Effect of Total Body Irradiation, Busulfan-Cyclophosphamide, or Cyclophosphamide Conditioning on Inflammatory Cytokine Release and Development of Acute and Chronic Graft-Versus-Host Disease in H-2-Incompatible Transplanted SCID Mice

Abstract

AbstractIn a previous study, we found that total body irradiation (TBI) was essential to induce acute graft-versus-host disease (GVHD) after allogeneic H-2-incompatible splenocyte (SP) transplantation in SCID mice. SCID mice (H-2d) conditioned with cyclophosphamide and transplanted intravenously (IV) with 5 × 107 C57BL/6 (H-2b) SP developed chronic GVHD within 3 months posttransplant without any evidence of preceding acute GVHD. In this study, SCID mice were conditioned with 4 Gy TBI or non-TBI regimens, either BuCy2 (bu-sulfan 4 mg/kg/d + cyclophosphamide 100 mg/kg/d for 2 days) or Cy5 (cyclophosphamide 100 mg/kg/d for 5 days), and then transplanted IV with 5 × 107 SP. The TBI-conditioned mice were further divided into three transplant groups: (1) TBI and SP administered the same day (TBI + DO SP), (2) SP administered 4 days post-TBI (TBI + D4 SP), and (3) SP administered 7 days post-TBI (TBI + D7 SP). The severity of GVHD was compared among these groups by clinical and histologic grading. Twenty-eight of 28 mice treated with TBI + DO SP died of acute GVHD, with overwhelming diarrhea by day 15 posttransplantation. Sixteen mice treated with either TBI + D4 SP or TBI + D7 SP developed acute GVHD, but none of them died of this disorder during 30 days posttransplantation. The mice conditioned with non-TBI regimens developed chronic GVHD within 3 months without showing any detectable signs of acute GVHD. Serum and in situ colonic cytokines were determined by enzyme-linked immunosorbent assay and immunohistology respectively. TBI itself significantly increased both serum and colonic tumor necrosis factor-α (TNF-α), interleukin-1a (IL-1α), and IL-6 when compared with non-TBI regimens and normal controls. TNF-α appeared in the serum and colon 4 hours post-TBI and peaked in 24 hours, followed by increasing IL-1α and then IL-6 levels. TNF-α and IL-1α decreased rapidly within 3 to 5 days post-TBI if no allogeneic cells were transplanted. Histoincompatible transplantation augmented cytokine release, which remained elevated on day 10 in these animals. Mice treated with TBI + DO SP developed the most severe acute GVHD and had the highest levels of TNF-α, IL-1α, and IL-6. The BuCy2-condm’oned mice had the lowest cytokine levels and developed no acute GVHD. When the mice transplanted with TBI + DO SP were treated immediately with recombinant soluble human TNF receptor (rhuTNFR:Fc) 100 µg/d intraperitoneally and for the subsequent 15 days acute GVHD mortality was significantly reduced from 100% to 50% (P < .001). Although all surviving mice later developed subacute or chronic GVHD, they survived with the chronic GVHD to the follow-up at 6 months. We conclude that the agents used for conditioning are an important variable in the etiology of acute GVHD, and that those that cause the release of high levels of inflammatory cytokines predispose to acute GVHD. We further propose that the interaction of inflammatory cytokines released by conditioning with histoincompatible donor cells is a critical process in the pathogenesis of acute GVHD.