Myelosuppression in the Pig (Sus scrofa): Uteroferrin Reduces the Myelosuppressive Effects of 5-Fluorouracil in Young Pigs

Comp. Biochem. Physiol. Vol. 116A, No. 4, pp. 369–377, 1997 Copyright  1997 Elsevier Science Inc.

ISSN 0300-9629/97/$17.00 PII S0300-9629(96)00368-4

Myelosuppression in the Pig (Sus scrofa): Uteroferrin Reduces the Myelosuppressive Effects of 5-Fluorouracil in Young Pigs Jamie C. Laurenz,* Michael Hadjisavas, Greg W. Chovanic, and Fuller W. Bazer Department of Animal Science and Center for Animal Biotechnology, Institute of Biosciences and Technology, Texas A&M University, College Station, TX 77843-2471 U.S.A. ABSTRACT. The present study investigated the ability of uteroferrin to modulate the myelosuppressive effects of 5-fluorouracil (5-FU) in young pigs (Sus scrofa). Pigs (28–35 days of age; n 5 6 per treatment) were infused with equal amounts of 5-FU on days 0 and 1 of the experimental period (37.5 mg/kg cumulative dose). Uteroferrin (100 µg/kg in 0.9% NaCl) or control (equivalent volume of 0.9% NaCl) was administered to pigs as intramuscular injections twice daily (08:00 and 20:00 hr) on days 1 through 21. Peripheral blood cell number, composition and progenitor cells were determined over 28 days. Treatment of pigs with 5-FU resulted in a rapid dose-dependent (P , 0.05) leukocytopenia. Concurrent treatment of pigs with uteroferrin reduced (P , 0.05) the rate of 5-FU-induced leukocytopenia (44 vs 77 6 7% decline from baseline on day 3) and enhanced (P , 0.05) the recovery from 5-FU on days 10 and 12 postinfusion. The positive effect of uteroferrin on leukocytes resulted primarily from a protection and/or enhanced recovery of neutrophils and monocytes. In addition, uteroferrin attenuated (P , 0.05) the suppression of red blood cell numbers after 5-FU administration (6.9 vs 6.1 6 0.2 3 106 cells/µl on day 3), an affect reflected in increased hematocrit and hemoglobin concentrations. The effects of uteroferrin appeared to result from enhancement of the proliferation and/or differentiation of primitive pluripotent stem cells resistant to 5-FU, as concurrent treatment of pigs with uteroferrin resulted in a protection and/or enhanced recovery (P , 0.05) of CFU-GEMM, CFU-GM and BFU-E progenitor cells in the peripheral blood. These results are the first to demonstrate that uteroferrin can reduce the myelosuppressive effects of 5FU in the pig and suggest that uteroferrin has hematopoietic growth factor activity in vivo. Copyright  1997 Elsevier Science Inc. comp biochem physiol 116A;4:369–377, 1997. KEY WORDS. Pigs, hematopoiesis, myelosuppression, 5-fluorouracil, uteroferrin, type 5-tartrate resistant acid phosphatase, CFU-GM, CFU-GEMM, BFU-E

INTRODUCTION 5-Fluorouracil (5-FU) is a widely used cytoreductive cancer chemotherapeutic whose mechanisms of action include inhibition of thymidylate synthetase leading to the attenuation of DNA synthesis and incorporation of 5-FU into RNA (19). Hematopoietic toxicity induced by 5-FU administration is frequently a dose-limiting complication, resulting in an increased risk for bacterial infections (3,15,21). A number of positive regulators of hematopoiesis (i.e., various interleukins, colony-stimulating factors and/or stem cell factor) protect or stimulate recovery of the myeloid and Address reprint requests to: F.W. Bazer, Department of Animal Science, 442D Kleberg Center, Texas A&M University, College Station, TX 77843-2471. Tel. (409) 862-2659; Fax (409) 862-2662; E-mail: [email protected]. *Current address: Department of Wildlife Sciences, Texas A&M University–Kingsville, Kingsville, TX 78363. Abbreviations—CFU, colony forming unit; GEMM, granulocyte-erythrocyte-monocyte/macrophage megakaryocyte; GM, granulocytemonocyte/macrophage; BFU-E, burst-forming unit-erythroid. Received 18 April 1996; revised 26 August 1996; accepted 6 September 1996.

