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Communicated by Edward A. Boyse, February 9, 1987. ABSTRACT ...... Herrmann, F., Cannistra, S. A. & Griffin, J. D. (1986) J. Immunol. 136, 2856-2861. 42.
Proc. Natl. Acad. Sci. USA Vol. 84, pp. 3871-3875, June 1987 Medical Sciences

Synergistic myelopoietic actions in vivo after administration to mice of combinations of purified natural murine colony-stimulating factor 1, recombinant murine interleukin 3, and recombinant murine granulocyte/macrophage colony-stimulating factor HAL E. BROXMEYER*tt§, DOUGLAS E. WILLIAMS*, GIAo HANGOC*, SCOrr COOPER*, STEVEN RICHARD K. SHADDUCKII, AND DAVID C. BICKNELL*

GILLIS$,

(Hematology/Oncology), tMicrobiology and Immunology, and the tWalther Medical Research Institute, Indiana University School of Medicine, Indianapolis, IN 46223; ¶Department of Research and Development, Immunex Corporation, Seattle, WA 98101; and I'Department of Medicine, Montefiore Hospital, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213

Departments of *Medicine

Communicated by Edward A. Boyse, February 9, 1987

ABSTRACT Combinations of low dosages of purified murine hematopoietic colony-stimulating factors (CSFs}-Lcell CSF type 1 (CSF-1), recombinant interleukin 3 (IL-3), and recombinant granulocyte/macrophage CSF (GM-CSF)-were compared with single CSFs for their influence on the cycling rates and numbers of bone marrow granulocyte/macrophage, erythroid, and multipotential progenitor cells in vivo in mice pretreated with human lactoferrin. Lactoferrin was used to enhance detection of the stimulating effects of exogenously administered CSFs. Concentrations of CSFs that were not active in vivo when given alone were active when administered together with other types of CSF. The concentrations of CSF-1, IL-3, and GM-CSF needed to increase progenitor cell cycling rates were reduced by factors of 40-200, 10-50, and 40->400, respectively; the concentrations needed to increase progenitor cell numbers were reduced by factors of 40-500 (CSF-1), 20-80 (IL-3), and >40->200 (GM-CSF) when these forms of CSFs were administered in combination with low dosages of one of the other forms of CSFs. The results demonstrate that different CSFs can synergize when administered in vivo to increase the cycling rates and numbers of marrow hematopoietic progenitor cells. These findings may be of relevance physiologically to the regulation of myeloid blood cell production by CSFs. The hematopoietic colony-stimulating factors (CSFs) were originally defined by their capacity to stimulate colony formation in vitro from hematopoietic progenitor cells (1, 2), although the CSFs also influence the functional activities of more mature cells (3). CSFs include interleukin 3 (IL-3), also referred to as multi-CSF, CSF-1, a macrophage CSF, granulocyte/macrophage (GM)-CSF, granulocyte (G)-CSF, and erythropoietin, a stimulator of erythrocyte production (1, 3). The CSFs have been purified and the cDNAs and genes for them have been cloned and expressed (1, 3). Their actions in vitro have been well documented (1-3) and various CSFs can synergize with each other in vitro to stimulate hematopoietic cell colony formation (4-8). Recent reports have demonstrated that individual CSFsIL-3 (9-13), CSF-1 (12, 14), GM-CSF (12, 15), and G-CSF (16)-have myelopoietic stimulating activity when administered to animals. The present report evaluates the actions in vivo of combinations of the CSFs-purified recombinant murine IL-3, purified murine L-cell CSF-1, and purified recombinant murine GM-CSF-in mice pretreated with purified iron-saturated human lactoferrmn (LF). LF decreases release of factors from monocytes and macrophages in vitro, which directly (17-19) and/or indirectly (20-22) stimulate The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

colony formation of hematopoietic progenitors, and decreases myelopoiesis and release of hematopoietic stimulating factors in vivo (12, 17, 23). Human LF has been useful as a means of enhancing detection in vivo of the myelopoietic stimulating actions of single injections of relatively low concentrations of either IL-3, CSF-1, or GM-CSF (12). The results presented in this report demonstrate that combinations of CSFs can synergize in vivo to stimulate the percentage of cells in cycle and absolute numbers of bone marrow granulocyte/macrophage (granulocyte/macrophage colonyforming unit; CFU-GM), erythroid (erythroid burst-forming unit; BFU-E), and multipotential (granulocyte/erythrocyte/ macrophage/monocyte/megakaryocyte colony-forming unit; CFU-GEMM) progenitor cells at concentrations of CSFs that are lower by several factors than those that are active when the CSFs are administered as single agents to LF-pretreated mice.

MATERIALS AND METHODS Mice. (C57BL/6 x DBA/2)F1 (BDF1) mice, 6-8 weeks old, were purchased from Cumberland View Farms (Clinton, TN). Recombinant Molecules and Native Factors. Murine IL-3 and GM-CSF were expressed from cloned cDNAs prepared from RNA extracted from LBRM-33-5A4 cells. The two cDNA clones had nucleotide sequences that were essentially identical to those reported by others for their respective factors (24, 25). The factors were produced in a yeast expression system that used the prepro a-factor promoter and leader sequence to direct secretion of the mature forms of the factors (24, 25). The expressed IL-3 and GM-CSF were purified using reversed-phase high-performance liquid chromatography. The IL-3 had a specific activity of 109 units/mg of protein when titered against the IL-3-dependent murine cell line FDC-P2 (12, 24). The GM-CSF had a specific activity of 108 units/mg of protein based upon mouse bone marrow colony formation (12, 25). L-cell CSF (CSF-1) was produced by the growth of murine L cells in serum-free CMRL 1066 medium as described (26). A 10-liter pool of conditioned medium (CM) was purified by affinity chromatography (27) and had a specific activity of 2.3 x 107 units/mg of protein when assayed by the in vitro growth of murine bone marrow colonies (12, 27). This material was subjected to sucrose Abbreviations: CSF, colony-stimulating factor; GM-CSF, granulocyte/macrophage CSF; IL-3, interleukin 3, LF, lactoferrin; CFU-

GM, granulocyte/macrophage colony-forming unit(s); BFU-E, erythroid burst-forming unit(s); CFU-GEMM, granulocyte/erythrocyte/macrophage/monocyte/megakaryocyte colony-forming unit(s). §To whom reprint requests should be addressed.

