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Activation of the G-CSF and Flt-3 receptors protects hematopoietic stem cells from lethal irradiation
Experimental Hematology 31 (2003) 1119–1125
Activation of the G-CSF and Flt-3 receptors protects hematopoietic stem cells from lethal irradiation Philip R. Streeter, Layla Z. Dudley, and William H. Fleming BMT Program, Division of Hematology and Medical Oncology, Department of Medicine, Oregon Health & Science University, Portland, Ore, USA (Received 19 May 2003; revised 24 July 2003; accepted 30 July 2003)
Objective. Synergy between Flt-3 ligand and G-CSF produces a marked expansion of hematopoietic progenitor cells and mobilizes large numbers of stem cells into the peripheral blood. To determine if the activation of the Flt-3 and G-CSF receptors enhances the regenerative capacity of the hematopoietic compartment, we evaluated whether activation of these receptors augments stem cell recovery following lethal doses of radiation. Methods. C57BL/6 mice received a single injection of the bi-functional Flt-3 and G-GSF agonist progenipoietin-1, 24 hours prior to exposure to 1100 cGy of gamma radiation. Survival, hematopoietic reconstitution, and competitive repopulation potential were evaluated. Results. All cytokine-treated mice survived for up to 9 months. Radioprotected recipients exhibited stable multilineage hematopoiesis and recovered normal numbers of T cells, B cells, and myelomonocytic cells in the blood, bone marrow, and thymus. Between 2 and 3 weeks following radiation, cytokine-treated mice demonstrated threefold higher serum hemoglobin levels, 10-fold higher nucleated blood cell counts, and 20-fold higher platelet counts compared to controls. Radioprotection of self-renewing hematopoietic stem cells was revealed by multilineage hematopoietic reconstitution following transplantation in a competitive repopulation assay. To further evaluate the extent of cytokine-induced radioprotective activity, a cohort of mice received a second cycle of cytokine treatment and a second exposure to radiation (1100 cGy). Survival of this serially irradiated group was 70% and analysis of the peripheral blood revealed sustained multilineage hematopoiesis. Conclusion. These results demonstrate that activation of both the Flt-3 and G-CSF receptors provides a high degree of radioprotection to the hematopoietic progenitor cell and stem cell compartment. 쑖 2003 International Society for Experimental Hematology. Published by Elsevier Inc.
Protection of the hematopoietic stem cell (HSC) compartment from the effects of lethal doses of radiation provides a model system to study the biology of stem cell regeneration. Pioneering studies by Smith et al. demonstrated that bacterial endotoxins were able to enhance the hematopoietic recovery and the survival of irradiated mice [1,2]. These findings led to the hypothesis that the stimulation of cytokine production may protect and repair the stem cell compartment. The administration of cytokines following exposure to sublethal doses of radiation typically enhances the survival of hematopoietic progenitor cells in the bone marrow and accelerates the
Offprint requests to: William H. Fleming, M.D., Ph.D., BMT Program, Division of Hematology and Medical Oncology, Department of Medicine Oregon Health & Science University (UHN73C), 3181 SW Sam Jackson Park Rd., Portland, OR 97239-3089; E-mail: [email protected]
0301-472X/03 $–see front matter. Copyright doi: 1 0. 10 1 6 / j .e x p he m.2 0 03 .0 7 .0 0 3
recovery of peripheral blood cells [3,4]. However, this approach is often unsuccessful when bone marrow lethal doses of radiation are administered. Consequently, cytokine administration prior to irradiation has been evaluated in an effort to protect the hematopoietic stem cell and progenitor cell compartments. Pretreatment of recipients with interleukin-1 (IL-1) [5,6], tumor necrosis factor-α (TNF-α) [7], granulocyte colony-stimulating factor (G-CSF) [8], c-kit ligand (SCF) [9], and Flt-3 ligand (Flt-3L) [10,11] provides some degree of radioprotection. However, maintaining high levels of circulating red blood cells and platelets, the primary determinants of short-term survival remains an elusive goal. Progenipoietin (ProGP) is a dual-receptor agonist of the Flt-3 and G-CSF receptors, with a broad spectrum of multilineage hematopoietic activity [12]. This cytokine has the capacity to mobilize large numbers of hematopoietic stem
쑖 2003 International Society for Experimental Hematology. Published by Elsevier Inc.
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cells into the peripheral blood [13], expand functional dendritic cells [14], suppress apoptosis in hematopoietic cells [15], and attenuate the development of graft-vs-host disease [16]. As the combination of Flt-3L and G-CSF has been demonstrated to have synergistic effects on hematopoiesis [17], we evaluated the radioprotective activity of this bifunctional receptor agonist. Our results show that a single injection of ProGP prior to treatment with bone marrow lethal doses of radiation (1100 cGy) rescues irradiated recipients, restores long-term multilineage hematopoiesis, and protects self-renewing HSC. Remarkably, the majority of radioprotected mice survive when they are again pretreated with a single dose of ProGP and challenged with a second 1100-cGy dose of radiation. These findings indicate that pretreatment with ProGP has the potential to serially protect the hematopoietic compartment from the effects of lethal doses of radiation.
