Administration of the stress protein gp96 prolongs rat cardiac allograft survival, modifies rejection-associated inflammatory events, and induces a state of peripheral T-cell hyporesponsiveness

Abstract

High-dose gp96 has been shown to inhibit experimental autoimmune disease by a mechanism that appears to involve immunoregulatory CD4+ T cells. This study tested the hypothesis that high-dose gp96 administration modifies allograft rejection and associated inflammatory events. Wistar cardiac allografts were transplanted into Lewis recipient rats and graft function was monitored daily by palpation. Intradermal administration of gp96 purified from Wistar rat livers (100 microg) at the time of transplantation and 3 days later significantly prolonged allograft survival (14 vs 8 days in phosphate-buffered saline [PBS]-treated recipients; P = 0.009). Rejected allografts from gp96-treated animals were significantly less enlarged than allografts from their PBS-treated counterparts (2.8 vs 4.3 g; P < 0.004). Gp96 was also effective when administered on days 1 and 8 (13 vs 7 days), but not if it was derived from recipient (Lewis) liver tissue or administered on days 0, 3, and 6. In parallel studies, CD3+ T cells from gp96-treated untransplanted animals secreted less interleukin (IL)-4, IL-10, and interferon (IFN)-gamma after in vitro polyclonal stimulation than CD3+ T cells from PBS-treated animals. Gp96 administration might therefore influence the induction of immunity to coencountered antigenic challenges and inflammatory events by inducing what appears to be a state of peripheral T-cell hyporesponsiveness.

Fig 1.

Gp96 administration prolongs cardiac allograft survival and preserves cardiac function. Upper panel: Effect of Wistar gp96 administration (100 μg intradermally on days 0 and 3) on the survival of Wistar cardiac allografts transplanted into Lewis recipients. Lower panel: Cardiac graft function (heart rate) in isografted animals, untreated Lewis recipients of Wistar allografts, and Wistar gp96–treated (100 μg, days 0 and 3) and PBS-treated (days 0 and 3) Lewis recipients of Wistar allografts in the first 8 days after transplantation. Data at later time points are not presented because of the much reduced number of animals in the untreated and PBS-treated groups as a result of rejection-mediated graft loss. Inset: Cardiac graft function (heart rate) in Wistar gp96–treated (100 μg, days 0 and 3) Lewis recipients of Wistar allografts on days 9 to 14 after transplantation. Long-term allograft survival was observed in 1 gp96-treated recipient. The axes are the same as those indicated in the main figure. In all instances, data are means + SEM from groups of 5 animals

Fig 2.

Change in cardiac graft function (heart rate) in PBS-treated (days 0 and 3) and Wistar gp96–treated (100 μg, days 0 and 3) recipients of Wistar allografts in the first 8 days after transplantation. Data are expressed as a percentage of the heart rates that were recorded approximately 2 hours after surgery. Data at later time points are not presented because of the much reduced number of animals in the untreated and PBS-treated groups as a result of rejection-mediated graft loss. Inset: Change in cardiac graft function (heart rate) in Wistar gp96–treated (100 μg, days 0 and 3) Lewis recipients of Wistar allografts on days 9 to 14 after transplantation. The axes are the same as those indicated in the main figure. In all instances, data are means ± SEM (as indicated) from groups of 5 animals

Fig 3.

Effect of gp96 treatment on the inflammatory events induced by allograft rejection. Upper panel: (A) Rejected allograft from untreated animal, (B) rejection allograft from PBS-treated animal, (C) rejected allograft from Wistar gp96–treated animal (100 μg administered on days 0 and 3), (D) 35-day isograft from untreated recipient, (E) native heart. Lower panel: Internal architecture of rejected allograft from PBS-treated animal (left) and rejected allograft from Wistar gp96–treated animal (100 μg administered on days 0 and 3; right)

Fig 4.

Weights of Wistar cardiac isografts and rejected allografts. (A) Recipients received 100 μg of gp96 intradermally on days 0 and 3 (n = 5); (B) recipients received 100 μg of gp96 intradermally on days 1 and 8 (n = 3); and (C) recipients received 200 μg of gp96 intradermally on days 0 and 3 (n = 5). Data are means + SEM from the indicated number of experiments

Fig 5.

IL-4, IL-10, and IFN-γ secretion by CD3⁺ T cells from PBS-treated and gp96-treated animals after 5 days of stimulation in vitro with immobilized anti-CD3 mAb and soluble anti-CD28 mAb. Each box and whisker plot shows the median (horizontal line), quartiles (box), and extreme values (lines). Data are generated from 5 independent experiments. Although the levels of cytokines generated in different experiments varied, the comparative responses of CD3⁺ T cells from PBS-treated and gp96-treated animals in individual experiments were consistent in all experiments. Outliers for IL-4 and IFN-γ levels secreted by CD3⁺ T cells from PBS-treated animals (117 and 60 191 pg/mL, respectively) are not presented in the plots but are included in the analyses. IL-4, IL-10, and IFN-γ levels in the culture supernatants of cells that were cultured in the absence of CD28 mAb were below the detection limit of the relevant assay. The differences in cytokine secretion by cells from PBS- and gp96-treated animals were not of statistical significance