MODULATION OF HEME OXYGENASE (HSP-32) BY COBALT-PROTOPORPHYRIN ABROGATES DEVELOPMENT OF ACUTE GVHD IN A PARENT TO F1 MOUSE MODEL

Abstract

637 Graft versus host disease (GVHD) remains a significant cause of morbidity and mortality after bone marrow transplantation. Recently, we demonstrated that elevated expression of heme oxygenase-1 (HO-1 or Hsp-32), an enzyme responsible for the degradation of hemoglobin to biliverdin and carbon-monoxide, results in the modulation of several immune effector functions. Based on these observations we evaluated if Cobalt-protoporphyrin(CoPP), a compound well known to induce HO-1 expression can prevent the development of acute GVHD. Acute GVHD was initiated by injection of unfractionated spleen cells from C57BL/6 into B6D2/F1 mice. Subsequently, recipients were given a single intravenous injection of saline or CoPP at 2.5 or 5 mg/kg. Administration of CoPP resulted in increased survival: 85% of CoPP treated animals (6 out of 7) survived for more than 30 days compared to only 29% of control animals (2 out of 7, p < 0.1). To determine if the protective effect of CoPP therapy was due to immunomodulation of the recipient or the donor cells, donor mice were treated with different doses of CoPP three days before harvesting their spleen cells. 80% of recipients (4 out of 5) injected with spleen cells from CoPP-treated donors (20 mg/kg) survived for more than 100 days compared to none of the mice (0 out of 5) injected with spleen cells from vehicle treated donors. No significant weight change was observed in animals injected with spleen cells from CoPP treated animals. The immunocompetence of injected animals was assessed on days 2, 15 and 100. Two days after injection of vehicle-treated donor-spleen cells, an increased spontaneous proliferation of lymphocytes could be measured in vitro. Such proliferation was not seen with recipients injected with CoPP-treated donor-spleen cells. Cytokine analysis revealed elevated IFN-γ production by spleen cells isolated from mice with GVHD, while spleen cells isolated from protected recipients secreted significantly lower levels of IFN-γ. Mice with active GVHD demonstrated a defective lymphoproliferative response to alloantigens or ConA. However, spleen cells isolated from survivors (on day 100) responded normally. Furthermore, anti-host cytotoxic T cell activity was reduced in protected mice compared to mice with GVHD. Flow cytometric analysis of splenic T cell populations revealed a majority of donor type (H2b+) cells in mice with active GVHD versus a majority of recipient (H2b+, H2d+) cells in protected mice on day 45. On day 100, the spleen cell population of three out of four (75%) long-term survivors showed a normal recipient phenotype. In conclusion, results from confirmed our previous observations that upregulation of HO-1 activity is associated with down-regulation of certain immune effector functions. This results in protection from acute GVHD in a parent into F1 mouse model.