Materials and methods Mice C57BL/6J (CD45.2) mice (Jackson Laboratory, Bar Harbor, ME, USA), aged 8 to 12 weeks, were used for radioprotection assays. C57BL/6J (CD45.1) mice were bred and maintained in the animal care facility at Oregon Health & Science University. For competitive repopulation assays, radioprotected survivors and C57BL/6J (CD45.1) mice were used as donors, and C57BL/6J (CD45.2) mice were used as recipients. All mice were maintained on acidified water (pH 2.2) for at least 1 week prior to irradiation. Radioprotection assay Progenipoietin-1(ProGP), a dual agonist of fetal liver tyrosine kinase-3 (Flt-3) and the granulocyte colony-stimulating factor receptor (G-GSF), was provided by G.D. Searle and Company (St. Louis, MO, USA). Treated mice received a single subcutaneous injection of ProGP (100 µg) 24 hours prior to irradiation. The cytokine was delivered in a vehicle of phosphate-buffered saline (PBS), and control mice received a single injection of PBS containing 0.1% bovine serum albumin (BSA). For radioprotection studies, mice were exposed to 1100 cGy of irradiation administered in two equal fractions delivered 3 hours apart using a J.L. Shepherd Co. cesium irradiator (rate 142 cGy/minute). Following radiation, mice were maintained on aqueous antibiotics (polymyxin B sulfate at 106 u/L and neomycin sulfate at 1.1 g/L) in their drinking water. Mice were monitored daily for a minimum of 50 days. Cell preparation Peripheral blood (PB) was collected in Microtainer tubes (BectonDickinson, Palo Alto, CA, USA) following retroorbital puncture and complete blood counts were obtained using a Cell-Dyn counter (Model #3500) calibrated for mouse cells. Differential counts were performed manually. PB for flow cytometric studies was collected in Hanks’ balanced salt solution (HBSS) containing 10 mM HEPES, 3% fetal bovine serum, and sodium heparin (50 u/mL). Nucleated PB cells were prepared by sedimenting erythrocytes using 3% dextran (T-500); residual red blood cells were removed by hypotonic lysis as described previously [18]. Bone marrow (BM) cells were obtained by flushing four long bones with tibias and
femurs with modified HBSS. Spleen and thymus cell suspensions free of connective tissue were obtained by passing cells through a wire mesh, then filtered with a 35-µm filter. Long-term reconstitution Radioprotected mice were evaluated for long-term hematopoietic reconstitution 6 months posttransplant. PB, BM, spleen, and thymus were harvested from radioprotected mice and cell suspensions were prepared as described above. Tissues from ProGP-protected mice were analyzed for hematopoietic lineage marker expression with the pan-hematopoietic marker (CD45, Ly5), myelomonocytic cells (Gr-1 and Mac-1), B cells (B220), and T cells (CD3). All antibodies were purchased from Pharmingen (San Diego, CA, USA). The expression of each of these markers was determined by 4-color flow cytometry using a FACScalibur (Becton-Dickinson, San Jose, CA, USA) and data analysis was performed using PaintA-Gate software (Becton-Dickinson). Competitive repopulation Recipient CD45.1 mice were irradiated to a dose of 1100 cGy as described above. Following irradiation 5 × 105 BM cells from ProGP-radioprotected (CD45.2) mice were combined with 1 × 105 BM cells from normal (CD45.1) donors. This mixture of cells was resuspended in modified HBSS and administered intravenously to anesthetized mice. Control recipient Ly5.2 mice received 5 × 105 BM cells from normal (Ly5.1) mice combined with 1 × 105 BM cells from normal (Ly5.2) donor mice. Transplant recipients were maintained on antibiotic water and monitored daily. The PB of recipient mice was analyzed for the presence of donor cells by flow cytometry. Secondary radioprotection Six months after initial radioprotection studies were performed, radioprotected mice were again pretreated with 100 µg of ProGP 24 hours before a second exposure to 1100 cGy. Age-matched, normal control mice received PBS containing 0.1% BSA 24 hours prior to irradiation. Mice were monitored daily for survival.
Results Pretreatment with ProGP protects mice from radiation-induced bone marrow failure To investigate the radioprotective properties of ProGP, a single dose was administered 24 hours prior to high-dose irradiation (1100 cGy). Control mice received a single injection of a vehicle control. In three independent experiments, 100% of ProGP-treated mice (n ⫽ 30) survived for up to 9 months. By contrast, all control mice died within 23 days of exposure to radiation (n ⫽ 20; Fig. 1). Within 3 days of irradiation, neutrophil and total mononuclear cell numbers were markedly decreased in both treatment groups; however, in ProGP-treated mice these cell populations recovered rapidly, approaching near-normal levels by day 17 postirradiation (Fig. 2A–C). There was no evidence of peripheral blood cell recovery in control mice. The number of platelets in the circulation of ProGP recipients was maintained above 100,000/µL, while platelets in control mice fell below 10,000/ µL (Fig. 2D). The most dramatic radioprotective effects were
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tissues examined and a comparable frequency of mature, lineage marker–positive hematopoietic cells in radioprotected mice.
Figure 1. Pretreatment of mice with a single dose of ProGP is radioprotective. Mice were injected 24 hours prior to radiation with either 100 µg ProGP in PBS [●] or 0.1% BSA in PBS [䊊]. A split dose of 1100 cGy (550 cGy × 2) radiation was given and survival was monitored daily for 50 days. The percentage of mice surviving over time is shown (n ⫽ 30 mice in the ProGP group, n ⫽ 20 mice in BSA control group).
