Reversal of the vasoconstrictor step of hyperacute xenogeneic rejection of the liver by endothelin antagonists

Abstract

The role of endothelin in the initial vasoconstrictor step of hyperacute xenogeneic rejection was investigated. Isolated rat livers were perfused in recirculation. Perfusion with human sera provided an ex vivo model of hyperacute rejection in a discordant combination. Perfusion of 10% xenogeneic serum induced a marked (70%) and sustained reduction of the liver flow and induced the release of endothelin into the perfusion medium. In contrast, perfusion of 10% allogeneic serum or of 10% decomplemented human serum induced a weak (25%) and transient reduction of the liver flow and induced the release of minimal amounts of endothelin. The simultaneous administration of BQ 123 and BQ 788, the respective antagonists of ET(A) and ET(B) endothelin receptors, or that of bosentan, a mixed ET(A)/ET(B) antagonist, antagonized the vasoconstrictor effect of 10% xenogeneic human serum, as well as that of 10(-9) M endothelin-1. The vasoconstrictor effects of xenogeneic serum on liver circulation are, at least partly, mediated through the release of endothelin by the graft.

Figure 1

Histological study. Minimal injury was constantly observed in the group perfused with xenogeneic serum. Only a few endothelial cells (<5%) of sinusoids and portal and central veins were stained after perfusion with trypan blue. Haematoxylin-eosin-safran staining. Bar=50 μm.

Figure 2

Isolated rat livers perfused with human serum (five experiments in each group). In the control group (perfusion with buffer solution alone), the liver flow remained stable and higher than 3 ml min⁻¹ g⁻¹ during the 65 min period of observation. After perfusion with 2.5, 5, 10 and 20% fresh human serum, the liver flow dropped by 23, 53, 66 and 80%, respectively. ^*:P<0.05 vs controls.

Figure 3

Isolated rat livers perfused with rat or decomplemented human serum (five experiments in each group). In the group perfused with 10% fresh rat serum, a transient liver vasoconstriction was induced. The liver flow recovered to basal value within 10 min, and remained stable until the end of the 65 min period of observation. In the group perfused with 10% decomplemented human serum, a transient liver vasoconstriction was also observed. ^*:P<0.05 vs controls; †:P<0.01 vs controls.

Figure 4

Isolated rat livers perfused with human serum. Effect of combined endothelin antagonists (five experiments in each group). The administration of 10⁻⁶ M BQ 123 and 10⁻⁶ M BQ 788, 10 min before the serum perfusion, partially reversed the effects of xenogeneic human serum. ^*:P<0.01 vs controls; †:P<0.05 vs livers perfused with fresh human serum alone.

Figure 5

Isolated rat livers perfused with human serum. Individual effects of endothelin antagonists (five experiments in each group). The administration of 10⁻⁶ M BQ 123 and 10 min before the serum perfusion, partially reversed the effects of xenogeneic human serum, whereas that of 10⁻⁶ M BQ 788 had only marginal effect. ^*:P<0.01 vs controls; †:P<0.05 vs livers perfused with fresh human serum alone.

Figure 6

Isolated rat livers perfused with human serum. Effect of bosentan (five experiments in each group). The administration of 10⁻⁵ M bosentan, 10 min before the serum perfusion, completely reversed the effects of xenogeneic human serum. ^*:P<0.01 vs controls; †:P<0.05 vs livers perfused with fresh human serum alone.

Figure 7

Isolated rat livers perfused with rat serum. Effect of endothelin antagonists (five experiments in each group). The administration of 10⁻⁶ M BQ 123 and 10⁻⁶ M BQ 788, 10 min before the serum perfusion, inhibited the vasoconstrictive effect of rat serum. ^*:P<0.05 vs controls.

Figure 8

Endothelin production in the isolated perfused rat liver experiments (five experiments in each group). Adding fresh human xenogeneic serum into the perfusate produced a marked endothelin production after 5 min, which was significantly higher than during perfusion of decomplemented human serum or allogeneic serum. ^*:P<0.05 vs controls perfused with buffer solution.

Figure 9

Immunohistochemistry in the isolated perfused rat liver experiments. On liver biopsies taken 5 and 25 min after adding xenogeneic serum, immunofluorescence studies showed the deposition of human IgM (a) and factor Bb (b) along sinusoidal endothelial cells (patterns obtained from one representative experiment out of six). After adding decomplemented human serum, only IgM deposits were observed (c). Bar=10 μm. Deposits are indicated by arrows.