Research ArticleRadiation Toxicity

Bactericidal/Permeability-Increasing Protein (rBPI21) and Fluoroquinolone Mitigate Radiation-Induced Bone Marrow Aplasia and Death

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Science Translational Medicine  23 Nov 2011:
Vol. 3, Issue 110, pp. 110ra118
DOI: 10.1126/scitranslmed.3003126

Abstract

Identification of safe, effective treatments to mitigate toxicity after extensive radiation exposure has proven challenging. Only a limited number of candidate approaches have emerged, and the U.S. Food and Drug Administration has yet to approve any agent for a mass-casualty radiation disaster. Because patients undergoing hematopoietic stem cell transplantation undergo radiation treatment that produces toxicities similar to radiation-disaster exposure, we studied patients early after such treatment to identify new approaches to this problem. Patients rapidly developed endotoxemia and reduced plasma bactericidal/permeability-increasing protein (BPI), a potent endotoxin-neutralizing protein, in association with neutropenia. We hypothesized that a treatment supplying similar endotoxin-neutralizing activity might replace the BPI deficit and mitigate radiation toxicity and tested this idea in mice. A single 7-Gy radiation dose, which killed 95% of the mice by 30 days, was followed 24 hours later by twice-daily, subcutaneous injections of the recombinant BPI fragment rBPI21 or vehicle alone for 14 or 30 days, with or without an oral fluoroquinolone antibiotic with broad-spectrum antibacterial activity, including that against endotoxin-bearing Gram-negative bacteria. Compared to either fluoroquinolone alone or vehicle plus fluoroquinolone, the combined rBPI21 plus fluoroquinolone treatment improved survival, accelerated hematopoietic recovery, and promoted expansion of stem and progenitor cells. The observed efficacy of rBPI21 plus fluoroquinolone initiated 24 hours after lethal irradiation, combined with their established favorable bioactivity and safety profiles in critically ill humans, suggests the potential clinical use of this radiation mitigation strategy and supports its further evaluation.

Introduction

The U.S. Congress appropriated funds to create a therapeutics stockpile to counter biological and chemical threats in 1988. Subsequently reconfigured as the Strategic National Stockpile (SNS) and managed by the Department of Health and Human Services, SNS is a “national repository of antibiotics, chemical antidotes, antitoxins, life-support medications… designed to supplement and resupply state and local public health agencies in the event of a national emergency anywhere and at anytime” (1). Such an emergency may arise from a nuclear event, which, as the accidents at Chernobyl and Fukushima demonstrate, can present enormous challenges even without detonation-related toxicities. Rapid exposure of extensive body surface area to significant doses of penetrating radiation results in acute radiation syndrome (ARS) (24). ARS—which can affect the hematopoietic, gastrointestinal (GI), central nervous, and cardiovascular systems—can manifest within minutes and last for weeks. To date, only five agents in the SNS address radiation exposure. Three are intended to promote clearance of internal radiation: calcium and zinc DTPA (diethylenetriamine pentaacetic acid) (chelating agents for the transuranium elements plutonium, americium, and curium) and Prussian blue (an ion exchanger for cesium-137 and thallium-201). The fourth, potassium iodide, blocks thyroid uptake of radioactive iodine. Only one agent, G-CSF (granulocyte colony-stimulating factor), used to stimulate hematopoietic recovery after intensive cancer treatment, has the potential to mitigate toxicity after radiation exposure, as inferred from human cancer therapy and animal radiation models (5, 6). G-CSF is not approved for radiation-induced neutropenia and can be obtained from SNS only with Emergency Use Authorization under an Investigational New Drug application (1).

In humans and mice, both radiation dose and host characteristics determine the extent of injury after exposure. At doses that are frequently fatal within weeks (4 to 10 Gy), hematopoietic toxicity (hematopoietic syndrome) contributes to mortality as demonstrated by the success of bone marrow shielding, hematopoietic cell transplant (HCT), and G-CSF administration in supporting recovery (3, 4, 6, 7). At higher doses, death occurs earlier and even HCT does not reduce mortality (24, 6). Concomitant thermal and skin injuries compromise survival at all doses (3, 7, 8).

Postradiation bacteremia contributes to total body irradiation (TBI) toxicity (9). Radiation alters both GI mucosal integrity, resulting in translocation of bacteria and bacterial products to the systemic circulation (10, 11), and the GI microbiome, favoring predominance of Gram-negative bacteria associated with increased mortality (9). In experimental animal models, administration of antibiotics active against Gram-negative bacteria, including fluoroquinolones, generally reduces radiation-induced mortality, although efficacy varies widely depending on the model used (9, 12). Consistent with a potentially detrimental role of endogenous flora, gnotobiotic (germ-free) mice show better survival than mice with conventional microflora at equivalent radiation dose, survive longer after lethal irradiation, require higher radiation doses to induce GI histopathologic changes, and more effectively repair radiation enteritis (10, 13).