found within the red cell compartment. The hemoglobin levels in ProGP recipients transiently declined from a mean of 14 g/dL to 11.5 g/dL, while in controls hemoglobin levels declined to a mean of 3.6 g/dL (Fig. 2E). This very modest decline in circulating erythroid cells in ProGP-treated recipients is most consistent with substantial radioprotection of committed erythroid progenitor cells. Radioprotected recipients exhibit long-term multilineage hematopoietic recovery Hematopoietic stem cells have the capacity to give rise to long-term multilineage hematopoiesis. At 6 months postirradiation, multilineage hematopoietic engraftment was evaluated in cytokine-protected mice. Hematopoietic recovery of the PB, BM, and spleen was determined by differential cell counts and flow cytometry. The PB of radioprotected mice revealed no significant differences in total nucleated cells, neutrophils, monocytes, lymphocytes, or platelet counts compared to age-matched control mice (Fig. 3A). Similarly, hemoglobin levels in radioprotected and control mice were within the normal range (data not shown). When total cellularity of the spleen, BM, and thymus was evaluated, no significant differences were observed between radioprotected mice and normal controls (Fig. 3B). Multilineage hematopoietic reconstitution was analyzed by determining the frequency of CD45⫹ cells that expressed B cell (B220), T cell (CD3), and myelomonocytic cell (Mac1/GR-1) markers. Representative multilineage reconstitution of the PB of a radioprotected mouse is shown in Figure 4A. Evaluation of hematopoietic reconstitution of cells from the PB, BM, and spleen did not reveal any differences between radioprotected and age-matched control mice (Fig. 4B–D). Similar frequencies of thymocyte subsets were also observed in radioprotected and control mice (data not shown). These results demonstrate normal cellularity in all hematopoietic
Engraftment potential of radioprotected bone marrow in a competitive repopulation assay Hematopoietic stem cell activity is demonstrated by the capacity to reconstitute the hematopoietic compartment of lethally irradiated recipients. To evaluate stem cell activity in the BM of ProGP radioprotected mice, lethally irradiated C57BL/6J (Ly5.2) mice were injected with (5 × 105) BM cells from ProGP-protected mice (Ly5.1) combined with (1 × 105) carrier host-type BM cells (Ly5.2) [19]. A second cohort of irradiated Ly5.2 mice received 5 × 105 BM cells from normal (Ly5.1) mice combined with 1 × 105 host-type BM cells. At 3 months posttransplant, the ratio of donor (Ly5.1) to host (Ly5.2) hematopoietic cells was determined by flow cytometry. Recipients of normal bone marrow exhibited the expected host-to-donor-cell ratio of 1:5. In our first experiment, recipients of BM from a ProGP radioprotected mouse demonstrated a host-to-donor-cell ratio of 2.3:1. In
Figure 2. ProGP pretreatment prevents anemia and thrombocytopenia, and enhances recovery of peripheral blood leukocytes. Mice were injected 24 hours prior to irradiation with either 100 µg of ProGP in PBS [●] or 0.1% BSA in PBS [䊊]. Peripheral blood was analyzed from cohorts of 5 mice on days 3, 10, 17, 20, and 28 after radiation. Each cohort of mice was analyzed once to prevent the effects of serial phlebotomy. (A): Total leukocytes (WBC); (B): Neutrophils; (C): Total mononuclear cells (lymphocytes plus monocytes); (D): Platelet counts; (E): Hemoglobin concentration. The mean and SEM for each treatment group is shown. (Note log scale used in A–D.)
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(Fig. 6B) and a normal serum hemoglobin concentration (data not shown) were found. The total number of nucleated cells in the blood, however, declined from a mean of 7200/µL to a mean of 1500/µL due primarily to a 10-fold decrease in lymphocytes. A twofold to 2.5-fold decrease in the frequency of monocytes and neutrophils was also observed (Fig. 6B). These results indicate that pretreatment with ProGP is sufficient to protect the hematopoietic compartment from a second BM lethal dose of radiation.
Figure 3. Long-term hematopoietic recovery in ProGP-protected mice. Six months after exposure to 1100 cGy irradiation, ProGP-protected mice (䊐; n ⫽ 10) and age-matched untreated C75BL/6J mice (■; n ⫽ 4)were analyzed for hematopoietic cell recovery in the peripheral blood (A—Note log scale; there were no significant differences between groups using the Student’s t-test). (B): Cellularity of the PB, thymus, spleen, and BM (4 long bones). The mean and SEM of each group is shown.
Discussion The data presented in this study demonstrate that coactivation of the Flt-3 and G-CSF receptors with the dual-receptor agonist ProGP consistently protects the hematopoietic stem/ progenitor cell compartment from the effects of lethal doses of radiation. Previous studies have shown that cytokineinduced radioprotection is dependent on both the cytokine dose and the administration schedule. Cytokine treatment given after radiation exposure typically enhances hematopoietic recovery following sublethal doses of irradiation but has relatively modest effects on recipient survival after bone marrow lethal doses of radiation. The radioprotective effects
a second experiment utilizing an independent radioprotected donor, the host-to-donor-cell ratio was 4.5:1 (Fig. 5A). Despite the reduced absolute number of cells derived from ProGP-radioprotected BM, a similar frequency of multilineage hematopoietic reconstitution of the B cell, T cell, and myelomonocytic cell compartments was consistently derived from the BM of ProGP-radioprotected mice (Fig. 5B). These results indicate that the multilineage competitive repopulation activity of ProGP-radioprotected BM is only ∼ twofold to fourfold lower than normal bone marrow despite exposure to BM lethal doses of radiation. Pretreatment with ProGP protects mice from serial doses of lethal radiation Although the competitive repopulation assay described above demonstrates that radioprotected bone marrow has reduced competitive repopulating activity compared to normal BM, surviving hematopoietic stem/progenitor cells continued to give rise to large numbers of circulating hematopoietic cells. To further address the extent of regenerative capacity of these BM cells, mice radioprotected for 6 months were again given a 100-µg dose of ProGP followed 24 hours later by a second exposure to 1100 cGy. Remarkably, 70% of ProGP-protected mice survived a second BM lethal dose of irradiation (Fig. 6A). Two months following the second radiation exposure, the peripheral blood of radioprotected mice was analyzed. Near-normal numbers of platelets
Figure 4. Long-term multilineage hematopoiesis in ProGP-protected mice. Six months after exposure to 1100 cGy irradiation, hematopoietic tissues of ProGP-protected mice were analyzed for the presence of different cell lineages. Flow cytometry was used to determine the frequency of B cells (B220), T cells (CD3), myelomonocytic cells (Mac-1/Gr-1), and red blood cell progenitors (Ter119). Age-matched C75BL/6J untreated mice were used as controls. Multilineage hematopoiesis in the PB of a representative ProGP radioprotection survivor is shown in (A). Expression of lineagespecific markers on Ly5.1⫹ hematopoietic cells in the PB (B), spleen (C), and BM (D). The mean and SEM of ProGP-protected (䊐; n ⫽ 4) and control mice (■; n ⫽ 3) is shown. (There were no significant differences between groups using the Student’s t-test.)