lymphoid systems during chemotherapy resulting in both a reduced period of myelosuppression and a potential increased antitumor efficacy through the administration of more frequent or higher doses of the chemotherapeutic (13,18). Uteroferrin from pig uterus and human placenta has in vitro hematopoietic activity (2), suggesting it may be useful in attenuating the myelosuppressive effects of 5-FU. Uteroferrin, a progesterone-induced glycoprotein containing two molecules of iron, is secreted by the uterine endometrial epithelium of pigs (25). It exists as a 35,000 molecular weight (Mr) polypeptide but can be found as a heterodimer (Mr 80,000) associated with one of three uteroferrin-associated proteins having high sequence homology with serine protease inhibitors (14,25). Uteroferrin is a tartrate-resistant acid phosphatase that shares many properties with the type 5 tartrate-resistant acid phosphatase in human placenta, the chondrocytes of osteoclastic bone tumors, spleens of patients with hairy cell leukemia and Gaucher’s and Hodgkin’s diseases (11). Uteroferrin also has characteristics similar to those of splenic purple acid phosphatases in normal cattle, rats, mice and pigs and is a normal con-

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stituent of cow and horse uterine secretions and rat bone (11). During pregnancy in the pig, uteroferrin is secreted by the endometrial glandular epithelium and is taken up by the yolk sac as early as day 17 of gestation when hematopoiesis is initiated in the blood islands (1,4). Later in gestation, uteroferrin is transported from uterine glands into the fetal/ placental circulation by placental areolae (23). Through its mannose residues, uteroferrin is targeted to reticuloendothelial cells of the fetal liver (28), as well as to corresponding cells in spleen and bone marrow (5,6). Targeting of uteroferrin to the sequential sites of hematopoiesis in the fetal animal (26) suggested a role in hematopoiesis. We reported that treatment of hematopoietic stem cells from human and pig bone marrow and neonatal pig liver with uteroferrin results in dose-dependent colony forming unit (CFU) activity for granulocyte-monocyte/macrophages (CFU-GM), erythroid burst forming (BFU-E) and granulocyte-erythrocytemonocyte/macrophage-megakaryocytes (CFU-GEMM) (2). This activity was independent of the ability of uteroferrin to provide iron to the assay medium and suggested that uteroferrin may be a hematopoietic growth factor. In the present report, we provide evidence that uteroferrin can modulate the myelosuppressive effects of 5-fluorouracil in vivo, perhaps through expansion of primitive multipotential stem cells such as CFU-GEMM. MATERIALS AND METHODS Materials Uteroferrin was purified to homogeneity from the uterine secretions of pseudopregnant pigs using ion exchange and gel filtration chromatography (2) and was dialyzed (Spectrum Medical Industries, Inc., Los Angeles, CA) into 0.9% NaCl. The 5-FU was provided by Diana WorthingtonWhite (College of Medicine, University of Florida) and was reconstituted in 0.9% NaCl (pH 7.8). Solutions were filter sterilized (0.2 µm) and determined to be free of endotoxin by the Limulus amebocyte lysate assay (lower detection limit 0.06 ng/ml; ICN Biomedicals, Aurora, OH) before infusion into pigs. Tissue culture flasks were obtained from Corning (Corning, NY) and 35-mm culture dishes from Falcon (Bedford, MA). Iscove’s modified Dulbecco’s medium (IMDM), α-minimum essential medium (α-MEM), RPMI1640, and fetal bovine serum (FBS) were obtained from GIBCO BRL (Gaithersburg, MD). Ficoll-Hypaque and recombinant human erythropoietin (rhEPO) were from Sigma (St. Louis, MO). Recombinant human granulocytemonocyte/macrophage colony stimulating factor (rhGMCSF) and recombinant human interleukin-3 (rhIL-3) were from R&D systems (Minneapolis, MN). Pigs and 5-FU Treatment Prepubertal female pigs (28–35 days of age; 15–20 kg) were offspring of Yorkshire X Landrace sows and Duroc X Hamp-

