PROSTAGLANDINS ENDOTHELIAL CELLS AND INFLAMMATION : THE ROLE OF PAF, MONOKINES AND CIRCULATING CELLS F. Bussolino, E. Dejana, G. Camussi, F. Breviario, G. Garbariono, D. Ghigo and A. Bosia Dipartimento di Genetica, Biologia e Chimica Medica, Torino (Italy) lnstituto Mario Negri, Milan0 (Italy) Dept. of Pathology, Buffalo (USA) Stimulated endothelial cells (ECs) synthetize and partially release PAF. We studied the possible biological role of PAF produced by ECs after stimulation with interleukin 1 (ILI) and tumor necrosis factor (TNF). We have previously shown that IL1 stimulated EC to produce PAF in a dose-dependent manner (l-50 u/ml) after a lag phase of 2 h. The maximal PAF production was reached after 6 h and declined to the basal value after 18 h (J. Clin. Invest 77, 2027, 1986). IL1 stimulates the acetyltransferase and the deacylation I reacetylation cycle of alkylacyl glycero phospholipids with specific release of arachidonate, with a time course similar to that of PAF production. The PAF production induced by TNF is transient, concentration-dependent (1 O-l 000 U/ml), and involves a deacylation / reacetylation cycle. Both IL1 and TNF induce a 20-30 % release of PAF. IL1 and TNF act independently of ECs. ECs incubated with IL1 are refractory to IL1 restimulation, but can be restimulated by TNF to produce PAF and vice versa. The pretreatment of primary or passaged (8 passages) EC culture with IL1 (l-lo/U ml for 6 h) results in increased adhesion of neutrophils (PMN). The preincubation of PMN with PAF (desensitization) or with PAF-antagonists (BN 52021, CV-3988) reduces the adhesion (1525 %). However, only primary culture EC cultures at the first passage produce PAF after IL1 stimulation, indicating that PAF is not the unique mediator of the adhesion process. A considerable amount of PAF is detectable upon addition of PMN to five times passaged ECs stimulated with IL1 but not to unstimulated ECs. Neutrophil adhesion to primary cultures of ECs was maintained after ECs fixation with paraformaldehyde. In these conditions, no PAF is extractable from ECs unstimulated or stimulated with ILl. However, the addition of PMN to ILlstimulated and fixed ECs, but not to unstimulated and fixed ECs, enables detection of PAF, indicating that neutrophils are the source of the mediator. Furthermore, the incubation of PMN with the PAF containing supernatant of primary cultures of ECs stimulated with ILl, as well as with synthetic PAF, enhances the adhesion to unstimulated and passaged ECs. These experiments suggest that IL1 promotes changes in ECs membrane able to induce the PAF production from neutrophils. Both PAF released from neutrophils and from ECs, but not PAF associated to ECs, can directly stimulate PMN adhesiveness. 172 AUGUST 1987 VOL. 34 NO. 2

Endothelial cells and inflammation: The role of PAF, monokines and circulating cells

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PROSTAGLANDINS

ENDOTHELIAL CELLS AND INFLAMMATION : THE ROLE OF PAF, MONOKINES AND CIRCULATING CELLS

F. Bussolino, E. Dejana, G. Camussi, F. Breviario, G. Garbariono, D. Ghigo and A. Bosia

Dipartimento di Genetica, Biologia e Chimica Medica, Torino (Italy) lnstituto Mario Negri, Milan0 (Italy) Dept. of Pathology, Buffalo (USA)

Stimulated endothelial cells (ECs) synthetize and partially release PAF. We studied the possible biological role of PAF produced by ECs after stimulation with interleukin 1 (ILI) and tumor necrosis factor (TNF). We have previously shown that IL1 stimulated EC to produce PAF in a dose-dependent manner (l-50 u/ml) after a lag phase of 2 h. The maximal PAF production was reached after 6 h and declined to the basal value after 18 h (J. Clin. Invest 77, 2027, 1986). IL1 stimulates the acetyltransferase and the deacylation I reacetylation cycle of alkylacyl glycero phospholipids with specific release of arachidonate, with a time course similar to that of PAF production. The PAF production induced by TNF is transient, concentration-dependent (1 O-l 000 U/ml), and involves a deacylation / reacetylation cycle. Both IL1 and TNF induce a 20-30 % release of PAF. IL1 and TNF act independently of ECs. ECs incubated with IL1 are refractory to IL1 restimulation, but can be restimulated by TNF to produce PAF and vice versa. The pretreatment of primary or passaged (8 passages) EC culture with IL1 (l-lo/U ml for 6 h) results in increased adhesion of neutrophils (PMN). The preincubation of PMN with PAF (desensitization) or with PAF-antagonists (BN 52021, CV-3988) reduces the adhesion (1525 %). However, only primary culture EC cultures at the first passage produce PAF after IL1 stimulation, indicating that PAF is not the unique mediator of the adhesion process. A considerable amount of PAF is detectable upon addition of PMN to five times passaged ECs stimulated with IL1 but not to unstimulated ECs. Neutrophil adhesion to primary cultures of ECs was maintained after ECs fixation with paraformaldehyde. In these conditions, no PAF is extractable from ECs unstimulated or stimulated with ILl. However, the addition of PMN to ILlstimulated and fixed ECs, but not to unstimulated and fixed ECs, enables detection of PAF, indicating that neutrophils are the source of the mediator. Furthermore, the incubation of PMN with the PAF containing supernatant of primary cultures of ECs stimulated with ILl, as well as with synthetic PAF, enhances the adhesion to unstimulated and passaged ECs. These experiments suggest that IL1 promotes changes in ECs membrane able to induce the PAF production from neutrophils. Both PAF released from neutrophils and from ECs, but not PAF associated to ECs, can directly stimulate PMN adhesiveness.

172 AUGUST 1987 VOL. 34 NO. 2