Bacteria-derived components that engage the innate immune system influence the sequelae of radiation (10, 1317). Endotoxin, found uniquely in the outer leaflet of the outer membrane of Gram-negative bacteria such as the Enterobacteriaceae that colonize the human intestinal tract, translocates into the bloodstream in both bacteria-associated and free forms after radiation-induced mucosal injury (11). Humans are exquisitely sensitive to even picogram amounts of endotoxin, which promotes activation of host defenses in extravascular tissue but produces cardiovascular and pulmonary instability, dysregulated coagulation, and systemic inflammation during endotoxemia. Endotoxin sensitivity can be modified by changes in expression of host endotoxin-recognition proteins that promote either endotoxin activity or detoxification and enhanced clearance of endotoxin (1820). Of these host proteins, BPI (bactericidal/permeability-increasing protein), a 50- to 55-kD cationic antimicrobial protein found primarily in the azurophilic granules of human neutrophils, has the highest affinity (picomolar to nanomolar) for endotoxin (21). Most BPI is intracellular, but plasma concentrations of BPI rise with neutrophil activation and degranulation. BPI binding to endotoxin is anti-infective, promoting killing and opsonization of Gram-negative bacteria (21). Although conventional antibiotics can kill bacteria, antibiotics do not promote opsonization, nor do they mitigate the inflammatory responses induced by cell-free components derived from dead or dying bacteria. Because BPI binds both bacteria-associated and cell-free endotoxin, it effectively inhibits endotoxin-induced inflammation and apoptosis by precluding endotoxin ligation of the cellular proinflammatory endotoxin receptor complex composed of mCD14, MD-2, and TLR4 (Toll-like receptor 4) (21).

Herein, we demonstrate that humans undergoing myeloablative HCT become deficient in the endotoxin-neutralizing BPI protein, concurrent with treatment-related endotoxemia. Using a murine model, we then demonstrate that BPI supplementation of standard antibiotic treatment is an effective radiation mitigation strategy.

Results

Effect of myeloablative HCT on neutropenia, endotoxemia, BPI, and host responses to endotoxin in patients

We examined endotoxin and plasma BPI concentrations in an observational cohort study of patients undergoing myeloablative conditioning for HCT (table S1). As expected, myeloablative therapy followed by allogeneic hematopoietic stem cell (HSC) infusion resulted in a fall and recovery in the number of peripheral blood neutrophils (Fig. 1A). By the completion of myeloablative conditioning on day 0 (D0), endotoxemia was readily detectable (Fig. 1B). Simultaneously, plasma BPI concentrations declined rapidly (median decrease of 71-fold on D7; interquartile range, 9- to 193-fold; Fig. 1B), correlating with the absolute neutrophil count (ANC; Spearman r = 0.66; P < 0.001). At the ANC nadir (D7), plasma BPI was undetectable (<100 pg/ml) in 37 of 48 (77%) patients, and 8 of the 10 patients evaluated by our endotoxin activity assay were endotoxemic.

Fig. 1

Changes in peripheral blood counts and endotoxin-related parameters after myeloablative HCT in humans. (A) Pattern of ANC and platelet (PLT) fall and recovery after myeloablative conditioning treatment. Severe neutropenia (n = 46) and thrombocytopenia occurred (n = 48, nadir D7). Data represent geometric means ± SEM of log-transformed values labeled in original units. (B) Effect of myeloablative HCT on plasma endotoxin and BPI concentrations. Plasma endotoxin was evaluated in 18 patients by endotoxin activity (EA) assay at baseline (B; n = 17) and for D0 (n = 17), D7 (n = 10), D14 (n = 15), D21 (n = 15), and D28 (n = 3) after myeloablation. The horizontal dashed line (at 0.4 U) indicates the lower limit of detection. Plasma BPI concentrations (in picograms per milliliter) were assessed by ELISA at B (n = 48), D0 (n = 46), D7 (n = 48), D14 (n = 48), D21 (n = 47), and D28 (n = 33). The dotted line indicates the lower limit of detection for the BPI ELISA (<100 pg/ml). Samples below the lower limit of detection were assigned a value of 50% of this limit. (C) Effect of myeloablative HCT on endotoxin receptor components expressed on monocytes. Monocyte mCD14 and TLR4 surface expression by flow cytometry reached a concurrent nadir for mCD14 (n = 10) and peak for TLR4 at D0 (n = 9). Data represent mean fluorescence intensity (MFI) (mCD14) or binding index (TLR4) geometric means ± SEM of log-transformed values labeled in original units. (D) Effect of myeloablative HCT on plasma IL-6 (n = 37) and fever (n = 48). IL-6 data represent geometric means ± SEM of log-transformed values labeled in original units.

The mCD14 and TLR4 components of the TLR endotoxin receptor on peripheral blood monocytes exhibited decreased and increased surface expression, respectively, on D0 consistent with exposure to bioactive endotoxin (Fig. 1C) (20, 21). Subsequent elevation of interleukin-6 (IL-6) and fever, well described as frequent sequelae of endotoxemia (22), was maximal at the BPI nadir (D7, Fig. 1D). Intrapatient changes in IL-6 levels were positively correlated with the endotoxin activity values (Spearman r = 0.48; P = 0.01). Higher IL-6 concentrations were inversely correlated with BPI concentrations (Spearman r = −0.30; P < 0.0001). Although fever and BPI levels showed no association on D7, perhaps because nearly 80% of patients had undetectable BPI, on D14, patients with fever had lower BPI concentrations than afebrile patients (medians: undetectable versus 3475 pg/ml, P = 0.01). Notably, lower plasma BPI concentrations on D0, immediately before HSC infusion, were associated with slower neutrophil engraftment (Spearman r = −0.33; P = 0.03).

These findings suggested that BPI deficiency coupled with endotoxemia could exacerbate endotoxin-related toxicity after myeloablation and might impair engraftment, raising the possibility that BPI supplementation could attenuate these toxicities. HSC administration is not a readily deployable strategy for mass radiation exposure. Because mitigation of total body irradiation (TBI) without HSC treatment cannot be addressed experimentally in humans, we used a murine model to test our hypothesis.