shire boars. Pigs were housed in a metabolism barn with a controlled environment and allowed ab libitum access to feed and water. Baseline values for hematopoietic parameters were established for individual pigs from blood samples collected on 3 consecutive days before 5-FU treatment. Pigs were administered 5-FU by intravenous infusion into an ear vein on days 0 and 1 of each experimental period. To determine the optimal concentration of 5-FU for myelosuppression, pigs (n 5 3 per concentration) were infused with 5FU at either 0, 3, 6 or 12 mg/kg (experiment 1) and 25, 50, 75 or 100 mg/kg (experiment 2). Blood samples were subsequently obtained on days 0, 3, 6, 8 and 11 by jugular venipuncture. White blood cell counts were performed and the percentage change from baseline values calculated. To assess the effect of uteroferrin on myelosuppression, 12 gilts were assigned randomly to one of two treatments (n 5 6 per treatment). Pig weights were obtained 1 day before the initiation of treatments. Pigs were weighed at weekly intervals and treatment dosages adjusted accordingly. Myelosuppression was achieved through the intravenous administration of 5-FU (37.5 mg/kg cumulative dose). Treatments were administered to pigs on days 1 through 21 as intramuscular injections twice daily (08:00 and 20:00 hr) of uteroferrin (100 µg/kg in 0.9% NaCl) or an equivalent volume of 0.9% NaCl (2–3 ml; control). Blood samples were obtained three times per week (days 0 through 28) and blood cell and differential counts were performed. In addition, at one-week intervals (days 0 through 28) the number of hematopoietic progenitor cells in peripheral blood was ascertained by conducting CFU assays on isolated, nonadherent, mononuclear cells. All experiments were carried out in accordance with the guidelines of the National Research Council for the care and use of animals and were approved by Texas A&M University’s Institutional Animal Care Committee. Hematology Pigs were manually restrained (,2 min) and blood samples taken by jugular venipuncture on the indicated days with vacutainer tubes containing EDTA. Blood cell and differential counts were conducted at the Clinical Pathology Laboratory at the Texas Veterinary Medical Center, Texas A& M University. Peripheral blood white blood cell (WBC) counts, red blood cell (RBC) counts and hemoglobin concentrations (Hgb) were determined using a Coulter S 1 4 Counter (Coulter Electronics, Hialeah, FL). Differential counts were determined by examination of blood smears stained with Wright Giemsa. Neutrophil, lymphocyte, monocyte and eosinophil numbers were calculated from the total and differential WBC counts. Hematocrits were determined after centrifugation in an IEC microhematocrit centrifuge (Needham, MA). Based on RBC counts, hematocrit and Hgb concentration, mean cell volume (MCV), mean cell hemoglobin (MCH) and mean cell hemoglobin concentration (MCHC) were calculated as follows (30):

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MCV (fl) 5 [hematocrit (%) 3 10]/[RBC 3 106] MCH (pg) 5 [total blood hemoglobin (g/dl)]/ [RBC 3 106] MCHC (g/dl) 5 [total blood hemoglobin (g/dl)]/ [hematocrit (%)] CFU Assays At weekly intervals, peripheral blood mononuclear cells were obtained by density gradient centrifugation using Ficoll-Hypaque (specific gravity 5 1.080). Interface cells were collected, washed twice with Hank’s balanced salt solution and resuspended in RPMI-1640 containing 20% FBS. Adherent cells were removed by incubation in plastic Petri dishes at 37°C for 16 hr. The nonadherent cells were washed with RPMI-1640 and resuspended in RPMI-1640 containing 20% FBS before use in CFU assays (2). For CFU assays, 2 3 105 cells per dish were plated in 35-mm dishes in 0.3% agar (16). CFU-GM assays were performed in αMEM containing 20% FBS and 100 U/ml rhGM-CSF. Assays for BFU-E and CFU-GEMM were performed in IMDM containing 30% FBS, 1 3 1025 M β-mercaptoethanol and 1 U/ml rhEPO (BFU-E) or 1 U/ml rhEPO, 30 ng/ ml rhIL-3 and 100 U/ml rhGM-CSF (CFU-GEMM). Cultures were incubated at 37°C in a humidified incubator containing 5% CO2 and 95% air. Colonies were counted after 14 days of culture, with a colony being defined as an aggregate of at least 50 cells.

FIG. 1. Percentage change in white blood cell number

(WBC) in response to increasing concentrations of 5-fluorouracil. Pigs were infused with 5-fluorouracil on days 0 and 1 of the experimental period. Blood samples were collected on the indicated days and WBC number determined. Data are presented as the percentage change from baseline values and represent the means 6 SEM of n 5 3 animals/5-fluorouracil concentration. Dose-dependent changes in WBC number were analyzed by regression analysis and are presented as the regression line with the highest order fit ( P , 0.05). Regression equations are for day 3, % change in WBC 5 22.5 2 0.50x (R 2 5 0.45); for day 6, % change in WBC 5 3.2 2 0.95x (R 2 5 0.88); for day 8, % change in WBC 5 11.2 2 2.38x 1 0.013x 2 (R 2 5 0.87).