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[11]. By contrast, Flt3-L administered 2 hours prior to radiation in this study was not protective. These results confirm that Flt-3L pretreatment ∼20 hours prior to radiation confers radioprotective effects similar to those observed with TNF [21,22], IL-1 [5], and SCF [9]. A remarkable feature of ProGP-induced radioprotection is the high degree of protection of the erythroid and megakaryocytic cell lineages (Fig. 2). Hemoglobin levels only modestly decreased by 7 days (11.5 g/dL), then promptly returned to normal in ProGP-treated mice, while control animals demonstrated severe progressive anemia with hemoglobin levels falling to less than 3.5 g/dL. Similarly, the platelet counts in ProGP-treated mice were maintained at greater than 100,000/µL compared to less than 10,000/µL in controls. By contrast, the study by Hudak et al. evaluating pretreatment with Flt-3L alone demonstrated a ∼66% drop in circulating erythroid cells with platelet counts decreasing to less than 40,000/µL [11]. Both prolonged anemia and slow platelet recovery were observed in these recipients, and these findings correlated with the modest effects of Flt-3L alone on the erythroid and megakaryocytic lineages
Figure 5. Competitive repopulation studies reveal the engraftment potential of radioprotected BM. Six months after 1100 cGy irradiation, the BM from ProGP-protected mice was harvested and evaluated in competitive repopulation studies. Ly5.1 BM cells (5 × 105) from ProGP-protected or normal donors were combined with BM cells (1 × 105) from host Ly5.2 donors and transplanted into lethally irradiated Ly5.2 recipients. At 3 months posttransplant, the PB of recipient mice was evaluated for lineage-specific donor cells by flow cytometry. (A): The percentage of donor (Ly5.1; G) and host (Ly5.2; ■) cells in mice transplanted with either normal BM or ProGP-protected BM. Two independent experiments utilizing two different radioprotected BM donors and eight recipients are shown. (B): The frequency of T cells (CD3), B cells (B220), and myelomonocytic (Mac-1/Gr1) donor cells (Ly5.1) in the PB of competitive repopulation recipients of either normal BM (■) or ProGP-protected BM (G). The mean and SEM for each group is indicated.
of pretreatment dosing with G-CSF alone are modest, with 10% survival at a dose of 200 µg/kg, increasing to 27 to 62% survival at a dose of 2000 µg/kg [8,20]. This higher dose of G-CSF is similar to the dose used in our studies (ProGP 100 µg contains the equivalent of 2000 µg/kg G-CSF), yet the survival observed in these studies was considerably less, indicating that most recipients were not radioprotected by GCSF alone. In a previous study, treatment of recipients with Flt3-L and G-CSF for 14 days beginning 2 days prior to irradiation enhanced the survival of rabbits exposed to high does of radiation; however, the effect of cytokine pretreatment alone was not evaluated in this study [10]. Hudak and colleagues demonstrated that maximal survival (70–80%) was provided by the pretreatment of mice with Flt-3L at 20 hours and 2 hours before irradiation, while a single dose of Flt3-L administered 20 hours prior to irradiation rescued 65% of recipients
Figure 6. ProGP radioprotects mice from a second exposure to 1100 cGy irradiation. Six months after initial protection from 1100 cGy of radiation, mice were injected with 100 µg of ProGP [●] 24 hours prior to receiving a split dose of 1100 cGy (550 cGy × 2) irradiation (n ⫽ 10). Control mice were pretreated with 0.01% BSA in PBS [䊊]. (A) shows the percentage of mice surviving over time. (B) shows the recovery of hematopoietic cells in the PB at 50 days (G; n ⫽ 7). Values from age-matched normal mice are indicated (■; n ⫽ 5). The mean and SEM for each group is shown on a log scale. (Student’s t-test, ∗p ⱕ 0.001, ∗∗p ⱕ 0.01, NS ⫽ not significant.)
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[23,24]. Taken together with our results, these findings indicate that concurrent activation of the Flt3 and G-CSF receptors provides a greater level of protection to both erythroid and megakaryocytic lineages than administration of Flt-3L alone. The long-term multilineage hematopoietic recovery observed following ProGP pretreatment of lethally irradiated recipients may be the result of protecting of both longlived hematopoietic progenitor cells [25,26] and HSC. To determine if primitive HSC were radioprotected by ProGP, BM was harvested 6 to 8 months after ProGP treatment and exposure to lethal irradiation. Unfractionated, radioprotected BM (1 × 105 cells) was transplanted into irradiated CD45 congenic recipients along with 1 × 105 host-type BM cells. This competitive repopulation assay [19] was used to determine the relative repopulating activity of radioprotected donor BM compared to normal BM. The results of two independent experiments demonstrated only a ∼twofold to fourfold lower frequency of radioprotected BM-derived cells in the blood compared to cells derived from normal BM (Fig. 5). Analysis of the progeny of these radioprotected BM-derived cells revealed multilineage hematopoiesis with contributions to both the lymphoid and myelomonocytic cell lineages. These results are consistent with only a ∼twofold to fourfold reduction in the activity of functional HSC in ProGP-protected BM compared to normal untreated BM. This represents a high degree of recovery of the HSC compartment as normal bone marrow is typically depleted of HSC activity following exposure to high doses of radiation [27]. Whether or not ProGP treatment also enhances the survival of a population of long-term hematopoietic progenitors that read out in this competitive repopulation assay remains to be determined. The observation that significant numbers of transplantable HSC were radioprotected by ProGP prompted us to further evaluate the extent of the radioprotective potential of this bifunctional receptor agonist. Cohorts of radioprotected mice were again treated with a single dose of ProGP and exposed to a second BM lethal dose of radiation. Survival of these serially irradiated mice was 70%. This total dose of 2200 cGy is twofold higher than the LD100/30, indicating a remarkable degree of protection of the hematopoietic compartment. Indeed, at this high dose of total-body irradiation, the 30% mortality observed may in part be attributable to toxicity to nonhematopoietic tissues such as lung or liver. Not surprisingly, hematopoietic reconstitution of serially radioprotected mice was not complete. The peripheral blood demonstrated significantly reduced numbers of circulating neutrophils, lymphocytes, and monocytes. By contrast, only modest decreases in the hemoglobin and platelet counts were observed, suggesting a preferential recovery of erythroid and megakaryocytic progenitor cells in these supralethally irradiated recipients. In summary, our results show that pretreatment with an agonist of the Flt-3 and G-CSF receptors protects the hematopoietic compartment from high doses of radiation at the
level of the HSC. It remains to be determined if cytokine pretreatment prevents the initial radiation damage or alternatively enhances the function of critical cellular repair mechanisms. In either case, identifying the molecular mechanisms underlying radioprotection may have important implications for our understanding of HSC regeneration and repair.