Characterization of the toxicity of 7-Gy single-fraction TBI in BALB/c mice

To model potentially lethal radiation exposure in mice, we defined a dose of single-fraction TBI associated with bone marrow aplasia, GI damage, and substantial early mortality in BALB/c mice. A single-fraction dose of 7 Gy caused 95 to 100% mortality by D30 (LD95/30) in 12-week-old male BALB/c (Fig. 2A). The lethality of 7-Gy exposure was highly reproducible: Only 5 of 110 (4.5%) 7 Gy–irradiated mice survived to D30, and median survival in separate experiments ranged from 12 to 15 days. After 7-Gy TBI, colonic epithelial apoptosis increased and peaked at D3, and was associated with a parallel fall in plasma citrulline levels, which reflects reduced functional GI enterocyte mass (23) (Fig. 2B). Both of these GI mucosal parameters improved by D6 to D9. Endotoxemia was also detectable by D3 and persisted until peaking higher just before death (fig. S1). By D3, the bone marrow was aplastic (Fig. 2C), with an about two-log fall in bone marrow mononuclear cell content, including a decrement in progenitor (LK, LinSca-1c-Kit+) and hematopoietic stem (LSK, LinSca-1+c-Kit+) cells (Fig. 2, D to F).

Fig. 2

Bone marrow ablation and mortality after treatment of BALB/c mice with 7 Gy. (A) D30 mortality after 6-Gy (n = 9), 6.5-Gy (n = 19), and 7-Gy (n = 20) TBI differed by dose (P < 0.001, Mantel-Cox log-rank). (B) Rapid onset of mucosal damage documented by peak colonic epithelial apoptosis, measured as per Materials and Methods and reported as apoptoses per 40× microscopic field (Apopt/40×), at D3 after TBI (n = 5), coincident with nadir in plasma citrulline levels (n = 11), depicted normalized to the mean value in unirradiated mice (n = 12; D0 = 100%). Data represent means ± SEM. **P < 0.01, ***P < 0.001 (comparisons versus D0). (C) Representative H&E-stained femur section demonstrates bone marrow ablation D3 after 7-Gy TBI. Scale bar, 1 mm. (D) Number of bone marrow mononuclear cells (BM MNC) counted after 0 Gy (normal controls, n = 3 per time point), 6.5 Gy (n = 8 per time point), and 7 Gy (n = 8 on D3 and D10, n = 6 on D15 due to greater mortality). Data are the means ± SD of individual counts. Fewer bone marrow mononuclear cells were present after 7 Gy than after 6.5 Gy (D3, P = 0.05; D10, P = 0.0002; D15, P = 0.02). (E and F) Flow cytometry analysis of LK (E) and LSK (F) cells in the bone marrow of the same mice indicated that 7 Gy produced prolonged reduction in progenitor and HSC numbers. By D15, 6.5-Gy mice had greater LK and LSK cell numbers than 7-Gy mice (P = 0.01 for both LK and LSK). Each symbol represents the absolute number of LK or LSK cells within bone marrow from one limb of an individual animal. Median values are indicated by horizontal bars. Data in (B) and (D) to (F) were analyzed by Mann-Whitney test.

To ensure that the model was adequately myeloablative, we compared the hematopoietic effects of 7 and 6.5 Gy. Despite identical early histologic aplasia, mice had significantly fewer bone marrow mononuclear cells after 7 Gy on D3, D10, and D15 than after 6.5 Gy (Fig. 2D). On day 15, LK and LSK cell numbers were significantly lower in the 7-Gy group (Fig. 2, E and F). Subsequent experiments were performed with 7 Gy.

Effect of rBPI21 and enrofloxacin administration on TBI-related mortality

The combination (rBPI21/ENR) of rBPI21 and oral ENR (enrofloxacin), a veterinary fluoroquinolone antibiotic analogous to ciprofloxacin (24), initiated 24 hours after 7-Gy TBI and continued through D30, significantly improved D30 survival when compared to the effect of VEH/ENR (VEH denotes the formulation buffer for rBPI21), ENR, or 7 Gy alone (Fig. 3A and fig. S2). Moreover, only 4 deaths of 53 at-risk rBPI21/ENR-treated mice occurred after D14, whereas losses in the other groups ranged from 38 to 83% of at-risk animals. Seventeen irradiated, rBPI21/ENR-treated mice in two separate studies, performed about 4 months apart, were followed beyond D30. As of D242 and D131, respectively, all are alive and appear healthy. Three irradiated, ENR-treated mice from the first of the two studies were also followed past D30. One died at D68 and two healthy-appearing mice are still alive at D242.

Fig. 3

Effect of rBPI21 in combination with ENR on survival of BALB/c mice after 7-Gy TBI. (A) Survival of mice irradiated with 7 Gy given ENR plus rBPI21 or VEH, ENR alone, or no treatment (denoted 7 Gy) 24 hours after irradiation and continuing for 30 days. In a composite analysis of three replicate experiments, survival of rBPI21/ENR-treated mice exceeded that of the other groups (P < 0.0001 by Mantel-Cox log-rank, n = 70 mice per arm). Survival of the rBPI21/ENR group also exceeded that of VEH/ENR, ENR, and 7 Gy (P < 0.0001, 0.008, and <0.0001, respectively, by pairwise Mantel-Cox log-rank). (B) Survival of mice irradiated with 7 Gy given rBPI21 or VEH (continued for either 14 or 30 days) plus ENR (continued for 30 days) or no treatment (denoted 7 Gy) 24 hours after irradiation. Survival was unaffected by duration of rBPI21 treatment. Data were analyzed by pairwise Mantel-Cox log-rank (n = 20 mice per group).