Statistical Analysis Data were subjected to analysis of variance using the General Linear Models (GLM) procedures of the Statistical Analysis System (27). Partitioned sources of variation included treatment, day, animal (treatment) and treatment by day. Due to the large variability found among pigs for baseline WBC, neutrophil, lymphocyte, monocyte and eosinophil numbers, data were analyzed as the percentage change from individual mean baseline values. For all data, the results are presented as the least squares means 6 SEM. Within day, treatment-related differences in blood cell numbers, Hgb, hematocrit, MCV, MCH, MCHC and hematopoietic progenitor cell numbers were identified using orthogonal contrasts. RESULTS 5-FU Dose-Response Limited information is available regarding the effects of 5FU on hematopoietic parameters in the pig. Therefore, initial experiments investigated the relationship between increasing concentrations of 5-FU and the percentage change in WBC numbers. After the establishment of baseline, pigs (n 5 3 per concentration) were infused with 5-FU (0–100

mg/kg cumulative dose) and WBC number determined from peripheral blood on days 0, 3, 6, 8 and 11 postinfusion. Treatment of pigs with high concentrations of 5-FU resulted in substantial mortality. All pigs receiving the 75and 100-mg/kg doses and 33% of the pigs receiving 50 mg/ kg 5-FU died 8–14 days postinfusion. For the 100-mg/kg group, significant morbidity was apparent within 4–5 days postinfusion and precluded subsequent sampling. In those pigs that recovered, nadir was reached by day 8; therefore, data analyses were confined to days 0–8 postinfusion. Treatment of pigs with 5-FU resulted in a rapid dose-dependent (P , 0.05) leukocytopenia (Fig. 1). On days 3 and 6, 5-FU treatment resulted in linear decreases (P , 0.05) in WBC number, with 75 mg/kg causing maximal decreases of 42 and 69 6 4%, respectively. By day 8, WBC numbers in pigs receiving 50 and 75 mg/kg 5-FU continued to decrease (P , 0.05) and resulted in similar depressions in WBC (83 6 12 and 93 6 4%, respectively). In contrast, pigs receiving 5-FU concentrations below 50 mg/kg had reached nadir in WBC number or had began recovery. The combination of these effects resulted in a change in WBC number best described by a second order regression equation. Based on

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these results, subsequent experiments were conducted using a cumulative dose of 37.5 mg/kg 5-FU to obtain adequate myelosuppression without causing significant mortality. Effect of 5-FU and Uteroferrin on WBC Parameters Mean baseline WBC numbers did not differ (P . 0.05) between treatment groups and were 16.9 6 2.2 and 15.2 6 1.9 3 104 cells/µl for control and uteroferrin-treated pigs, respectively (range 12.3 to 22.7 3 104 cells/µl). Treatment of pigs with 37.5 mg/kg 5-FU resulted in mortality in both the control (2/6 pigs) and uteroferrin (1/6 pigs) treatment groups. Analysis was confined to those pigs that recovered from 5-FU treatment. Regression analysis indicated the pattern of change in WBC number for control pigs was best described by a sixth order regression equation. Physiologically, this curve appears to represent at least three distinct phases. The first phase (suppressive phase) is characterized by a rapid leukocytopenia with nadir by day 7 postinfusion (22 6 7% of baseline) (Fig. 2). This is followed by a recovery phase in which a sustained leukocytosis occurs (days 14– 21; maximum 167 6 7% of baseline on day 17). The final phase appears to be a readjustment of baseline values (days 22–28). Although it did not significantly reduce (P . 0.05) the extent of suppression (30 6 7% of baseline), uteroferrin did afford protection against the myelosuppressive effects of 5-FU, indicated by a reduced (P , 0.05) leukocytopenia on day 3 (44 vs 77 6 7% decline from baseline for control

