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Comparative Study
. 1999 Aug;104(4):459-67.
doi: 10.1172/JCI6896.

Differential roles of IL-1 and TNF-alpha on graft-versus-host disease and graft versus leukemia

G R Hill  1 , T Teshima, A Gerbitz, L Pan, K R Cooke, Y S Brinson, J M Crawford, J L Ferrara
Affiliations
Comparative Study

Differential roles of IL-1 and TNF-alpha on graft-versus-host disease and graft versus leukemia

G R Hill et al. J Clin Invest. 1999 Aug.

Abstract

We demonstrate an increase in graft-versus-host disease (GVHD) after experimental bone marrow transplant (BMT) when cyclophosphamide (Cy) is added to an otherwise well-tolerated dose (900 cGy) of total body irradiation (TBI). Donor T cell expansion on day +13 was increased after conditioning with Cy/TBI compared with Cy or TBI alone, although cytotoxic T lymphocyte (CTL) function was not altered. Histological analysis of the gastrointestinal tract demonstrated synergistic damage by Cy/TBI and allogeneic donor cells, which permitted increased translocation of LPS into the systemic circulation. TNF-alpha and IL-1 production in response to LPS was increased in BMT recipients after Cy/TBI conditioning. Neutralization of IL-1 significantly reduced serum LPS levels and GVHD mortality, but it did not affect donor CTL activity. By contrast, neutralization of TNF-alpha did not prevent GVHD mortality but did impair CTL activity after BMT. When P815 leukemia cells were added to the bone marrow inoculum, allogeneic BMT recipients given the TNF-alpha inhibitor relapsed at a significantly faster rate than those given the IL-1 inhibitor. To confirm that the role of TNF-alpha in graft versus leukemia (GVL) was due to effects on donor T cells, cohorts of animals were transplanted with T cells from either wild-type mice or p55 TNF-alpha receptor-deficient mice. Recipients of TNF-alpha p55 receptor-deficient T cells demonstrated a significant impairment in donor CTL activity after BMT and an increased rate of leukemic relapse compared with recipients of wild-type T cells. These data highlight the importance of conditioning in GVHD pathophysiology, and demonstrate that TNF-alpha is critical to GVL mediated by donor T cells, whereas IL-1 is not.