rBPI21 has a 3-hour half-life in mice when administered by intravenous bolus or subcutaneous injection. Because optimal continuous or 6-hour interval intravenous or subcutaneous injection regimens were not feasible, we elected to use twice-daily subcutaneous administration, initiating all treatments 24 hours after TBI and continuing through D30. As illustrated in Fig. 3A, VEH/ENR, the control for the rBPI21/ENR regimen, was associated with worse D30 survival than oral ENR alone (P = 0.0002, by pairwise Mantel-Cox log-rank), suggesting that repetitive handling and trauma entailed by subcutaneous administration caused significant toxicity. We therefore explored a curtailed schedule, stopping injections after 14 days [denoted rBPI21(14) and VEH(14) in Fig. 3B]. Reasoning that oral antibiotic treatment could be more readily deployed in a mass-casualty setting, we did not change the ENR schedule. rBPI21(14)/ENR provided the same survival advantage as the longer schedule (Fig. 3B). Six irradiated mice that had received rBPI21(14)/ENR were followed beyond D30, and five of six mice remained alive and healthy-appearing at D131.

Characterization of mitigation treatment on GI mucosal mass and plasma levels of inflammatory cytokines early after TBI

Neither rBPI21/ENR nor VEH/ENR had a significant effect on the radiation-induced decrease in GI mucosal mass, as reflected by plasma citrulline levels, or subsequent rapid mucosal recovery (fig. S3). Treatment with ENR, VEH/ENR, or rBPI21/ENR had little differential effect on systemic concentrations of multiple cytokines and chemokines frequently altered early after TBI, although increases in CCL2 and IL-6 concentrations on D6 were significantly greater in rBPI21/ENR-treated mice (table S2; see Discussion).

Mitigation of hematopoietic toxicity by rBPI21/ENR administration after TBI

To characterize effects on hematopoiesis, we enumerated bone marrow mononuclear cells retrieved from flushed bone marrow cavities (Fig. 4). At D10, all irradiated groups exhibited fewer bone marrow mononuclear cells than unirradiated, age-matched normals (P < 0.0001). However, rBPI21/ENR-treated mice had significantly more bone marrow mononuclear cells than any other irradiated cohort at D10, D15, and D19 (Fig. 4, left-hand panels). On D19, rBPI21/ENR-treated irradiated mice had 80 to 90% bone marrow cellularity, in contrast to ~20%, <5%, and 10 to 50% in the 7 Gy–alone, ENR, and VEH/ENR groups, respectively (Fig. 4, right-hand panels). Although all surviving mice demonstrated improved cellularity at D30, including the rare survivor of 7 Gy alone, rBPI21/ENR-treated mice continued to demonstrate greater cellularity (Fig. 5, A to E) and significantly more bone marrow mononuclear cells than the other groups (Fig. 5F). Trilineage hematopoiesis was observed in all surviving mice (fig. S4).

Fig. 4

Effect of rBPI21/ENR on hematopoietic recovery after TBI-induced aplasia. BALB/c bone marrow mononuclear cell count (one hindlimb) and histopathology (contralateral hindlimb) were assessed at D10, D15, and D19. (A and B) Data shown for (A) untreated, unirradiated, age-matched controls (normal) or (B) 7 Gy–irradiated mice. (C to E) Other mice received both 7-Gy TBI and the following treatments started 24 hours after irradiation: (C) ENR, (D) VEH/ENR, or (E) rBPI21/ENR. (Left panels) Each graph shows counts (means ± SD) of the number of bone marrow mononuclear cells flushed from a hind leg of eight individual mice per group, except (B) where n = 2 to 8 per time point due to early mortality observed after 7 Gy alone. rBPI21/ENR resulted in improved bone marrow cellularity compared to 7 Gy, ENR, and VEH/ENR on D10 (P = 0.0003, 0.001, and <0.0001, respectively), D15 (P = 0.0007, 0.001, and 0.001, respectively), and D19 (P = 0.0006, <0.0001, and <0.0001, respectively). At each time point, all irradiated groups had fewer bone marrow mononuclear cells than controls (P < 0.001). Data were analyzed by Mann-Whitney. Data are aggregated from two replicate studies. (Right panels) Representative D19 H&E-stained femur sections show the close correlation of bone marrow mononuclear cell counts with bone marrow histology.

Fig. 5

Effect of rBPI21/ENR treatment on bone marrow cellularity on D30 after irradiation. (A to E) Bone marrow histopathology (one hindlimb) in mice surviving to D30. Representative femur histology is shown for (A) untreated, age-matched normal or (B) 7 Gy–irradiated mice. Other mice received both 7-Gy TBI and the following treatments started 24 hours after irradiation: (C) ENR, (D) VEH/ENR, or (E) rBPI21/ENR. (F) Mononuclear cell count (contralateral hindlimb) was assessed by flushing cells from a hind leg of individual mice. Bars show means ± SD for n = 4, 3, 12, 7, and 16 mice per group, respectively. The early mortality of 7 Gy alone and VEH/ENR-treated mice limited the cohort sizes. Only rBPI21/ENR treatment resulted in bone marrow mononuclear cell counts statistically indistinguishable from 0 Gy. rBPI21/ENR mononuclear cell counts also differed from counts in 7 Gy, ENR, and VEH/ENR (P = 0.01, 0.0002, and 0.001, respectively). Data from two replicate studies are shown. Data were analyzed by Mann-Whitney. ns, not significant.