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and uteroferrin, respectively). Uteroferrin also enhanced (P , 0.05) recovery from 5-FU on days 10 and 12 (75 and 120 vs 54 and 87 6 7% of baseline on days 10 and 12 for uteroferrin and control, respectively). Consistent with their half-life in peripheral blood, individual WBC types demonstrated a hierarchy of sensitivity to 5-FU. In control pigs, monocytes and a subset of lymphocytes were most sensitive, declining (P , 0.05) by 100% and 58 6 10% from baseline by day 5 postinfusion, respectively (Fig. 3). These changes were followed by dramatic declines (P , 0.05) in neutrophils and eosinophils (100% declines from baseline by day 7, respectively). Treatment of pigs with uteroferrin did not effect (P . 0.05) the decline in lymphocytes and monocytes but caused an initial expansion and/or protection (P , 0.05) of neutrophils and eosinophils (132 6 22% and 149 6 16% of baseline by day 3 postinfusion). Combined, these changes are probably responsible for the initial protective effect of uteroferrin on WBC number. During the recovery phase, the most obvious change in control pigs was an increase (P , 0.05) in neutrophils to 248 6 22% of baseline on day 17. In contrast, lymphocyte number returned to baseline (114 6 10% of baseline) on day 17 and remained near baseline thereafter. Uteroferrin enhanced (P , 0.05) the recovery and extent of neutrophil expansion (309 6 22% of baseline on day 17) but had little effect on the lymphocyte population relative to control. Uteroferrin also modulated changes in monocyte numbers. In control pigs, monocytes returned to baseline values by day 17 (88 6 21% of baseline) and remained near baseline thereafter, whereas eosinophils were generally elevated (P , 0.05) above baseline from days 17 to 28 (maximum 324 6 16% of baseline on day 21). Treatment with uteroferrin resulted in a much earlier recovery of monocytes (103 6 21% of baseline on day 10) with monocyte numbers exceeding baseline (P , 0.05) on day 12 (159 6 21%). With the exception of an earlier peak (335 6 16% of baseline on day 19), uteroferrin did not substantially alter the recovery of eosinophils from 5-FU. These results suggest that expansion of the neutrophil population is primarily responsible for the observed leukocytosis during the recovery phase and this effect is enhanced in pigs treated with uteroferrin. Effect of 5-FU and Uteroferrin on RBC Parameters

FIG. 2. The effect of uteroferrin on white blood cell (WBC)

number in myelosuppressed pigs. Pigs were infused with 5fluorouracil (37.5 mg/kg) on days 0 and 1 of the experimental period. Uteroferrin (100 mg/kg in 0.9% NaCl; n 5 5) or control (0.9% NaCl; n 5 4) were administered as i.m. injections on days 1 through 21. Blood samples were collected three times per week between days 0 through 28 and WBC number determined. Values are expressed as the percentage change from baseline and represent the least-squares means 6 SEM. *Within day differs from control ( P , 0.05).

Mean baseline RBC, hematocrit and Hgb were similar (P . 0.05) between treatments (6.8 6 0.2 3 106 cells/µl, 33 6 1% and 9.8 6 0.3 g/dl vs 7.0 6 0.2 3 106 cells/µl, 34 6 1% and 10.6 6 0.3 g/dl for control and uteroferrin, respectively). The effects of 5-FU on RBC number, hematocrit and Hgb are presented in Fig. 4. In control pigs, treatment with 5-FU resulted in a moderate (22%; P , 0.05) decline in RBC number with nadir at day 10 (5.3 6 0.2 3 106 cells/µl). Although not effecting the overall extent of RBC suppression, uteroferrin did provide a protective effect

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FIG. 3. The effect of uteroferrin on (A) neutrophil, (B) lymphocyte, (C) monocyte and (D) eosinophil numbers in myelosuppressed pigs. Pigs were infused with 5-fluorouracil (37.5 mg/kg) on days 0 and 1 of the experimental period. Uteroferrin (100 mg/kg in 0.9% NaCl; n 5 5) or control (0.9% NaCl; n 5 4) were administered as i.m. injections on days 1 through 21. Blood samples were collected three times per week, total WBC and WBC differential counts were performed and neutrophil, lymphocyte, monocyte and eosinophil numbers calculated. Values are expressed as the percentage change from baseline and represent the least-squares means 6 SEM. *Within day differs from control ( P , 0.05).

for RBC indicated by a higher RBC number on day 3 (6.9 vs 6.1 6 0.2 3 106 cells/µl for uteroferrin and control, respectively), and an increased (P , 0.05) time to nadir (5.6 6 0.2 3 106 cells/ µl on day 17). The 5-FU-induced suppression of RBC number was supported by corresponding decreases in hematocrit and Hgb (26 6 1% and 8.1 6 0.3 g/ dl on days 10 in control pigs). As with RBC number, uteroferrin provided a protective effect as indicated by higher (P . 0.05) hematocrit and Hgb on days 3, 5 and 10. In control pigs, Hgb and hematocrit recovered to baseline values by day 19 and 21, respectively, and recoveries were not affected by uteroferrin. In contrast, RBC number remained below baseline values throughout the experiment in both treatment groups. The apparent dichotomy between the recovery of RBC, hematocrit and Hgb suggested an increase in RBC size. As indicated by Fig. 5, treatment with 5-FU caused a progressive increase in MCV. In control pigs, MCV began to increase (P , 0.05) on day 19 and was maximum on day 28 (47 vs 59 6 1 fl on days 0 and 28, respec-