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Figures

Figure 1
Figure 1
GVHD severity is significantly increased after a Cy/TBI conditioning regimen. B6D2F1 recipients received allogeneic B6 donor bone marrow and T cells after 1 of 3 conditioning regimens: Cy (dotted line; n = 11), TBI (900 cGy; dashed line; n = 10), or Cy/TBI (solid line; n = 35). Syngeneic recipients received B6D2F1 bone marrow and T cells after Cy/TBI (dashed line broken by dots; n = 15). (a) Mortality was significantly (P < 0.0001) greater in allogeneic Cy/TBI group than in all others. (b) GVHD severity was determined by clinical score (as described in Methods) and was significantly greater (P < 0.001) after Cy/TBI conditioning than after all other regimens. Allogeneic groups were significantly different (P < 0.001) from syngeneic recipients from day 14 forward. Results are from 3 similar experiments.
Figure 2
Figure 2
Cy/TBI conditioning accelerates T-cell expansion. (a) Thirteen days after BMT, the numbers of CD4+ and CD8+ cells were determined in the spleens of allogeneic BMT recipients after conditioning with Cy (dotted bars; n = 9), TBI (filled bars; n = 10), or Cy/TBI (hatched bars; n = 13). CD4+ and CD8+ numbers (×106) in syngeneic BMT recipients after Cy/TBI (n = 6) were 3.2 ± 0.7 and 1.8 ± 0.3, respectively (P < 0.03 for Cy/TBI vs. Cy and TBI allogeneic and Cy/TBI syngeneic groups). (b) CTL activity of splenic T cells 13 days after BMT subsequent to conditioning with Cy (dotted line), TBI (dashed line), or Cy/TBI (solid line). Spleens from 3–4 animals in each allogeneic group were pooled and used as effectors in standard 5-hour 51Cr release assays against host-type P815 targets (top curves) or donor-type EL4 targets (bottom curves). Results represent average curves from 3 similar experiments.
Figure 3
Figure 3
Cy/TBI conditioning enhances GVHD of the GI tract and subsequent endotoxemia. (a) GI tract injury was determined 7 days after BMT by a semiquantitative histological scoring system as described in Methods. The severity of injury was significantly increased (P < 0.02) in allogeneic BMT recipients after Cy/TBI conditioning (hatched bars; n = 13) compared with recipients transplanted with syngeneic marrow after Cy/TBI conditioning (open bars; n = 6) or allogeneic recipients transplanted after TBI alone (filled bars; n = 8). (b) Serum LPS levels 7 days after BMT were significantly increased (P < 0.03) in allogeneic BMT recipients after Cy/TBI conditioning (n = 17) compared with recipients transplanted with syngeneic marrow after Cy/TBI conditioning (n = 9) or allogeneic recipients transplanted after TBI alone (n = 23). (c) Elevated levels of serum LPS 7 days after BMT correlate with the severity of GI tract injury (P < 0.01). Cy/TBI allogeneic: filled squares (n = 13); Cy/TBI syngeneic: filled circles (n = 3); TBI allogeneic: open circles (n = 2).
Figure 4
Figure 4
Cy/TBI conditioning amplifies macrophage TNF-α and IL-1β responses to LPS. Peritoneal macrophages were pooled from 4 animals per group and stimulated in vitro with 0.1 μg/mL of LPS. TNF-α (a) and IL-1β (b) were determined in the supernatant by ELISA at 4 and 48 hours, respectively, after conditioning with Cy (stippled bars), TBI (filled bars), or Cy/TBI (hatched bars). Results are expressed as mean ± SD of quadruplicate wells and represent 1 of 3 similar experiments. Macrophages from syngeneic BMT recipients transplanted after Cy/TBI conditioning produced 484 ± 41 pg of TNF-α and 8 ± 1 pg of IL-1β. P < 0.05 for Cy/TBI vs. Cy and TBI.
Figure 5
Figure 5
Cy/TBI conditioning amplifies IL-1–mediated GVHD mortality. Animals were transplanted with allogeneic bone marrow and T cells after Cy/TBI conditioning as in Figure 1. (a) Animals received TNF blockade (dashed line; n = 19) with 100 μg/dose of recombinant human TNFR:Fc on days –3, –2, –1, and 0 and then on alternate days to day +16. (b) Animals received IL-1 blockade (dotted line; n = 19) with 200 μg/dose of the hamster anti-mouse IL-1 receptor antibody (anti–IL-1R) in the same dose schedule. Control-treated animals (solid line; n = 19) received 200 μg/dose of human or hamster IgG. P < 0.01 for IL-1 blockade vs. control; P = 0.70 for TNF blockade vs. control. All control-treated syngeneic BMT recipients survived the period of observation (data not shown). Data are pooled from 2 similar experiments.
Figure 6
Figure 6
TNF-α blockade impairs GVL activity after allogeneic BMT. Allogeneic BMT recipients were transplanted with TNF (dashed line; n = 30) or IL-1 blockade (dotted line; n = 19) as in Figure 5, with the addition of 5 × 104 host-type P815 leukemic cells to the donor inoculum. Syngeneic BMT recipients (dashed line broken by dots; n = 15) were used as positive controls. (a) Overall survival; (b) leukemic relapse. P < 0.002 and P < 0.05 for survival and relapse between allogeneic arms.
Figure 7
Figure 7
Optimal CTL generation after allogeneic BMT requires TNF-α signaling through the TNF p55 receptor. (a) Spleen lytic activity (LU20) was determined 14 days after BMT in animals transplanted with wild-type T-cell–depleted bone marrow and either wild-type T cells (hatched bar) or p55 TNFR–/– T cells (solid bar). Results are from 3 experiments. P < 0.05 between groups. (b) The rate of relapse was determined after allogeneic BMT with 5 × 104 P815 cells and wild-type T-cell–depleted bone marrow and wild-type T cells (solid line; n = 30), or wild-type T-cell–depleted bone marrow and p55 TNFR–/– T cells (dashed line; n = 20). P < 0.001 between groups.
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