Although less than that of unirradiated normal mice at each time point, the absolute number of both LSK and LK cells per hindlimb in rBPI21/ENR-treated mice was significantly greater at both D10 and D19 than in other irradiated groups (Fig. 6). Only rBPI21/ENR treatment was associated with a difference from the untreated, irradiated group. At D30, there was no difference in LSK or LK cell number among surviving mice between treatment groups or between treatment and normal controls.

Fig. 6

Effect of rBPI21/ENR treatment on expansion of early hematopoietic cells after 7-Gy TBI. Flow cytometry was used to quantify LK (left panels) and LSK (right panels) cells contained within bone marrow mononuclear cells of age-matched untreated controls (0 Gy) or mice administered 7 Gy and initiated on no treatment (7 Gy), ENR, VEH/ENR, or rBPI21/ENR treatments 24 hours thereafter. Results from D10, D19, and D30 are shown. Box and whisker graphs depict the range, 25th and 75th percentiles, and median number of LK or LSK phenotype cells within bone marrow from one hindlimb of each animal in each treatment group. n = 4 for 0-Gy controls at all time points. n = 8 mice per treatment on D10. n = 6 to 8 mice per treatment at D19. Unequal D30 survival resulted in n = 3 (7 Gy), 12 (ENR), 7 (VEH/ENR), and 16 (rBPI21/ENR) mice per group. Compared to 7 Gy, ENR, or VEH/ENR, rBPI21/ENR treatment was associated with greater numbers of LK and LSK cells at early time points (P = 0.004 for all comparisons on D10 and P = 0.004, 0.0003, and 0.0001 on D19, respectively). D30 LK and LSK content of all groups, including normals, was equivalent. Data from two replicate experiments are shown. Data were analyzed by Mann-Whitney.

Bone marrow cellularity changes correlated with changes in peripheral blood counts, which demonstrated rapid and prolonged depression after 7 Gy (Table 1). By D19, rBPI21/ENR-treated mice showed greater recovery of white blood cell, neutrophil, monocyte, and platelet numbers than did any other group; recovery of median white blood cell, neutrophil, and monocyte numbers to normal values occurred only in rBPI21/ENR-treated mice. The white blood cell and neutrophil counts of rBPI21/ENR-treated mice remained significantly greater than in ENR only–treated animals at D30. Equivalent mitigation of hematopoietic toxicity was observed with the shorter rBPI21(14)/ENR schedule (figs. S5 and S6).

Table 1

Peripheral blood counts after TBI by treatment group. Data are shown as median (range). Bold values indicate that the median falls in the normal range of 0-Gy controls. All P < 0.05. Aggregate data from two replicate experiments. Zero-gray data pooled from D15 to D30, n = 11. On D15/19/30, 7 Gy n = 5/2/3, ENR n = 7/4/12, VEH/ENR n = 5/7/7, and rBPI21/ENR n = 5/6/13.

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Discussion

By studying a patient cohort undergoing myeloablative HCT, we identified molecular and cellular changes potentially related to the toxicity of TBI. The neutropenia that routinely follows myeloablative treatment was associated with rapid depletion of plasma BPI, a neutrophil-derived protein with potent endotoxin-neutralizing activity (19, 21), concurrent with endotoxemia. These changes paralleled cellular (mCD14 and TLR4 surface expression), plasma (IL-6), and physiologic (fever) alterations consistent with increased systemic endotoxin activity (1820, 22, 25). We also observed that lower plasma BPI concentrations at the time of HSC infusion correlated with slower myeloid engraftment, suggesting that endotoxin might directly or indirectly exert a negative influence on HSC at the time of infusion and for a period thereafter. We therefore explored the hypothesis that exogenous BPI supplementation might mitigate the toxicity of exposure to TBI doses that produce mucosal injury, endotoxemia, and prolonged bone marrow aplasia.

We selected rBPI21 and a fluoroquinolone antibiotic (ENR) as components of a mitigation strategy that could be readily deployed; both agents have biologic activity and highly favorable safety profiles in healthy and ill humans, including those with multiorgan compromise (22, 2632). Using an LD95/30 single-fraction myeloablative TBI model in BALB/c mice, we demonstrated that treatment with the rBPI21/ENR combination, initiated 24 hours after radiation, was associated with survival rates of 65 to 80%, significantly greater than the 0 to 25% observed with VEH/ENR treatment. rBPI21 alone did not improve survival. Fluoroquinolones have shown variable efficacy as radiation mitigators, potentially due to differences in experimental design (9, 12). In our hands, despite the repetitive injury by rBPI21 injection to the irradiated, myeloablated mice, survival after ENR-alone treatment was significantly worse, about half or less, than observed after rBPI21/ENR treatment. Most striking, surviving mice on ENR or VEH/ENR treatment exhibited markedly delayed and inferior hematopoietic recovery than did those treated with rBPI21/ENR.

Hematopoietic syndrome contributes significantly to the morbidity and mortality of ARS in humans (24, 7, 33), underscoring the relevance of the observed rBPI21/ENR effects on hematopoiesis. Radioprotectants (that is, agents administered before TBI) and potential radiation mitigation strategies generally share some capacity to improve hematologic recovery (24, 14, 15, 3438). Hematopoietic syndrome mitigation by resource-intensive allogeneic HCT is unlikely to be rapidly, successfully implemented during a mass radiation exposure (6, 7, 33). Although the efficacy of specific mitigation agents to date has generally been dependent on administration within minutes to hours after radiation exposure (24, 12, 3539), strategies that can be delayed for at least 24 hours are highly desirable.