Acknowledgments G.D. Searle & Co. (St. Louis, MO, USA) provided Progenipoietin1 and financial support for these studies. P.R. Streeter was a scientist at G.D. Searle when these studies were initiated.
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Activation of the G-CSF and Flt-3 receptors protects hematopoietic stem cells from lethal irradiation Philip R. Streeter, Layla Z. Dudley, and William H. Fleming BMT Program, Division of Hematology and Medical Oncology, Department of Medicine, Oregon Health & Science University, Portland, Ore, USA (Received 19 May 2003; revised 24 July 2003; accepted 30 July 2003)
Objective. Synergy between Flt-3 ligand and G-CSF produces a marked expansion of hematopoietic progenitor cells and mobilizes large numbers of stem cells into the peripheral blood. To determine if the activation of the Flt-3 and G-CSF receptors enhances the regenerative capacity of the hematopoietic compartment, we evaluated whether activation of these receptors augments stem cell recovery following lethal doses of radiation. Methods. C57BL/6 mice received a single injection of the bi-functional Flt-3 and G-GSF agonist progenipoietin-1, 24 hours prior to exposure to 1100 cGy of gamma radiation. Survival, hematopoietic reconstitution, and competitive repopulation potential were evaluated. Results. All cytokine-treated mice survived for up to 9 months. Radioprotected recipients exhibited stable multilineage hematopoiesis and recovered normal numbers of T cells, B cells, and myelomonocytic cells in the blood, bone marrow, and thymus. Between 2 and 3 weeks following radiation, cytokine-treated mice demonstrated threefold higher serum hemoglobin levels, 10-fold higher nucleated blood cell counts, and 20-fold higher platelet counts compared to controls. Radioprotection of self-renewing hematopoietic stem cells was revealed by multilineage hematopoietic reconstitution following transplantation in a competitive repopulation assay. To further evaluate the extent of cytokine-induced radioprotective activity, a cohort of mice received a second cycle of cytokine treatment and a second exposure to radiation (1100 cGy). Survival of this serially irradiated group was 70% and analysis of the peripheral blood revealed sustained multilineage hematopoiesis. Conclusion. These results demonstrate that activation of both the Flt-3 and G-CSF receptors provides a high degree of radioprotection to the hematopoietic progenitor cell and stem cell compartment. 쑖 2003 International Society for Experimental Hematology. Published by Elsevier Inc.
Protection of the hematopoietic stem cell (HSC) compartment from the effects of lethal doses of radiation provides a model system to study the biology of stem cell regeneration. Pioneering studies by Smith et al. demonstrated that bacterial endotoxins were able to enhance the hematopoietic recovery and the survival of irradiated mice [1,2]. These findings led to the hypothesis that the stimulation of cytokine production may protect and repair the stem cell compartment. The administration of cytokines following exposure to sublethal doses of radiation typically enhances the survival of hematopoietic progenitor cells in the bone marrow and accelerates the
Offprint requests to: William H. Fleming, M.D., Ph.D., BMT Program, Division of Hematology and Medical Oncology, Department of Medicine Oregon Health & Science University (UHN73C), 3181 SW Sam Jackson Park Rd., Portland, OR 97239-3089; E-mail: [email protected]
0301-472X/03 $–see front matter. Copyright doi: 1 0. 10 1 6 / j .e x p he m.2 0 03 .0 7 .0 0 3
recovery of peripheral blood cells [3,4]. However, this approach is often unsuccessful when bone marrow lethal doses of radiation are administered. Consequently, cytokine administration prior to irradiation has been evaluated in an effort to protect the hematopoietic stem cell and progenitor cell compartments. Pretreatment of recipients with interleukin-1 (IL-1) [5,6], tumor necrosis factor-α (TNF-α) [7], granulocyte colony-stimulating factor (G-CSF) [8], c-kit ligand (SCF) [9], and Flt-3 ligand (Flt-3L) [10,11] provides some degree of radioprotection. However, maintaining high levels of circulating red blood cells and platelets, the primary determinants of short-term survival remains an elusive goal. Progenipoietin (ProGP) is a dual-receptor agonist of the Flt-3 and G-CSF receptors, with a broad spectrum of multilineage hematopoietic activity [12]. This cytokine has the capacity to mobilize large numbers of hematopoietic stem
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cells into the peripheral blood [13], expand functional dendritic cells [14], suppress apoptosis in hematopoietic cells [15], and attenuate the development of graft-vs-host disease [16]. As the combination of Flt-3L and G-CSF has been demonstrated to have synergistic effects on hematopoiesis [17], we evaluated the radioprotective activity of this bifunctional receptor agonist. Our results show that a single injection of ProGP prior to treatment with bone marrow lethal doses of radiation (1100 cGy) rescues irradiated recipients, restores long-term multilineage hematopoiesis, and protects self-renewing HSC. Remarkably, the majority of radioprotected mice survive when they are again pretreated with a single dose of ProGP and challenged with a second 1100-cGy dose of radiation. These findings indicate that pretreatment with ProGP has the potential to serially protect the hematopoietic compartment from the effects of lethal doses of radiation.
