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Influence of rhG-CSF Scheduling on Megakaryocytopoietic Recovery following 5-Fluorouracil-Induced Hematotoxicity in Splenectomized B₆D₂F₁ Mice

Division of Hematology and Oncology, Wayne State University School of Medicine, Detroit, Michigan, USA;

Key Words. Megakaryocytopoiesis • rhG-CSF • Mice • 5-Fluorouracil

Dr. Alexander Nakeff, Division of Hematology and Oncology, Wayne State University, School of Medicine, 550 East Canfield Avenue, Lande MRB 15, Detroit, MI 48201, USA.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Recombinant human granulocyte colony-stimulating factor, rhG-CSF, is widely applied to ameliorate neutropenia following chemotherapy. However, rhG-CSF can exert negative effects on megakaryocytopoiesis that might cause a delay of megakaryocyte recovery. Therefore, the present study was designed to test different rhG-CSF administration protocols with regard to their megakaryocytic inhibitory potential in a 5-fluorouracil (5-FU)-induced experimental model system. Splenectomized B₆D₂F₁ mice received a single injection of 5-FU (150 mg/kg) on day 0 followed by 50 µg/kg/day rhG-CSF given daily for either zero, four, or eight days. Five days after 5-FU, bone marrow and blood hematopoiesis were reduced significantly when compared with controls, independent of whether or not animals received rhG-CSF. However, nine days after 5-FU, granulopoietic recovery from 5-FU-induced toxicity was faster for rhG-CSF-treated versus untreated mice as demonstrated by higher values for colony forming unit-granulocyte macrophage (CFU-GM) and granulocytes (CFU-GM: 7.2 ± 0.4 versus 5 ± 0.6 x 10⁴/femur, granulocytes: 4.3 ± 2 versus 1.4 ± 0.4 x 10⁵/ml, respectively). Furthermore, significant mobilization of CFU-megakaryocyte (CFU-Meg) and CFU-GM into the peripheral blood was induced by the eight-day administration of rhG-CSF following 5-FU (day 9: 911 ± 102 CFU-Meg/ml, 2330 ± 152 CFU-GM/ml). However, megakaryocytic cells in these same mice were considerably lower when compared with those of animals receiving no rhG-CSF (CFU-Meg: 2.7 ± 0.2 x 10³ versus 4.2 ± 0.2 x 10³/femur; small acetylcholinesterase positive (SAChE⁺) cells: 4.9 ± 0.3 x 10³ versus 7.3 ± 0.9 x 10³/femur; megakaryocytes: 2.5 ± 0.2 x 10³ versus 4.1 ± 0.7 x 10³/femur; platelets: 2.67 ± 0.5 x 10⁹ versus 3.1 ± 0.5 x 10⁹/ml, respectively). On the other hand, the shortening of the rhG-CSF treatment from eight to four days caused a rapid granulopoietic recovery comparable to animals receiving eight days of G-CSF with no significant delay in megakaryocytic recovery when compared with mice treated with 5-FU alone; however, with four days of rhG-CSF, the mobilization of CFU into the peripheral blood was significantly less effective. Taken together, the results showed that a shortening of rhG-CSF treatment after chemotherapy is capable of ameliorating neutropenia without negatively affecting megakaryocytopoietic recovery. If, however, maximum recruitment of CFU into the peripheral blood circulation by rhG-CSF for subsequent harvest and transplantation is needed, any shortening of rhG-CSF administration is not advisable.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Recombinant human granulocyte colony-stimulating factor (rhG-CSF) has been shown to be an agent of significant therapeutic value for shortening the chemotherapy-induced period of blood neutropenia. Other applications for rhG-CSF have included the amelioration of congenital, acquired, and cyclic neutropenias [1-3]. Recently, rhG-CSF has been used widely for mobilization of peripheral blood progenitor cells (PBPC) for use in autologous as well as allogeneic stem cell transplants [4].

