Graft-versus-host-disease-associated donor cell engraftment in an F1 hybrid model is dependent upon the Fas pathway

Abstract

The graft-versus-host disease (GVHD) generated in BDF1 mice by the injection of spleen cells from the C57BL/6 parental strain induces a direct cell-mediated attack on host lymphohaematopoietic populations, resulting in the reconstitution of the host with donor cells. We examined Fas-Fas ligand (FasL) interactions in donor and host haematopoietic cells over a prolonged period of parental-induced GVHD. Fas expression on bone marrow cells of both donor and host origin increased at 2 weeks. Host cell incubation with anti-Fas antibody induced apoptosis, and the number of haematopoietic progenitor cells decreased. Fas-induced apoptosis by the repopulating donor cells, however, did not increase until 12 weeks, when more than 90% of the cells were donor cells. The expression of various cytokines, such as interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha), and FasL gene expression in the bone marrow increased concomitantly. To examine directly whether FasL has a major role in the development of donor cell engraftment, FasL-deficient (gld) mice were used as donors. Injection of B6/gld spleen cells induced significantly less host lymphohaematopoietic depletion, resulting in a failure of donor cell engraftment. Furthermore, injection of IFN-gamma gene knockout (gko) B6 spleen cells failed to augment Fas and FasL expression in recipient mice, resulting in a failure of donor cell engraftment. This suggests that the induction of apoptosis by Fas-FasL interactions in host cells may contribute to a reconstitution of the host with donor cells and that donor-derived IFN-gamma plays a significant role for Fas-FasL interactions in host cells during parental-induced GVHD.

Figure 1

Flow cytometric analysis of bone marrow cells from BDF₁ mice injected with B6 spleen cells. BDF₁ mice were injected i.v. with 50 × 10⁶ B6 spleen cells. At 2 weeks, host bone marrow cells were harvested and donor–host chimerism and Fas‐expressing cells in the bone marrow were determined by two‐colour flow cytometry. BDF₁ host cells were distinguished from B6 donor cells by anti‐H‐2K^d mAb. Similar results were observed in five additional experiments.

Figure 2

(a) Effect of GVHD on haematopoiesis. Mice were treated as described in the experiment shown in Fig. 1, and the numbers of bone marrow cells and CFU‐GM in the bone marrow were determined as described in the Materials and Methods. Results represent mean±SD for three to four mice. (b) Anti‐Fas mAb effects on haematopoietic colony formation. The numbers of CFU‐GM in the bone marrow cells preincubated with either anti‐Fas mAb (1 µg/ml) or control mAb (1 µg/ml) for 3 hr were determined as described in the Materials and Methods. Results represent mean±SD for three to four mice and are expressed as the percentage of the numbers of CFU‐GM treated by control mAb. The percent reduction of the number of CFU‐GM : normal F₁ versus GVHD, P‐values < 0·05. Anti‐mouse αβ T‐cell receptor mAb (clone H57‐597) was used as isotype‐matched control mAb.

Figure 3

(a) Time–course of donor–host chimerism in Fas‐expressing cells over the first 12 weeks of GVHD. The frequency of Fas‐expressing cells in the GVHD bone marrow cells was determined by FCM analysis. BDF₁ host cells were distinguished from B6 donor cells by anti‐H‐2K^d mAb. Similar results were observed in two additional experiments. (b) Anti‐Fas mAb effects on donor haematopoietic cell colony formation. Mice were treated as described in the experiment shown in Fig 1, and 4 weeks after GVHD induction donor cells were enriched by treatment of the GVHD bone marrow cells with saturating concentrations of anti‐H‐2K^d mAb (clone SF1‐1.1) and Lo‐Tox rabbit complement. The numbers of CFU‐GM in the donor bone marrow cells preincubated by either anti‐Fas mAb (1 µg/ml) or control mAb (1 µg/ml) for 3 hr were determined as described in the Materials and Methods. The effect of anti‐Fas mAb on the numbers of CFU‐GM in the bone marrow cells at 12 weeks after GVHD induction was also determined. Results represent mean±SD for three to four mice and are expressed as the percentage of the numbers of CFU‐GM treated with control mAb. (c) Induction of apoptosis in bone marrow cells from GVHD mice with anti‐Fas mAb; 2 × 10⁶ T‐cell‐depleted bone marrow cells were cultured for 24 hr in the presence of either 1 µg/ml of anti‐Fas mAb or isotype‐matched control mAb, and DNA was extracted as described in the Materials and Methods, and agarose gel electrophoresis was performed. Lane 1, GVHD donor bone marrow cells treated with control mAb; lane 2, GVHD donor bone marrow cells treated with anti Fas mAb; lane 3, GVHD bone marrow cells treated with control mAb; and lane 4, GVHD bone marrow cells treated with anti‐Fas mAb. Anti‐mouse αβ T‐cell receptor mAb (clone H57‐597) was used as an isotype‐matched control mAb.

Figure 4

(a) Time–course of donor–host chimerism in Fas‐expressing cells over the first 12 weeks of B6/gld GVHD. Mice were injected by 50 × 10⁶ parental spleen cells as described in the experiment shown in Fig. 1, and the frequency of Fas‐expressing cells in the GVHD bone marrow cells was determined by FCM analysis. BDF₁ host cells were distinguished from B6/gld donor cells by anti‐H‐2K^d mAb. Similar results were observed in two additional experiments. (b) Effect of B6/gld GVHD on haematopoiesis. Mice were injected with 50 × 10⁶ parental spleen cells as described in the experiment shown in Fig. 1, and at 4 weeks, the numbers of bone marrow cells were determined as described in the Materials and Methods. Results represent mean±SD for three to four mice.

Figure 5

(a) Time–course of donor–host chimerism in Fas‐expressing cells over the first 12 weeks of IFN‐γ gko GVHD. Mice were treated as described in the experiment shown in Fig. 1, and the frequency of Fas‐expressing cells in the GVHD bone marrow cells was determined by FCM analysis. BDF₁ host cells were distinguished from IFN‐γ gkoB6 donor cells by anti‐H‐2K^d mAb. Similar results were observed in two additional experiments. (b) Effect of IFN‐γ gko GVHD on haematopoiesis. Mice were treated as described in the experiment shown in Fig. 1, and the numbers of bone marrow cells were determined as described in the Materials and Methods. Results represent mean±SD for three to four mice. (c) Semi‐quantitative analysis of cytokines and FasL mRNA expression in the bone marrow cells from IFN‐γ gko GVHD mice. BDF₁ mice were injected i.v. with 50 × 10⁶ B6 IFN‐γ gko spleen cells. At 2 weeks after GVHD induction, bone marrow cells were harvested and RT‐PCR was performed as described in the Materials and Methods. Data represent relative expression of cytokines and FasL PCR products to β‐actin internal control. Similar results were observed in three additional experiments.