Materials and methods Mice C57BL/6J (CD45.2) mice (Jackson Laboratory, Bar Harbor, ME, USA), aged 8 to 12 weeks, were used for radioprotection assays. C57BL/6J (CD45.1) mice were bred and maintained in the animal care facility at Oregon Health & Science University. For competitive repopulation assays, radioprotected survivors and C57BL/6J (CD45.1) mice were used as donors, and C57BL/6J (CD45.2) mice were used as recipients. All mice were maintained on acidified water (pH 2.2) for at least 1 week prior to irradiation. Radioprotection assay Progenipoietin-1(ProGP), a dual agonist of fetal liver tyrosine kinase-3 (Flt-3) and the granulocyte colony-stimulating factor receptor (G-GSF), was provided by G.D. Searle and Company (St. Louis, MO, USA). Treated mice received a single subcutaneous injection of ProGP (100 µg) 24 hours prior to irradiation. The cytokine was delivered in a vehicle of phosphate-buffered saline (PBS), and control mice received a single injection of PBS containing 0.1% bovine serum albumin (BSA). For radioprotection studies, mice were exposed to 1100 cGy of irradiation administered in two equal fractions delivered 3 hours apart using a J.L. Shepherd Co. cesium irradiator (rate 142 cGy/minute). Following radiation, mice were maintained on aqueous antibiotics (polymyxin B sulfate at 106 u/L and neomycin sulfate at 1.1 g/L) in their drinking water. Mice were monitored daily for a minimum of 50 days. Cell preparation Peripheral blood (PB) was collected in Microtainer tubes (BectonDickinson, Palo Alto, CA, USA) following retroorbital puncture and complete blood counts were obtained using a Cell-Dyn counter (Model #3500) calibrated for mouse cells. Differential counts were performed manually. PB for flow cytometric studies was collected in Hanks’ balanced salt solution (HBSS) containing 10 mM HEPES, 3% fetal bovine serum, and sodium heparin (50 u/mL). Nucleated PB cells were prepared by sedimenting erythrocytes using 3% dextran (T-500); residual red blood cells were removed by hypotonic lysis as described previously [18]. Bone marrow (BM) cells were obtained by flushing four long bones with tibias and
femurs with modified HBSS. Spleen and thymus cell suspensions free of connective tissue were obtained by passing cells through a wire mesh, then filtered with a 35-µm filter. Long-term reconstitution Radioprotected mice were evaluated for long-term hematopoietic reconstitution 6 months posttransplant. PB, BM, spleen, and thymus were harvested from radioprotected mice and cell suspensions were prepared as described above. Tissues from ProGP-protected mice were analyzed for hematopoietic lineage marker expression with the pan-hematopoietic marker (CD45, Ly5), myelomonocytic cells (Gr-1 and Mac-1), B cells (B220), and T cells (CD3). All antibodies were purchased from Pharmingen (San Diego, CA, USA). The expression of each of these markers was determined by 4-color flow cytometry using a FACScalibur (Becton-Dickinson, San Jose, CA, USA) and data analysis was performed using PaintA-Gate software (Becton-Dickinson). Competitive repopulation Recipient CD45.1 mice were irradiated to a dose of 1100 cGy as described above. Following irradiation 5 × 105 BM cells from ProGP-radioprotected (CD45.2) mice were combined with 1 × 105 BM cells from normal (CD45.1) donors. This mixture of cells was resuspended in modified HBSS and administered intravenously to anesthetized mice. Control recipient Ly5.2 mice received 5 × 105 BM cells from normal (Ly5.1) mice combined with 1 × 105 BM cells from normal (Ly5.2) donor mice. Transplant recipients were maintained on antibiotic water and monitored daily. The PB of recipient mice was analyzed for the presence of donor cells by flow cytometry. Secondary radioprotection Six months after initial radioprotection studies were performed, radioprotected mice were again pretreated with 100 µg of ProGP 24 hours before a second exposure to 1100 cGy. Age-matched, normal control mice received PBS containing 0.1% BSA 24 hours prior to irradiation. Mice were monitored daily for survival.
Results Pretreatment with ProGP protects mice from radiation-induced bone marrow failure To investigate the radioprotective properties of ProGP, a single dose was administered 24 hours prior to high-dose irradiation (1100 cGy). Control mice received a single injection of a vehicle control. In three independent experiments, 100% of ProGP-treated mice (n ⫽ 30) survived for up to 9 months. By contrast, all control mice died within 23 days of exposure to radiation (n ⫽ 20; Fig. 1). Within 3 days of irradiation, neutrophil and total mononuclear cell numbers were markedly decreased in both treatment groups; however, in ProGP-treated mice these cell populations recovered rapidly, approaching near-normal levels by day 17 postirradiation (Fig. 2A–C). There was no evidence of peripheral blood cell recovery in control mice. The number of platelets in the circulation of ProGP recipients was maintained above 100,000/µL, while platelets in control mice fell below 10,000/ µL (Fig. 2D). The most dramatic radioprotective effects were
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tissues examined and a comparable frequency of mature, lineage marker–positive hematopoietic cells in radioprotected mice.
Figure 1. Pretreatment of mice with a single dose of ProGP is radioprotective. Mice were injected 24 hours prior to radiation with either 100 µg ProGP in PBS [●] or 0.1% BSA in PBS [䊊]. A split dose of 1100 cGy (550 cGy × 2) radiation was given and survival was monitored daily for 50 days. The percentage of mice surviving over time is shown (n ⫽ 30 mice in the ProGP group, n ⫽ 20 mice in BSA control group).
found within the red cell compartment. The hemoglobin levels in ProGP recipients transiently declined from a mean of 14 g/dL to 11.5 g/dL, while in controls hemoglobin levels declined to a mean of 3.6 g/dL (Fig. 2E). This very modest decline in circulating erythroid cells in ProGP-treated recipients is most consistent with substantial radioprotection of committed erythroid progenitor cells. Radioprotected recipients exhibit long-term multilineage hematopoietic recovery Hematopoietic stem cells have the capacity to give rise to long-term multilineage hematopoiesis. At 6 months postirradiation, multilineage hematopoietic engraftment was evaluated in cytokine-protected mice. Hematopoietic recovery of the PB, BM, and spleen was determined by differential cell counts and flow cytometry. The PB of radioprotected mice revealed no significant differences in total nucleated cells, neutrophils, monocytes, lymphocytes, or platelet counts compared to age-matched control mice (Fig. 3A). Similarly, hemoglobin levels in radioprotected and control mice were within the normal range (data not shown). When total cellularity of the spleen, BM, and thymus was evaluated, no significant differences were observed between radioprotected mice and normal controls (Fig. 3B). Multilineage hematopoietic reconstitution was analyzed by determining the frequency of CD45⫹ cells that expressed B cell (B220), T cell (CD3), and myelomonocytic cell (Mac1/GR-1) markers. Representative multilineage reconstitution of the PB of a radioprotected mouse is shown in Figure 4A. Evaluation of hematopoietic reconstitution of cells from the PB, BM, and spleen did not reveal any differences between radioprotected and age-matched control mice (Fig. 4B–D). Similar frequencies of thymocyte subsets were also observed in radioprotected and control mice (data not shown). These results demonstrate normal cellularity in all hematopoietic
Engraftment potential of radioprotected bone marrow in a competitive repopulation assay Hematopoietic stem cell activity is demonstrated by the capacity to reconstitute the hematopoietic compartment of lethally irradiated recipients. To evaluate stem cell activity in the BM of ProGP radioprotected mice, lethally irradiated C57BL/6J (Ly5.2) mice were injected with (5 × 105) BM cells from ProGP-protected mice (Ly5.1) combined with (1 × 105) carrier host-type BM cells (Ly5.2) [19]. A second cohort of irradiated Ly5.2 mice received 5 × 105 BM cells from normal (Ly5.1) mice combined with 1 × 105 host-type BM cells. At 3 months posttransplant, the ratio of donor (Ly5.1) to host (Ly5.2) hematopoietic cells was determined by flow cytometry. Recipients of normal bone marrow exhibited the expected host-to-donor-cell ratio of 1:5. In our first experiment, recipients of BM from a ProGP radioprotected mouse demonstrated a host-to-donor-cell ratio of 2.3:1. In
Figure 2. ProGP pretreatment prevents anemia and thrombocytopenia, and enhances recovery of peripheral blood leukocytes. Mice were injected 24 hours prior to irradiation with either 100 µg of ProGP in PBS [●] or 0.1% BSA in PBS [䊊]. Peripheral blood was analyzed from cohorts of 5 mice on days 3, 10, 17, 20, and 28 after radiation. Each cohort of mice was analyzed once to prevent the effects of serial phlebotomy. (A): Total leukocytes (WBC); (B): Neutrophils; (C): Total mononuclear cells (lymphocytes plus monocytes); (D): Platelet counts; (E): Hemoglobin concentration. The mean and SEM for each treatment group is shown. (Note log scale used in A–D.)