tively). An increase in RBC size is further supported by an increase (P , 0.05) in MCH (142 vs 187 6 5 pg on day 0 vs 28, respectively), whereas MCHC did not differ (P . 0.05) from baseline at any time point. Treatment of pigs with uteroferrin did not substantially effect MCV, MCH and MCHC relative to control. Effect of 5-FU and Uteroferrin on Hematopoietic Progenitor Cells The effects of 5-FU and uteroferrin on the number of hematopoietic progenitor cells per ml of peripheral blood of pigs are presented as Fig. 6. Initial CFU-GM numbers were similar (P . 0.05) between treatments (1020 6 106 and 870 6 94 per ml of blood for control and uteroferrin treated pigs, respectively). As with WBC, CFU-GM progenitor cells in peripheral blood of control pigs declined (P . 0.05) rapidly and were 75 6 16 per ml of blood on day 7. This was followed by increases (P , 0.05) in CFU-GM on days 14 and

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FIG. 4. The effect of uteroferrin on (A) red blood cell (RBC)

number, (B) hematocrit and (C) hemoglobin (Hgb) in myelosuppressed pigs. Pigs were infused with 5-fluorouracil (37.5 mg/kg) on days 0 and 1 of the experimental period. Uteroferrin (100 mg/kg in 0.9% NaCl; n 5 5) or control (0.9% NaCl; n 5 4) were administered as i.m. injections on days 1 through 21. Blood samples were collected three times per week and RBC number, hematocrit and Hgb determined. Values represent the least-squares means 6 SEM. *Within day differs from control ( P , 0.05).

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FIG. 5. The effect of uteroferrin on (A) mean cell volume (MCV), (B) mean cell hemoglobin (MCH) and (C) mean cell hemoglobin concentration (MCHC) in myelosuppressed pigs. Pigs were infused with 5-fluorouracil (37.5 mg/ kg) on days 0 and 1 of the experimental period. Uteroferrin (100 mg/kg in 0.9% NaCl; n 5 5) or control (0.9% NaCl; n 5 4) were administered as i.m. injections on days 1 through 21. Blood samples were collected three times per week and RBC number, hematocrit and Hgb determined. The MCV, MCH and MCHC for individual pigs were calculated as described. Values represent the least-squares means 6 SEM. *Within day differs from control ( P , 0.05).

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21, resulting in a modest elevation (P , 0.05) above baseline on day 21 (1458 6 106 per ml blood). The CFU-GM in peripheral blood returned to basal values on day 28. Treatment of pigs with uteroferrin resulted in a similar decline in CFU-GM on day 7 but resulted in a more rapid recovery of CFU-GM than control pigs on day 14 (1442 6 98 vs 947 6 106 per ml of blood, respectively), possibly accounting for the reduced leukocytopenia and enhanced leukocytosis in these pigs. As with control pigs, CFU-GM numbers returned to initial values on day 21 and 28. Initial BFU-E colony numbers were 1797 6 75 and 1655 6 65 per ml of blood for control and uteroferrin treated pigs, respectively. As with RBC number, BFU-E in control pigs progressively decreased (P , 0.05) on days 7 and 14 of the experimental period (975 and 558 6 75 per ml of blood, respectively). As for CFU-GM, there was a moderate rebound in BFU-E over initial values on day 21 (2338 6 116 per ml of blood), with BFU-E returning to initial values on day 28. Treatment with uteroferrin provided a protective effect for BFU-E progenitor cells as indicated by the smaller decline relative to control on day 7 (1147 6 65 per ml of blood), which could partially account for the ability of uteroferrin to modulate the suppressive effects of 5-FU on RBC number. Yet treatment with uteroferrin did not effect the decline in BFU-E on day 14 and decreased (P , 0.05) BFUE relative to control on days 21 and 28. Initial CFU-GEMM colony numbers were 118 6 11 and 108 6 10 per ml of blood for control and uteroferrin treated pigs, respectively. In control pigs, CFU-GEMM was suppressed (P , 0.05) on day 7 (31 6 11 per ml of blood) but was dramatically increased (P , 0.05) above initial on day 14 (263 6 11 per ml of blood). Uteroferrin abolished the suppression of CFU-GEMM at day 7 (94 6 10 per ml of blood) yet reduced (P , 0.05) and delayed the increase in CFU-GEMM until day 21 (213 6 10). Protection of these more primitive progenitor cells and their concomitant differentiation could account for the protective effect of uteroferrin on CFU-GM and BFU-E progenitor cells. CFUGEMM subsequently returned to initial values in uteroferrin treated pigs on day 28. DISCUSSION