There is no established radiation dosimetry technology that can accurately triage exposed individuals and determine those most likely to require mitigation treatment, nor is there a human therapeutic application of TBI without HSC support in which to study the efficacy and toxicity of radiation mitigation agents. These limitations highlight the importance of selecting strategies unlikely to produce toxicity in either minimally affected or critically ill populations. The components of the strategy reported here meet this criterion. Ciprofloxacin, the human-use equivalent of the veterinary fluoroquinolone ENR, was Food and Drug Administration–approved in 1987. Fluoroquinolones have excellent oral bioavailability, are well tolerated (31, 32), and have been extensively used after myeloablative chemoradiotherapy. rBPI21 is available in a soluble form that is stable stored at 2 to 8°C, facilitating stockpiling. It can be administered subcutaneously, intravenously, or intraperitoneally. In addition to showing efficacy in animal models of pure endotoxemia and Gram-negative bacteremia, rBPI21 can abrogate the signs and symptoms of endotoxemia in humans, including associated cytokine dysregulation and coagulopathy (22, 29). rBPI21 has advantages as an endotoxin-neutralizing agent over anti-endotoxin antibodies, which have limited range and potency (40) and, in contrast to rBPI21 (28), failed to demonstrate benefit in sepsis studies (41). Moreover, no significant toxicity has been seen in phase 1 to 3 trials of more than 1100 normal volunteers and critically ill patients, including infants and subjects with meningococcemia or undergoing major operative procedures (22, 2630). In aggregate, these data suggest that rBPI21/ENR could be safely administered to individuals with poorly documented degrees of radiation exposure.

Given the relatively low sensitivity of mice to endotoxin in comparison to humans (19, 42), suboptimal dosing schedule, and relatively greater trauma of repetitive subcutaneous injection in mice (the readily accessible ventrolateral body surface area of a human is about 100-fold larger than that of an entire mouse), our results may underestimate the potential benefit of this combination treatment. The survival of mice receiving rBPI21/ENR treatment 24 hours after TBI exceeded that observed at the same time point in similar murine studies, in which mitigators were effective only when initiated 10 min to 4 hours (16, 36, 39, 43) or up to 20 hours (38) after radiation, and weighs heavily in its favor. Although direct comparison is difficult, our data also suggest that rBPI21/ENR treatment is associated with earlier and larger improvement in hematopoietic parameters than seen with other treatments (36, 38, 39, 43).

Endotoxin-mediated signaling through TLR4 can alter bone marrow HSC and progenitor numbers and function, with resulting myelosuppression (44). Nevertheless, the mechanism underlying the radiation mitigation effect of rBPI21/ENR may be multifactorial and extend beyond prevention of endotoxin-induced TLR4 signaling. In that vein, the enhancement of plasma IL-6 concentrations early after initiation of rBPI21/ENR treatment is particularly noteworthy, because IL-6 has been shown to contribute to hematopoietic recovery after TBI (45, 46). rBPI has been previously shown to reduce the IL-6 response to systemic endotoxin when coadministered with purified endotoxin (22, 25). Whether the unexpected effect of rBPI21 on IL-6 that we observed is a consequence of the temporal dissociation between rBPI21 administration and onset of endotoxemia, the difference between a radiation and a purified endotoxin challenge, the mitigation of radiation (endotoxin)–induced toxicity to IL-6–producing cells, and/or selective inhibition of endotoxin but not other microbial and/or endogenous immune system agonists requires further study. The enhancement of CCL2 levels when treatment of irradiated mice included rBPI21 could also contribute to hematopoietic recovery by reducing hematopoietic progenitor cycling (47) and susceptibility to further toxic insults, in a manner postulated to underlie the radiation mitigation activity of PD0332991, a CDK4/6 (cyclin-dependent kinase 4/6) inhibitor (38). Lastly, the antibacterial activity of rBPI21, in combination with a fluoroquinolone antibiotic, might also provide an anti-infective benefit and reduce the potential for emergence of resistant bacterial species in infection-prone, irradiated individuals. Thus, the aggregate effects of our proposed strategy may have the potential to limit the scope and duration of supportive care as well as improve survival.

Global concerns about radiation injury consequent to natural disasters, nuclear conflict or terrorism, or as an untoward consequence of intentional medical exposure have stimulated federal funding that supports development of potential mitigation agents. To date, these have been limited in number and do not yet have optimal characteristics. Given the obligate overtreatment necessitated by current limitations of radiation dosimetry and the human safety record of rBPI21 and fluoroquinolones in critically ill and/or myeloablated individuals, the approach outlined here has advantages in comparison to many agents, for which such human data are unlikely to be available before a catastrophic event. Moreover, the observed efficacy of our approach after a delay of 24 hours is particularly important for practical application. Further exploration of this approach, including optimization of the formulation and therapeutic regimen and standardized comparison to other potential mitigators, appears warranted.

Materials and Methods

Patient characteristics

Patients (n = 48) undergoing myeloablative allogeneic HCT from 2005 to 2009 at Children’s Hospital Boston (CHB) or Brigham and Women’s Hospital (BWH) were recruited prospectively onto an Institutional Review Board–approved study. All participants and/or legal guardians gave consent as appropriate. Patient and treatment characteristics are shown in table S1. Supportive care was per institutional routine (48, 49). Prophylactic oral nonabsorbable antibiotics were administered: bacitracin and polymyxin (BWH) or vancomycin (CHB). Blood counts and cultures were performed in clinical laboratories. Maximal temperature was recorded on the day of sample acquisition ± 1 day. Endotoxin Activity Assay measurements, which require fresh samples, could only be performed on patients enrolled after the assay became available.