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(Fig. 6B) and a normal serum hemoglobin concentration (data not shown) were found. The total number of nucleated cells in the blood, however, declined from a mean of 7200/µL to a mean of 1500/µL due primarily to a 10-fold decrease in lymphocytes. A twofold to 2.5-fold decrease in the frequency of monocytes and neutrophils was also observed (Fig. 6B). These results indicate that pretreatment with ProGP is sufficient to protect the hematopoietic compartment from a second BM lethal dose of radiation.
Figure 3. Long-term hematopoietic recovery in ProGP-protected mice. Six months after exposure to 1100 cGy irradiation, ProGP-protected mice (䊐; n ⫽ 10) and age-matched untreated C75BL/6J mice (■; n ⫽ 4)were analyzed for hematopoietic cell recovery in the peripheral blood (A—Note log scale; there were no significant differences between groups using the Student’s t-test). (B): Cellularity of the PB, thymus, spleen, and BM (4 long bones). The mean and SEM of each group is shown.
Discussion The data presented in this study demonstrate that coactivation of the Flt-3 and G-CSF receptors with the dual-receptor agonist ProGP consistently protects the hematopoietic stem/ progenitor cell compartment from the effects of lethal doses of radiation. Previous studies have shown that cytokineinduced radioprotection is dependent on both the cytokine dose and the administration schedule. Cytokine treatment given after radiation exposure typically enhances hematopoietic recovery following sublethal doses of irradiation but has relatively modest effects on recipient survival after bone marrow lethal doses of radiation. The radioprotective effects
a second experiment utilizing an independent radioprotected donor, the host-to-donor-cell ratio was 4.5:1 (Fig. 5A). Despite the reduced absolute number of cells derived from ProGP-radioprotected BM, a similar frequency of multilineage hematopoietic reconstitution of the B cell, T cell, and myelomonocytic cell compartments was consistently derived from the BM of ProGP-radioprotected mice (Fig. 5B). These results indicate that the multilineage competitive repopulation activity of ProGP-radioprotected BM is only ∼ twofold to fourfold lower than normal bone marrow despite exposure to BM lethal doses of radiation. Pretreatment with ProGP protects mice from serial doses of lethal radiation Although the competitive repopulation assay described above demonstrates that radioprotected bone marrow has reduced competitive repopulating activity compared to normal BM, surviving hematopoietic stem/progenitor cells continued to give rise to large numbers of circulating hematopoietic cells. To further address the extent of regenerative capacity of these BM cells, mice radioprotected for 6 months were again given a 100-µg dose of ProGP followed 24 hours later by a second exposure to 1100 cGy. Remarkably, 70% of ProGP-protected mice survived a second BM lethal dose of irradiation (Fig. 6A). Two months following the second radiation exposure, the peripheral blood of radioprotected mice was analyzed. Near-normal numbers of platelets
Figure 4. Long-term multilineage hematopoiesis in ProGP-protected mice. Six months after exposure to 1100 cGy irradiation, hematopoietic tissues of ProGP-protected mice were analyzed for the presence of different cell lineages. Flow cytometry was used to determine the frequency of B cells (B220), T cells (CD3), myelomonocytic cells (Mac-1/Gr-1), and red blood cell progenitors (Ter119). Age-matched C75BL/6J untreated mice were used as controls. Multilineage hematopoiesis in the PB of a representative ProGP radioprotection survivor is shown in (A). Expression of lineagespecific markers on Ly5.1⫹ hematopoietic cells in the PB (B), spleen (C), and BM (D). The mean and SEM of ProGP-protected (䊐; n ⫽ 4) and control mice (■; n ⫽ 3) is shown. (There were no significant differences between groups using the Student’s t-test.)
