Differential effects of proteasome inhibition by bortezomib on murine acute graft-versus-host disease (GVHD): delayed administration of bortezomib results in increased GVHD-dependent gastrointestinal toxicity

Abstract

We have recently demonstrated that the proteasome inhibitor, bortezomib, administered immediately following murine allogeneic bone marrow transplantation (BMT) resulted in marked inhibition of acute graft-versus-host disease (GVHD) with retention of graft-versus-tumor effects. We now assessed the effects of delayed bortezomib administration (5 or more days after BMT) on GVHD. Recipient C57BL/6 (H2b) mice were lethally irradiated and given transplants of bone marrow cells and splenocytes from major histocompatibility complex (MHC)-disparate BALB/c (H2d) donors. In marked contrast to the effects of bortezomib on GVHD prevention when administered immediately after BMT, delayed bortezomib administration resulted in significant acceleration of GVHD-dependent morbidity. No toxicity was observed following delayed bortezomib administration in models where donor T cells were not coadministered, indicating that these deleterious effects were critically dependent on GVHD induction. The increase in GVHD susceptibility even occurred when late administration of bortezomib was preceded by early administration. Pathologic assessment revealed that significant increases in gastrointestinal lesions occurred following delayed bortezomib administration during GVHD. This pathology correlated with significant increases of type 1 tumor necrosis factor alpha (TNF-alpha) receptor transcription in gastrointestinal cells and with significant increases of TNF-alpha, interleukin 1beta (IL-1beta), and IL-6 levels in the serum. These results indicate that the differential effects of proteasome inhibition with bortezomib on GVHD are critically dependent on the timing of bortezomib administration.

Figure 1.

The paradoxic effect of proteasome inhibition with bortezomib on the alteration of GVHD development and mortality as determined by the timing of bortezomib administration. (A-B) B6 (H2^b) recipients of BALB/c (H2^d) 15 million bone marrow and 20 million spleen cells were treated with or without 15 μg bortezomib per dose daily. Bortezomib protected mice from GVHD mortality when administration was daily from day 0 through +2 after BMT. Significant increases in survival were observed in early bortezomib-treated mice (•) compared with GVHD control (vehicle control-treated) mice (▪; panels A and B, P < .001). Conversely, bortezomib accelerated GVHD mortality when administration was from day +5 through +7 (A) or bortezomib treatment from day 0 through +2 and day +5 through +7 (B) after BMT. Significant decreases in survival were observed in late bortezomib-treated mice (•) compared with GVHD control (vehicle control-treated) mice (▪; panel A, P < .001) and in continuous bortezomib-treated mice (▴) compared with GVHD control (vehicle control-treated) mice (▪; panel B, P < .001). Results from 1 of 3 independent experiments are presented for panels A and B. Each experiment consists of 5 to 10 mice per treatment group. (C) C.B-17 SCID (H2^d) recipients of B6 (H2^b) 40 million spleen cells were treated with or without 15 μg bortezomib per dose for 1 day. Significant decreases in survival were observed in day +5 bortezomib-treated mice (▾) compared with GVHD control (vehicle control-treated) mice (▪; panel A, P < .001) and in day +12 bortezomib-treated mice (▴) compared with GVHD control (vehicle control-treated) mice (▪; panel B, P < .001). Results from 1 of 3 independent experiments are presented. Each experiment consists of 4 to 10 mice per treatment group.

Figure 2.

Dose response of donor spleen cells necessary for the increased GVHD mortality by delayed bortezomib administration. (A-B) B6 (H2^b) recipients of BALB/c (H2^d)15 million BMCs with or without 15 million spleen cells (SCs) were treated with 15 μg bortezomib per dose daily. Morbidity was not observed in mice that received BMCs but not SCs and delayed bortezomib administration from day +5 through +7 (A) or bortezomib treatment from day 0 through +2 and from day +5 through +7 (B) after BMT. Significant decreases in survival were observed in delayed bortezomib-treated mice with SCs (▾, ▴) compared with delayed bortezomib-treated mice without SCs mice (•; panels A and B, P < .001). (C-E) B6 (H2^b) recipients of BALB/c (H2^d) 15 million bone marrow with 15 million (C), 10 million (D), or 5 million (E) SCs were treated with or without 15 μg bortezomib per dose daily. Delayed bortezomib treatment from day +12 through +14 after BMT accelerates GVHD mortality. Significant decreases in survival were observed in bortezomib-treated mice (▾) compared with mice that received PBS (no bortezomib-treatment; ▪; P < .005). Results from 1 of 3 independent experiments are presented. Each experiment consisted of 5 to 10 mice per treatment group.