Blood collection and plasma preparation

Peripheral blood samples were drawn into K2-EDTA or sodium heparin Vacutainers (Becton-Dickinson) before conditioning (baseline), on the day of HCT (D0), and weekly ± 1 day, spun at 1200g for 5 min at 4°C, recovered, aliquoted, and stored in pyrogen-free tubes at −80°C.

Human BPI and IL-6 enzyme-linked immunosorbent assay

BPI was measured by enzyme-linked immunosorbent assay (ELISA) (HyCult), according to the manufacturer’s instructions. IL-6 was measured by flow cytometry (MoFlo, DakoCytomation) with antibody-coated fluorescent beads (BD Biosciences) and Summit v4.0 software (DakoCytomation).

Human endotoxin, TLR4, and mCD14 measurements

Endotoxin was measured by Endotoxin Activity Assay according to the manufacturer’s instructions (Spectral Diagnostics). Monocyte surface expression of CD14 and TLR4 was measured with antigen-specific or isotype control monoclonal antibodies (eBioscience) as described (50).

In vivo radiation mitigation studies with rBPI21 and ENR

Male BALB/c mice (Charles River) were acclimated before irradiation at age 12 weeks. Studies were conducted in accordance with Dana-Farber Cancer Institute’s Animal Care and Use Committee–approved protocols. Mice were placed into a Rad Disk rodent microisolation irradiation cage (Braintree Scientific) and administered a single 7-Gy dose by a Gammacell 40Exactor (Best Theratronics) cesium source irradiator. Twenty-four hours thereafter, mice were either left untreated or received one or more of the following treatments for 14 or 30 days: (i) rBPI21 (XOMA), 250 μl per injection of a stock (2 mg/ml) constituted in formulation buffer (denoted VEH) and administered subcutaneously twice daily, 6 to 8 hours apart (rBPI21 per mouse was ~42 mg/kg per day); (ii) 250 μl of VEH consisting of citric acid (0.33 g/liter), sodium citrate (1.01 g/liter), sodium chloride (8.76 g/liter), poloxamer 188 (2.0 g/liter), and polysorbate 80 (2.0 g/liter) (Sigma) dissolved in water for injection, pH adjusted to 5.0, and filter-sterilized; (iii) Baytril (ENR, MedVets) at 10 mg/kg per day by gavage via 25-gauge feeding needles (Cadence Science) for the first 5 to 7 days, after which mice continued to receive antibiotic ad libitum in water bottles until study termination or death. All mice were observed at least twice daily. Moribund mice were euthanized via CO2 asphyxiation. At scheduled time points, mice were killed humanely via isoflurane anesthetic overdose (IsoFlo, Abbott Labs).

Blood and tissue preparation

Blood counts were determined with a Hemavet 950 FS hematology analyzer (Drew Scientific) with EDTA-anticoagulated (Becton-Dickinson) cardiac blood. Plasma was obtained by mixing blood with pyrogen-free heparin (APP Pharmaceuticals), in pyrogen-free Eppendorf tubes (USA Scientific), and centrifugation at 14,000 rpm for 10 min. Single-use aliquots were stored at −80°C. In some studies, femurs and tibiae from one leg/animal were dissected, fixed for 24 hours in 10% neutral buffered formalin (Fisher Scientific), and processed, including coronal sectioning and hematoxylin and eosin (H&E) staining (Specialized Histopathology Services, Longwood). Contralateral femurs and tibiae were taken for bone marrow mononuclear cell enumeration and flow cytometry by flushing cells from medullary cavities with cold RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) (JRH Biosciences), l-glutamine, Hepes, penicillin/streptomycin, and gentamicin (all from Invitrogen). Red blood cells were lysed with hypotonic lysing buffer (Sigma). Bone marrow mononuclear cells were stained with trypan blue. Dye-excluding viable cells were counted; viability was typically >90 to 95%.

Citrulline determinations

Samples were analyzed with the MassTrak Amino Acid Analysis system (Waters) with AccQ-Tag derivatization and ultraviolet/visible detection.

Histopathologic evaluations

A board-certified hematopathologist (J.K.) assessed femoral bone marrow cellularity on decalcified, formalin-fixed, H&E-stained, paraffin-fixed sections with an Olympus BX51 microscope, an Olympus DP71 camera, and DP Capture software. Two slides with two fields per slide were scored per animal for the percent of bone marrow space occupied by hematopoietic cells. A board-certified pathologist (J.-A.V.) enumerated apoptotic bodies/40× field in triplicate samples of H&E-stained, paraffin-fixed colon sections. Samples from normal mice were identified, but all others were de-identified and presented for analysis in random order.

Murine endotoxin and cytokine measurements

Endotoxin was measured with the Limulus amebocyte lysate assay according to the manufacturer’s instructions (Charles River). Multicytokine analysis was conducted with a commercially available fluorescent bead-based Luminex assay (Millipore) per the manufacturer’s instructions. Data were acquired on a Bio-Plex 200 Analyzer (Bio-Rad).