Although the in vitro effects of rhG-CSF are lineage-specific for granulopoiesis, evidence (mainly from animal experimentation) exists that rhG-CSF treatment in vivo also affects non-granulopoietic cells. Several reports show that rhG-CSF can depress marrow erythropoiesis in mice which is effectively compensated by an enhanced splenic erythropoiesis [5-10]. Platelet levels were reported to be reduced by high doses of rhG-CSF in Syrian hamsters and mice [11, 12]. A transient, dose-dependent thrombocytopenia was observed in a phase I clinical study of rhG-CSF in 18 patients who had received chemotherapy and radiation therapy at least three weeks prior to receiving rhG-CSF [13]. In another clinical study, consistent severe thrombocytopenia in the rhG-CSF-treated group, as opposed to the non-rhG-CSF-treated group, was observed in patients receiving concurrent chemoradiotherapy for stage IIIb non-small cell lung cancer [14].

We have previously shown that rhG-CSF treatment of splenectomized B₆D₂F₁ mice caused a significant decrease of femoral CFU-Meg as well as a decrease of platelets [15, 16]. In these studies, following a single injection of 5-fluorouracil (5-FU) (150 mg/kg, day 0), rhG-CSF (50 µg/kg/day) administered on days 1 through 8 suppressed megakaryocytopoietic recovery as indicated by significantly lower CFU-Meg/femur and platelet numbers on day 9, whereas granulopoietic recovery was accelerated by rhG-CSF. When rhG-CSF treatment was started on day 5, no beneficial effect on granulopoietic recovery was observed, but again, platelet levels were significantly lower, indicating that within the first four days of rhG-CSF application, recruitment or lineage competition was not a critical event. The exact mechanism of this rhG-CSF-related inhibition of megakaryocytopoiesis is not known, but the development of strategies that would avoid the potential non-myeloid-specific inhibitory effects of rhG-CSF without compromising the beneficial effects on granulopoiesis are warranted, particularly for those clinical situations that carry a high potential for treatment-induced thrombocytopenia.

Therefore, this study was undertaken to test whether or not a shortening of rhG-CSF treatment after chemotherapy might be reasonable to avoid inhibition of megakaryocytopoiesis while accelerating granulopoietic recovery after 5-FU-induced hematotoxicity. Our data showed that this could be achieved by reducing the rhG-CSF treatment period from eight to four days. However, if progenitor cell recruitment is required, the shortening of rhG-CSF treatment (or the delayed administration of rhG-CSF [16]) is not appropriate.

	Materials and Methods

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Mice
Male B₆D₂F₁ mice (C57BL/6 x DBA/2), obtained from the NIH, were between 10 and 12 weeks of age at the beginning of all experiments. Acidified water (pH = 2.5) and mouse chow were provided ad libitum, and cages were covered by a filter hood. Splenectomy was performed under general anesthesia. Mice were allowed to recover for at least four weeks following splenectomy before being included in the subsequent protocols.

Drug Treatments
Mice received a single i.v. injection of 150 mg/kg 5-FU (Sigma; St. Louis, MO) on day 0. The 5-FU dose was chosen according to Radley et al. [17] and Radley and Scurfield [18]. Dilutions of rhG-CSF (AMGEN; Thousand Oaks, CA) were prepared in Dulbecco's phosphate buffered saline ([DPBS], GIBCO; Grand Island, NY) + 0.1% bovine serum albumin (BSA). Mice received 50 µg/kg rhG-CSF per day, delivered as two daily s.c. injections. The rhG-CSF dose was chosen according to our previously published results [15, 16]. One day after 5-FU injection, growth factor therapy with rhG-CSF was commenced for either four days (days 1 to 4) or eight days (days 1 to 8). One group of control animals received 5-FU and carrier solution and another control group received only carrier solution.

