; ***P < 0·001 compared with controls, i.e. cytotoxicity induced by IL-1β or TNF-α without prednisolone.

Fig. 4

Effect of prednisolone on polymorphonuclear neutrophils (PMN) [Ca^{2 +}]_i, as reflected by changes of Fura-2 fluorescence. Human umbilical vein endothelial cells (HUVEC), treated with 100 ng/ml of tumour necrosis factor-alpha (TNF-α) for 2 h with or without pretreatment by prednisolone 50 µm for 30 min, were added (arrows) to PMN loaded with Fura-2. The figure depicts one representative tracing out of three separate experiments run in duplicate with similar results.