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[11]. By contrast, Flt3-L administered 2 hours prior to radiation in this study was not protective. These results confirm that Flt-3L pretreatment ∼20 hours prior to radiation confers radioprotective effects similar to those observed with TNF [21,22], IL-1 [5], and SCF [9]. A remarkable feature of ProGP-induced radioprotection is the high degree of protection of the erythroid and megakaryocytic cell lineages (Fig. 2). Hemoglobin levels only modestly decreased by 7 days (11.5 g/dL), then promptly returned to normal in ProGP-treated mice, while control animals demonstrated severe progressive anemia with hemoglobin levels falling to less than 3.5 g/dL. Similarly, the platelet counts in ProGP-treated mice were maintained at greater than 100,000/µL compared to less than 10,000/µL in controls. By contrast, the study by Hudak et al. evaluating pretreatment with Flt-3L alone demonstrated a ∼66% drop in circulating erythroid cells with platelet counts decreasing to less than 40,000/µL [11]. Both prolonged anemia and slow platelet recovery were observed in these recipients, and these findings correlated with the modest effects of Flt-3L alone on the erythroid and megakaryocytic lineages
Figure 5. Competitive repopulation studies reveal the engraftment potential of radioprotected BM. Six months after 1100 cGy irradiation, the BM from ProGP-protected mice was harvested and evaluated in competitive repopulation studies. Ly5.1 BM cells (5 × 105) from ProGP-protected or normal donors were combined with BM cells (1 × 105) from host Ly5.2 donors and transplanted into lethally irradiated Ly5.2 recipients. At 3 months posttransplant, the PB of recipient mice was evaluated for lineage-specific donor cells by flow cytometry. (A): The percentage of donor (Ly5.1; G) and host (Ly5.2; ■) cells in mice transplanted with either normal BM or ProGP-protected BM. Two independent experiments utilizing two different radioprotected BM donors and eight recipients are shown. (B): The frequency of T cells (CD3), B cells (B220), and myelomonocytic (Mac-1/Gr1) donor cells (Ly5.1) in the PB of competitive repopulation recipients of either normal BM (■) or ProGP-protected BM (G). The mean and SEM for each group is indicated.
of pretreatment dosing with G-CSF alone are modest, with 10% survival at a dose of 200 µg/kg, increasing to 27 to 62% survival at a dose of 2000 µg/kg [8,20]. This higher dose of G-CSF is similar to the dose used in our studies (ProGP 100 µg contains the equivalent of 2000 µg/kg G-CSF), yet the survival observed in these studies was considerably less, indicating that most recipients were not radioprotected by GCSF alone. In a previous study, treatment of recipients with Flt3-L and G-CSF for 14 days beginning 2 days prior to irradiation enhanced the survival of rabbits exposed to high does of radiation; however, the effect of cytokine pretreatment alone was not evaluated in this study [10]. Hudak and colleagues demonstrated that maximal survival (70–80%) was provided by the pretreatment of mice with Flt-3L at 20 hours and 2 hours before irradiation, while a single dose of Flt3-L administered 20 hours prior to irradiation rescued 65% of recipients
Figure 6. ProGP radioprotects mice from a second exposure to 1100 cGy irradiation. Six months after initial protection from 1100 cGy of radiation, mice were injected with 100 µg of ProGP [●] 24 hours prior to receiving a split dose of 1100 cGy (550 cGy × 2) irradiation (n ⫽ 10). Control mice were pretreated with 0.01% BSA in PBS [䊊]. (A) shows the percentage of mice surviving over time. (B) shows the recovery of hematopoietic cells in the PB at 50 days (G; n ⫽ 7). Values from age-matched normal mice are indicated (■; n ⫽ 5). The mean and SEM for each group is shown on a log scale. (Student’s t-test, ∗p ⱕ 0.001, ∗∗p ⱕ 0.01, NS ⫽ not significant.)
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[23,24]. Taken together with our results, these findings indicate that concurrent activation of the Flt3 and G-CSF receptors provides a greater level of protection to both erythroid and megakaryocytic lineages than administration of Flt-3L alone. The long-term multilineage hematopoietic recovery observed following ProGP pretreatment of lethally irradiated recipients may be the result of protecting of both longlived hematopoietic progenitor cells [25,26] and HSC. To determine if primitive HSC were radioprotected by ProGP, BM was harvested 6 to 8 months after ProGP treatment and exposure to lethal irradiation. Unfractionated, radioprotected BM (1 × 105 cells) was transplanted into irradiated CD45 congenic recipients along with 1 × 105 host-type BM cells. This competitive repopulation assay [19] was used to determine the relative repopulating activity of radioprotected donor BM compared to normal BM. The results of two independent experiments demonstrated only a ∼twofold to fourfold lower frequency of radioprotected BM-derived cells in the blood compared to cells derived from normal BM (Fig. 5). Analysis of the progeny of these radioprotected BM-derived cells revealed multilineage hematopoiesis with contributions to both the lymphoid and myelomonocytic cell lineages. These results are consistent with only a ∼twofold to fourfold reduction in the activity of functional HSC in ProGP-protected BM compared to normal untreated BM. This represents a high degree of recovery of the HSC compartment as normal bone marrow is typically depleted of HSC activity following exposure to high doses of radiation [27]. Whether or not ProGP treatment also enhances the survival of a population of long-term hematopoietic progenitors that read out in this competitive repopulation assay remains to be determined. The observation that significant numbers of transplantable HSC were radioprotected by ProGP prompted us to further evaluate the extent of the radioprotective potential of this bifunctional receptor agonist. Cohorts of radioprotected mice were again treated with a single dose of ProGP and exposed to a second BM lethal dose of radiation. Survival of these serially irradiated mice was 70%. This total dose of 2200 cGy is twofold higher than the LD100/30, indicating a remarkable degree of protection of the hematopoietic compartment. Indeed, at this high dose of total-body irradiation, the 30% mortality observed may in part be attributable to toxicity to nonhematopoietic tissues such as lung or liver. Not surprisingly, hematopoietic reconstitution of serially radioprotected mice was not complete. The peripheral blood demonstrated significantly reduced numbers of circulating neutrophils, lymphocytes, and monocytes. By contrast, only modest decreases in the hemoglobin and platelet counts were observed, suggesting a preferential recovery of erythroid and megakaryocytic progenitor cells in these supralethally irradiated recipients. In summary, our results show that pretreatment with an agonist of the Flt-3 and G-CSF receptors protects the hematopoietic compartment from high doses of radiation at the
level of the HSC. It remains to be determined if cytokine pretreatment prevents the initial radiation damage or alternatively enhances the function of critical cellular repair mechanisms. In either case, identifying the molecular mechanisms underlying radioprotection may have important implications for our understanding of HSC regeneration and repair.
Acknowledgments G.D. Searle & Co. (St. Louis, MO, USA) provided Progenipoietin1 and financial support for these studies. P.R. Streeter was a scientist at G.D. Searle when these studies were initiated.
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