Number of Marrow and Blood Progenitor Cells
At times indicated, mice were bled retro-orbitally and killed by cervical dislocation the day after the last rhG-CSF injection. For every group, citrated blood was pooled from three animals. Peripheral blood mononuclear cells were isolated after centrifugation on a layer of Histopaque (1.083 g/ml; Sigma). The interphase was removed and washed three times with Hanks balanced salt solution (GIBCO) and resuspended in Leibovitz-15 (GIBCO) supplemented with 2% fetal calf serum ([FCS], GIBCO). Monodispersed cell suspensions of bone marrow were obtained by flushing of femurs with Leibovitz-15 medium + 2% FCS (GIBCO) at room temperature. The marrow was pooled from groups of three donor mice. Total nucleated blood and bone marrow cell counts were obtained using a hemacytometer, and fractions of the resulting cell suspension were used for assaying progenitor cells committed to the megakaryocyte (CFU-Meg), and granulocyte-macrophage (CFU-GM) lineage using a modified plasma culture technique [19, 20]. Briefly, unfractionated femoral or blood nucleated cells were cultured in Leibovitz-15 medium supplemented with 20% FCS (GIBCO), 56 µg/ml CaCl₂ (Sigma), 0.1 U/ml thrombin (Sigma), and either 10% L-cell-conditioned medium or 5% pokeweed mitogen-conditioned medium (Stem Cell Technologies; Vancouver, BC, Canada) for stimulation of CFU-GM and CFU-Meg growth, respectively. Clotting was induced by addition of 10% prescreened bovine citrated plasma (Sigma) and 0.5 ml clots were cultured in quadruplicate in Nunclon culture dishes (NUNC; Kamstrup, Denmark) at 37°C with 5% CO₂ and 5% O₂ for either seven (CFU-GM) or five days (CFU-Meg). Colonies consisting of >50 granulomacrophagocytic cells (hematoxylin-stained) were counted as CFU-GM; CFU-Meg were identified as >3 AChE⁺ cells, as defined previously [19].

Number of Megakaryocytopoietic Bone Marrow Cells
Small, acetylcholinesterase-positive (SAChE⁺) cells were quantitated in duplicate, and 20 µl samples of marrow dried and fixed on glass slides with 2% glutaraldehyde and then stained for 90 minutes at 37°C with acetylthiocholine iodide (Sigma) [19]. Preparations were counterstained with hematoxylin and mounted for microscopic examination. AChE-positive cells 129 µm² in area [21] and with diffuse staining were quantitated and expressed per femur. Femoral megakaryocytes were quantitated on the same slide.

Blood Cell Numbers
Total white blood cells (0.4% crystal violet staining) and platelets (in 1% ammonium oxalate, phase contrast) were counted by hemacytometer. For each group, blood smears were prepared in duplicate and stained with Wright-Giemsa for quantification of neutrophilic granulocytes.

Statistical Analysis
Results are presented as the mean ± the standard error (SE) of at least three separate experiments. Statistical analysis was performed using Student's t-test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effects of rhG-CSF Administration Alone on Megakaryoand Granulocytopoiesis
To characterize the effects of rhG-CSF on megakaryocyto- and granulopoiesis, we treated splenectomized B₆D₂F₁ mice with 50 µg/kg rhG-CSF per day for eight days. As shown in Table 1, rhG-CSF caused a significant decrease of femoral CFU-Meg to about 60% and 56% of carrier-treated animals after five and eight days of rhG-CSF, respectively. Furthermore, rhG-CSF-induced megakaryocytopoietic suppression was demonstrated by significantly lower numbers of SAChE⁺ cells (46% and 35% of those in carrier-treated controls at days 5 and 9, respectively) and bone marrow megakaryocytes (61% and 32% of those in carrier-treated controls at days 5 and 9, respectively). Neither the number of granulopoietic marrow progenitors nor the total number of femoral nucleated cells were significantly altered by rhG-CSF on day 9. Compared with carrier-treated control mice, rhG-CSF caused a significant rise of total nucleated blood cells (i.e., 1.8 ± 0.28 x 10⁷/ml in the case of carrier-treated mice compared with 4.1 ± 0.36 x 10⁷/ml for rhG-CSF-treated mice; p < 0.001) with granulocytes being significantly increased about ten- (day 5) and sevenfold (day 9). Platelets, however, were decreased to about 53% following eight days of rhG-CSF treatment. The rhG-CSF-induced recruitment of progenitors was documented by high levels of peripheral clonogenic cells after eight days of rhG-CSF administration with circulating CFU-Meg and CFU-GM being increased about 20- and 50-fold, respectively ( Table 1).

As shown in Table 2, the treatment with a single injection of 150 mg/kg 5-FU caused a significant reduction of bone marrow and blood hematopoiesis after five days as compared with control animals. At day 9, however, all megakaryocytopoietic parameters (femoral CFU-Meg, megakaryocytes, SAChE⁺ cells, and blood platelets) of 5-FU-treated mice were increased over controls, whereas femoral nucleated cells and CFU-GM, as well as circulating nucleated cells and granulocytes, remained below control values.