FIG. 6. The effect of uteroferrin on (A) CFU-GM, (B) BFU-

The present study is the first to document the effect of 5FU on hematopoiesis in the pig. Consistent with its hematotoxic effects in other species (3,15,21), treatment of pigs with 5-FU caused a rapid dose-dependent decrease in peripheral blood WBC. However, it appears that the pig is more sensitive to 5-FU than rodents for which LD50 estimates for 5-FU have ranged from 200–400 mg/kg (3,15) (i.e., in the pig doses of $75 mg/kg resulted in 100% mortality). The enhanced sensitivity to 5-FU could have resulted from the split dose regimen used; however, Harrison and Lerner (9) reported only slight decreases in marrow repopulation when split doses were administered 24 hr apart.

E and (C) CFU-GEMM in peripheral blood from myelosuppresssed pigs. Pigs were infused with 5-fluorouracil (37.5 mg/kg) on days 0 and 1 of the experimental period. Uteroferrin (100 mg/kg in 0.9% NaCl; n 5 5) or control (0.9% NaCl; n 5 4) were administered as i.m. injections on days 1 through 21. Peripheral blood mononuclear cells were isolated at weekly intervals and plated in medium containing 0.3% agar. CFU-GM assays were performed in a-MEM containing 20% FBS and 100 unit/ml rhGM-CSF. BFU-E and CFU-GEMM were performed in IMDM containing 1 unit/ ml rhEpo (BFU-E) or rhGM-CSF, rhEpo and 30 ng/ml rhIL3 (CFU-GEMM). Colonies were counted after 14 days of culture. Values are expressed as progenitor cells/ml of blood and represent the least-squares means 6 SEM. *Within day differs from control ( P , 0.05).

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Hence, the results probably reflect a species and/or genotypic (breed) difference in 5-FU toxicity. In control pigs, 5-FU caused suppression in both WBC and RBC numbers in the peripheral blood. Similar to mice, the effect of 5-FU on WBC number was transient (10,12,22,31) and was followed by a sustained leukocytosis comprised primarily of neutrophils. In contrast, 5-FU caused a persistent suppression in RBC numbers, which was not reflected by coordinate changes in hematocrit and Hgb. This was due to an increase in RBC size, as indicated by the increase in MCV from days 19 to 28. The concomitant increase in MCH in the absence of any change in MCHC further supports this conclusion. These results are in accord with previous reports that both adult and fetal animals treated with 5-FU may prematurely release reticulocytes into the peripheral blood (24,29), a known response to anemia in the neonatal pig (8). Significantly, concurrent treatment of pigs with uteroferrin attenuated the suppressive effects of 5-FU on both WBC and RBC and enhanced expansion of neutrophil and monocyte numbers during the recovery phase. Although the results from these experiments do not allow definitive conclusions concerning the direct and/or indirect mechanism(s) by which uteroferrin modulates the effects of 5-FU on hematopoiesis, the patterns of change in peripheral blood cell numbers and hematopoietic progenitor cells do suggest potential sites of action. In initial experiments with pigs we found that frequent bone marrow biopsies were associated with transient elevations in peripheral blood leukocytes (data not shown). Because the primary focus of this study was to determine the effect of 5-FU and uteroferrin on peripheral blood cell numbers, the effect of treatments on hematopoietic progenitor cells was confined to quantification of those occurring within peripheral blood. Normally peripheral blood progenitor cells are low in number and frequency relative to bone marrow (17). Yet the administration of chemotherapeutic agents alone or in combination with hematopoietic growth factors can result in a transient expansion and mobilization of progenitor cells into the peripheral circulation (7). In addition, the increases in peripheral blood progenitor cells occurs subsequent to recovery of bone marrow progenitor cells but precedes or occurs simultaneously with the recovery of peripheral blood cell numbers (20). Therefore, changes in the number of progenitor cells in the peripheral blood may reflect hematopoietic recovery and may also reflect the ability of cytokines to expand the progenitor cell pool size. In the present study, the numbers of CFU-GM and CFUGEMM progenitor cells in peripheral blood from control pigs declined more rapidly in response to 5-FU than BFUE progenitor cells. These results are consistent with WBC numbers reaching nadir more rapidly than RBC (day 6 vs day 12 for WBC and RBC numbers, respectively). During the recovery phase (days 14 and 21), CFU-GEMM and CFU-GM in peripheral blood increased dramatically and