Bone marrow flow cytometry analysis

Bone marrow cells were preincubated with 2% rat anti-mouse CD16/CD32 and 1% normal rat serum for Fc blocking before staining cells bearing hematopoietic lineage markers (CD3ε, CD45/B220, CD11b, Ly-6G/Ly-6C, TER 119) with a cocktail of allophycocyanin (APC)–conjugated lineage-specific antibodies, or equivalent concentration of APC-conjugated isotype control immunoglobulins, 1:20 dilutions of phycoerythrin (PE)–rat anti-mouse Sca-1 (clone D7), and peridinin chlorophyll protein (PerCP)–Cy5.5–rat anti-mouse c-Kit (clone 2B8) (all from Becton-Dickinson). Cells were stained for 25 min at 4°C, washed twice with cold Dulbecco’s phosphate-buffered saline, and resuspended in 0.4% paraformaldehyde. One hundred thousand events were acquired on a FACSCalibur flow cytometer (Becton-Dickinson) and analyzed with FlowJo v.7.0.5 (TreeStar) software. Cells negative for lineage marker expression were assessed for percentages of LinSca-1c-Kit+ (LK) and LinSca-1+c-Kit+ (LSK) in the bone marrow (fig. S7, gating strategy). Data from controls were consistent with published reports in naïve BALB/c mice (51).

Statistics

For the human HCT study, samples with undetectable analytes were assigned a value at half the lower limit of detection. For ANC, platelet, BPI, TLR4, and IL-6, data were analyzed after logarithmic transformation, which yielded distributions that were about normal. Geometric means and error bars indicating +1 SEM of the log values were then transformed back to original units and plotted on a logarithmic axis. The Wilcoxon signed-rank test for matched pairs was used when comparing values for the same patients at different time points, with values compared to baseline. Comparisons between subjects with or without fever were evaluated with the Mann-Whitney test. When assessing correlations between different parameters, within-subject correlations were calculated with the Spearman correlation coefficient and data from multiple time points. The calculated coefficients were averaged over the different subjects, and significance was tested with the signed-rank test. P values for Fig. 1C were one-sided. All other P values were two-sided. Statistical significance and graphic output were generated with Prism v. 4.0a (GraphPad Software) and SAS v. 9.1 (SAS Institute). Statistical analysis for murine experiments was performed with GraphPad Prism version 5. Mantel-Cox log-rank was used to compare survival curves. Two-tailed Mann-Whitney tests were performed throughout except for peripheral blood count and cytokine analyses in which unpaired t tests with Welch’s correction for unequal variances were performed. A P value of <0.05 was considered statistically significant. Where indicated in figures, *P < 0.05, **P < 0.01, ***P < 0.001.

Supplementary Material

www.sciencetranslationalmedicine.org/cgi/content/full/3/110/110ra118/DC1

Fig. S1. Seven-gray irradiation of BALB/c mice is associated with subsequent endotoxemia.

Fig. S2. The effect of rBPI21/ENR and VEH/ENR on survival of BALB/c mice after 7-Gy TBI is reproducible.

Fig. S3. rBPI21/ENR does not mitigate early mucosal damage after 7-Gy irradiation.

Fig. S4. Trilineage hematopoiesis is evident in bone marrow of rBPI21/ENR-treated mice.

Fig. S5. Effects of 14 and 30 days of rBPI21 plus ENR on bone marrow mononuclear cell, LSK, and LK cells are equivalent.

Fig. S6. Peripheral blood counts are equivalent after 14 or 30 days of rBPI21 plus ENR treatment.

Fig. S7. Gating strategy for determining LK and LSK cells in bone marrow from BALB/c mice by flow cytometry (FACS).

Table S1. Characteristics and treatment of patients undergoing myeloablative transplantation on the observational cohort study.

Table S2. Median fold difference in peripheral blood levels of chemokines and cytokines after radiation alone, radiation with indicated treatment, or no irradiation.

Footnotes

  • Present address: Infinity Pharmaceuticals, 780 Memorial Drive, Cambridge, MA 02139, USA.

  • Present address: Weizmann Institute, Rehovot 76100, Israel.

  • § Present address: Feinberg School of Medicine, Northwestern University, 420 East Superior Street, Chicago, IL 60611, USA.

  • || Present address: Georgia Institute of Technology; Atlanta, GA 30332, USA.

References and Notes

  1. Acknowledgments: We thank P. Scannon, M. Wessels, and L. Nadler for their insights and support, and L. Brennan and V. Russo for their many contributions. Funding: Defense Advanced Research Projects Agency HR001-08-1-0011, NIH 5R21 HL089659 and 5U19 AI067751, the Dana Foundation, the G. Green Foundation, and Shea Family Fund. Author contributions: E.C.G. and O.L. conceived the studies, executed the clinical study, designed experiments, analyzed and interpreted data, and drafted the article. C.M.B. designed experiments, developed methods, performed experiments and data analysis, and drafted and reviewed the manuscript. L.A.K. analyzed and interpreted data and provided critical manuscript revision. J.K. and J.-A.V. analyzed murine data and reviewed the manuscript. K.P. contributed to initial murine experimental design, data interpretation, and manuscript review. A.D. provided important resources for murine studies and reviewed the manuscript. G.C. performed experiments and manuscript review. E.E.S., L.S.-B., C.D.P., C.J.M., J.D.R., L.C.G., K.Z., and A.V. contributed to experiment planning, execution, analysis, and manuscript preparation. R.S. contributed to human study execution and manuscript review. J.P.W. reviewed and interpreted data and contributed critical manuscript revisions. Competing interests: O.L.’s laboratory received sponsorship and reagent support from XOMA (US) LLC and Spectral Diagnostics. The other authors declare that they have no competing interests.
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