View larger version (19K): [in this window] [in a new window]   Figure 1. Effects of rhG-CSF administration on megakaryocytopoietic recovery from 5-FU-induced hematopoietic injury. Mice received a single i.v. injection of 150 mg/kg 5-FU on day 0. Data are shown for day 5 and day 9 after 5-FU. Groups of mice either received twice-daily injections with carrier (open bars), 50 µg/kg/day rhG-CSF daily for up to four days (gray bars), or eight days (solid bars). Data are shown as mean ± SE. Significant differences between rhG-CSF-treated and non-rhG-CSF-treated groups are indicated as: *: p<0.05; ***: p<0.001.

View larger version (14K): [in this window] [in a new window]   Figure 2. Effects of rhG-CSF administration on femoral total nucleated cells and granulopoietic recovery from 5-FU-induced hematopoietic injury. Mice received a single i.v. injection of 150 mg/kg 5-FU on day 0. Data are shown for day 5 and day 9 after 5-FU. Groups of mice either received twice-daily injections with carrier (open bars), 50 µg/kg/day rhG-CSF daily for up to four days (gray bars), or eight days (solid bars). Data are shown as mean ± SE. Significant differences between rhG-CSF-treated and non-rhG-CSF-treated groups are indicated as: *: p < 0.05, **: p < 0.01, ***: p < 0.001.

View larger version (11K): [in this window] [in a new window]   Figure 3. Effects of rhG-CSF administration on the mobilization of megakaryocytopoietic (CFU-Meg) and granulopoietic (CFU-GM) progenitors during recovery from 5-FU-induced hematopoietic injury. Mice received a single i.v. injection of 150 mg/kg 5-FU on day 0. Data are shown for day 9 after 5-FU. Groups of mice either received twice-daily injections with carrier (open bars), 50 µg/kg/day rhG-CSF daily for up to four days (gray bars), or eight days (solid bars). Data are shown as mean ± SE. Significant differences between rhG-CSF-treated and non-rhG-CSF-treated groups are indicated as: *: p < 0.05, ***: p < 0.001.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Utilizing the well-described hematopoietic recovery following 5-FU injection [17, 18], we previously demonstrated that nine days following 150 mg/kg 5-FU, platelets and femoral CFU-Meg in rhG-CSF-treated splenectomized B₆D₂F₁ mice were significantly lower than in non-rhG-CSF-treated controls, indicating an inhibitory influence of rhG-CSF on megakaryocytopoietic recovery [16]. Interestingly, the beneficial effect of eight days of continuous rhG-CSF on granulopoiesis resulting in an accelerated recovery of blood granulocytes was not observed when rhG-CSF administration was delayed for five days. Furthermore, in this latter group (rhG-CSF starting on day 5 after 5-FU), platelet levels remained significantly reduced at day 9 as they did in the 5-FU group treated for eight days with rhG-CSF [16]. These previously reported results showed that within the first four days, neither recruitment nor lineage competition were critical events, although the first four days of rhG-CSF treatment were necessary for a rapid recovery of granulopoiesis [16].

These observations prompted us to test whether a shortening of rhG-CSF administration after chemotherapy might be able to ameliorate neutropenia but not suppress megakaryocytic recovery from 5-FU-induced hematotoxicity. The results reported in the present study clearly demonstrate that rhG-CSF administration initiated immediately after 5-FU but limited to four days is superior to the eight-day rhG-CSF regimen with regard to megakaryocytic recovery ( Fig. 1). Eight days of rhG-CSF significantly delayed bone marrow CFU-Meg, SAChE, and megakaryocyte recovery when compared with 5-FU only. No statistically significant differences were observed at day 9 for those animals receiving four days of rhG-CSF after 5-FU over the 5-FU-only group. On the other hand, four days of rhG-CSF was as efficacious as the eight-day regimen with regard to accelerating the recovery of granulopoiesis after 5-FU ( Fig. 2). Whereas eight-day rhG-CSF-induced inhibition of megakaryocytopoietic recovery was clearly observable at the bone marrow level, platelet numbers, at least within the time frame studied, were not different among the groups. One possible explanation for this finding might be that a compensatory increase in platelet production, i.e., an increase of platelets produced per megakaryocyte per time, effectively compensated for the decrease of bone marrow megakaryocytes, an effect that can also be observed within the first days of rhG-CSF administration in otherwise unperturbed mice ( Table 1).