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actually surpassed baseline values (day 14 and 21, respectively). Consistent with these results, it is hypothesized that during the recovery phase the cytokine milieu in control pigs caused an increase in CFU-GEMM proliferation and subsequently an increase in differentiation of CFU-GEMM to CFU-GM. This would account for the rapid increase in CFU-GEMM and the associated increases in CFU-GM on days 14 and 21. Additionally, because BFU-E arise from CFU-GEMM cells (18), an enhanced rate of CFU-GEMM differentiation to CFU-GM would explain the continued decrease in BFU-E at day 14. The concomitant differentiation of the CFU-GM progenitor cells may also explain the leukocytosis from days 14 to 21. The results also suggest that as in the mouse (10,12,22,31), the cytokine environment during the recovery phase results in a preferential differentiation of CFU-GM toward granulocytic cells and overexpansion of neutrophils. With the increased neutrophils and CFU-GM, feedback mechanisms must increase conversion of CFU-GEMM to BFU-E and allow recovery of BFU-E. Pigs treated with uteroferrin were provided modest protection against suppression in total WBC numbers that resulted primarily from either expansion and/or protection of neutrophils. Additionally, treatment of pigs with uteroferrin resulted in an enhanced recovery of CFU-GM progenitor cells in the peripheral blood, which may reflect the ability of uteroferrin to directly modulate the proliferation and/or differentiation of these cells. However, because subsequent repopulation of hematopoietic cells would be expected to occur through the proliferation of more primitive multipotential stem cells resistant to 5-FU (10,12,31), it is significant that uteroferrin provided protection of the CFUGEMM progenitor cells (day 7). The ability of uteroferrin to enhance the proliferation and/or differentiation (as indicated by the delay in CFU-GEMM increase at day 14) of a more primitive pool(s) of hematopoietic progenitor cells could account for the enhanced recovery of CFU-GM and BFU-E during the suppressive phase and explain why CFUGEMM do not increase above baseline until after hematopoietic recovery. In addition, the subsequent differentiation of CFU-GM and BFU-E progenitor cells could account for both the reduced leukocytopenia and the decreased rate of RBC suppression compared with control pigs. Consistent with this hypothesis, we have shown that uteroferrin stimulates the formation of CFU-GEMM, CFU-GM and BFU-E colonies in vitro (2). The enhanced recovery of CFU-GM progenitor cells and their subsequent differentiation may also cause the augmented leukocytosis occurring in the uteroferrin-treated pigs. In summary, the results from this study are the first to demonstrate that uteroferrin can reduce the myelosuppressive effects of 5-FU in the pig, presumably through its ability to stimulate primitive stem cells such as the CFU-GEMM. These results are consistent with uteroferrin’s demonstrated hematopoietic activity in vitro (2) and suggest that uteroferrin is a unique hematopoietic growth factor. In addition,

Uteroferrin Reduces 5-Fluorouracil-Induced Myelosuppression

the localization of uteroferrin to the major sites of fetal hematopoiesis (4,5,28) indicates that uteroferrin may play an important role in the proliferation and/or differentiation of hematopoietic progenitor cells during fetal hematopoiesis in the pig. Interestingly, the presence of the tartrate-resistant type 5 acid phosphatase, which has high amino acid sequence homology with uteroferrin, in adult humans is indicative of abnormal function of cells associated with hematopoietic tissues (11) and diseases such as hairy cell leukemia. An understanding of this paradoxical situation requires further studies to determine the precise role(s) of uteroferrin in fetal hematopoiesis and whether the increased levels of uteroferrin found under certain disease conditions is associated with and/or merely reflective of the pathology. We thank Dr. Wenbin Tuo, Tom Spencer and Shyh-hwa Liu for their assistance in sample collection. The statistical assistance of Dr. Frank Bartol, Auburn University, and the assistance of Ms. P. A. Benson of the Texas Veterinary Medical Center for blood cell and differential counts is gratefully acknowledged. This research was supported by NIH grant DK 46766 to Fuller W. Bazer.

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