Figure 3.

Effects of delayed bortezomib administration on pathologic damage to target organs of GVHD. Significant increases in histologic changes were observed in the small and large intestine, but not the liver, of mice treated with delayed bortezomib on day +12 or day +12 through day +13 at 15 μg/mouse (intravenously). Mice were assessed 16 hours after a single bortezomib or vehicle control (VC) injection (day +12) or 6 hours after a second injection (day +13) of bortezomib or VC, respectively. Three 3 to 6 mice per experimental group in each of 4 independent experiments were assessed. Tissues were assessed for histologic changes in 3 to 4 parameters as described in “Materials and methods” and the sum of these scores are represented for each tissue. Histologic changes for each parameter were assessed and graded where 0 = normal, 1 = minimal, 2 = mild, 3 = moderate, and 4 = severe. Comparison of cumulative histopathologic scores between the groups with splenocytes were analyzed by Mann-Whitney test (P < .05).

Figure 4.

Delayed administration of bortezomib significantly increases gut histopathologic damage in mice with GVHD. B6 (H2^b) recipients of BALB/c (H2^d) 10 million bone marrow and 15 million spleen cells were treated with or without 15 μg bortezomib per dose daily on day 12 after BMT. The next day (day 13) mice were humanely killed and gut tissue was collected, processed, and stained with hematoxylin and eosin. (A-C) Small intestine from mice without delayed bortezomib treatment (A) have hyperplastic crypts (c) but normal villi (v). In contrast, the small intestines from mice with delayed bortezomib treatment (B-C) have villous blunting and fusion (v), hyperplastic crypts (c), and an inflammatory infiltrate (arrow). Other areas of small intestine are ulcerated (u) with a base of inflammatory cells. (D-F) Colons from mice without delayed bortezomib treatment (D) display relatively normal morphology. In contrast, colons from mice with delayed bortezomib treatment have increased (E) goblet cell depletion and inflammatory cells (arrow) in the lamina propria. Other areas of colon (F) are ulcerated (u), have sloughed cells in crypt lumens (c), and inflammation in the ulcer base and the submucosa (Sm). Original magnification × 200.

Figure 5.

Up-regulation of TNFR1 mRNA expression in gut tissue following delayed bortezomib administration. B6 (H2^b) recipients of BALB/c (H2^d) 15 million bone marrow and 20 million spleen cells were treated with or without 15 μg bortezomib per dose daily from day +12 through +13. Small intestine and liver were collected at 6 hours after bortezomib administration (day +13) for RNA extraction. TNFR1 mRNA expression was analyzed by RPA. (A-B) Autoradiograph of TNFR1 and GAPDH bands from gut (A) and liver (B) RPA gels. (C) Quantitative levels of TNFR1 steady-state mRNA levels in the gut and liver tissue from bortezomib and vehicle control-treated animals. Quantitative levels of TNFR1 mRNA were determined by densitometric analysis and are expressed as a ratio of the band volumes of TNFR1 normalized to the GAPDH housekeeping gene. These data are representative of 2 (liver) and 4 (gut) independent experiments. NS indicates not statistically significant.

Figure 6.

Delayed bortezomib administration significantly increases serum cytokine levels in mice with GVHD. B6 (H2^b) recipients of BALB/c (H2^d) 15 million BMCs with or without 20 million spleen cells (SCs) were treated with or without 15 μg bortezomib per dose daily, day 12 to 13 after BMT. Serum was collected at 6 hours after bortezomib administration (day +13). Significant increases in TNF-α, IL-1β, and IL-6 were observed in delayed bortezomib-treated recipients with SCs compared with vehicle control-treated recipients with SCs (P < .05). Representative data from 1 of 3 independent experiments are presented, 5 serum samples per group.