According to the guidelines developed by the American Society of Clinical Oncology [23], rhG-CSF has been used increasingly within the last years to ameliorate neutropenia following conventionally dosed chemotherapy as well as PBPC-supported high-dose chemotherapy. Despite the increasing clinical importance of rhG-CSF, its potential to impair bone marrow megakaryocytopoiesis (as indicated by our present data), and its high costs in the current cost-constrained clinical environment, the optimization of rhG-CSF administration has only been poorly investigated, with the majority of the published studies considering rhG-CSF support of autologous stem cell transplantation. After autologous transplantation, a delayed start of rhG-CSF for up to seven days does not seem to negatively affect neutrophil recovery [24-27]. However, this generalization remains to be confirmed in appropriately sized and randomized phase III studies. With respect to conventional, non-stem-cell-supported chemotherapy, even less information is available. Morstyn et al. [28] reported a phase I study with three to four patients per group showing that rhG-CSF started eight days after single-agent melphalan treatment was as effective with regard to prevention of neutropenia as rhG-CSF started after two days. Soda et al. [29] recently compared three different rhG-CSF administration schedules for lung cancer patients receiving MVP chemotherapy (mitomycin C 8 mg/m² on day 1; vindesine 3 mg/m² on days 1 and 8, cisplatin 80 mg/m² on day 1). Here, the incidence of neutropenia was lowest in the group receiving rhG-CSF started at day 8 (last day of chemotherapy) when compared with initiation at day 2 or the "therapeutic group," with rhG-CSF initiated after the onset of neutropenia.

Our present results demonstrate that the scheduling of rhG-CSF is critical for the effective amelioration of neutropenia as well as amelioration of possible negative effects on non-granulopoietic cells—clearly, an important consideration for future clinical trials.

Another striking finding is the rhG-CSF-induced release of pluripotent hematopoietic stem cells and committed progenitors of all cell lineages into the circulation, which has been reported in both rodent models and humans [5, 7, 9, 30]. Recently, rhG-CSF has been used increasingly for mobilization of peripheral blood progenitor cells for autologous as well as allogeneic transplantation [4]. When compared to bone marrow, PBPC transplantation is linked to a faster neutrophil as well as platelet recovery, which in turn leads to reductions of infectious episodes and need for antibiotics as well as platelet transfusion [4]. Here, our data clearly show that granulopoietic and, importantly, megakaryocytic progenitor cells are effectively mobilized into the peripheral blood by rhG-CSF. Thus, a large number of mobilized megakaryocytic progenitor cells transplanted following high-dose therapy might contribute to the rapid platelet recovery observed following PBPC transplantation.

However, appropriate randomized clinical studies addressing the optimal rhG-CSF schedule for chemotherapy plus rhG-CSF-supported PBPC mobilization are missing. Our results showed that, if optimal PBPC mobilization is desired, rhG-CSF treatment should be commenced immediately after chemotherapy and administered for a prolonged period that exceeds the minimum time required to ameliorate neutropenia.

Taken together with our previous studies, the results of the present study provide additional evidence that the in vivo non-lineage effects of rhG-CSF include impairment of megakaryocytopoiesis and that, in situations with high platelet demand (such as that following chemoradiotherapy), this inhibitory effect might limit the efficacy of rhG-CSF to ameliorate treatment-induced myelosuppression. Furthermore, our results show that the negative rhG-CSF-induced effects on megakaryocytopoiesis might be overcome by choosing appropriate rhG-CSF administration protocols.

	Acknowledgments

 
This work was supported by funds from the Wayne State University Ben Kasle Trust for Cancer Research and a grant from the Deutsche Forschungsgemeinschaft, Germany (Sche364/1-1) awarded to SS.

	Footnotes

 
^a Present address: Medical Clinic II, University of Tübingen, Tübingen, Germany

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