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density gradient centrifugation as a means of removing any potential endotoxin (28). After centrifugation at 100,000 x g for 18 hr, the upper one-third of the sucrose gradient was aspirated and diluted in 0.1 M Tris-HCl containing 0.3% polyethylene glycol as a stabilizing agent. Purified human milk LF, purchased from Sigma, was fully iron-saturated (19) and depleted of endotoxin by removing the material that gelled in the presence of Limulus lysate (Sigma) (29). The concentrations of LF was measured by an immunoradiometric assay for LF (19). Injections of factors to mice were in volumes of 0.2 ml, and when more than one CSF were used, each CSF was injected separately within a few minutes of the other CSF. By using the Limulus lysate test for endotoxin, which has a sensitivity range down to 0.5 ng/ml, no endotoxin ( 0.05) on the cycling rates of the other progenitors (Table 1). Administration of 200 units of IL-3 significantly increased cycling of BFU-E-2, BFU-E-1, and CFU-GEMM but did not influence cycling of CFU-GM. Concentrations of 500-1000 units of CSF-1 or GM-CSF or 50-100 units of IL-3 had no significant effect on progenitor cell cycling rates. These results are consistent with those reported elsewhere (12). However, when combinations of different CSFs were administered in vivo, concentrations as low as 50 units of CSF-1 with 10 units of IL-3, 50 units of CSF-1 with 50 units of GM-CSF, or 25 units of IL-3 with 100 units of GM-CSF significantly enhanced the cycling rates of all of the progenitors. Concentrations as low as 10 units of IL-3 with 50 units of GM-CSF significantly enhanced the

Table 2. Influence of purified murine CSF-1, IL-3, and GM-CSF, alone or in combination, on numbers of bone marrow nucleated cells and hematopoietic progenitors in vivo after mice have been pretreated with purified human LF Nucleated cells, Test material given in vivo no. x 10-6 per femur CFU-GM Saline 18.0 ± 0.6 28.6 ± 2.0 CSF-1 (2000 U) 18.3 ± 0.5 25.8 ± 1.9 CSF-1 (1000 U) 18.3 ± 0.5 25.5 ± 1.6 CSF-1 (500 U) 21.0 ± 2.0 30.7 ± 3.0 IL-3 (200 U) 17.6 ± 0.5 21.9 ± 2.9 IL-3 (100 U) 17.3 ± 0.9 22.9 ± 2.9 IL-3 (50 U) 19.6 ± 1.1 27.7 ± 3.9 GM-CSF (2000 U) 15.7 ± 0.5 19.3 ± 3.6 GM-CSF (1000 U) 17.0 ± 0.7 21.0 ± 2.8 GM-CSF (500 U) 19.4 ± 1.1 30.7 ± 3.5 CSF-1 (500 U) + IL-3 (50 U) 17.4 ± 0.8 35.3 ± 2.0 (+23)* CSF-1 (100 U) + IL-3 (25 U) 16.8 ± 0.5 31.1 ± 2.4 CSF-1 (50 U) + IL-3 (10 U) 18.5 ± 1.2 27.8 ± 2.0 CSF-1 (10 U) + IL-3 (1 U) 16.5 ± 1.0 26.0 ± 1.5 CSF-1 (500 U) + GM-CSF (500 U) 18.6 ± 0.5 42.5 ± 1.7 (+49)t CSF-1 (100 U) + GM-CSF (100 U) 17.4 ± 0.9 31.8 ± 1.8 CSF-1 (50 U) + GM-CSF (50 U) 18.6 ± 1.0 31.2 ± 1.4 CSF-1 (10 U) + GM-CSF (10 U) 18.1 ± 0.9 28.5 ± 1.1 IL-3 (50 U) + GM-CSF (500 U) 18.6 ± 1.2 42.2 ± 2.7 (+48)t: IL-3 (25 U) + GM-CSF (100 U) 18.6 ± 0.9 31.6 ± 1.8 IL-3 (10 U) + GM-CSF (50 U) 20.0 ± 0.8 33.8 ± 2.1 IL-3 (1 U) + GM-CSF (10 U) 20.0 ± 0.7 31.7 ± 2.4 Protocol and numbers of mice assessed are the same as in Table 1. *Significant % increase from saline group, P at least < 0.05. tSignificant % increase from saline group, P at least < 0.0001. tSignificant % increase from saline group, P at least < 0.005. §Significant % increase from saline group, P at least < 0.0005. $Significant % increase from saline group, P at least < 0.01.

Colonies, no. x 10-3 per femur BFU-E-2 BFU-E-1 2.3 ± 0.3 3.7 ± 0.3 2.1 ± 0.3 3.7 ± 0.3 2.1 ± 0.2 3.7 ± 0.3 2.5 ± 0.4 3.8 ± 0.3 2.5 ± 0.3 3.1 ± 0.3 2.3 ± 0.3 3.0 ± 0.4 2.7 ± 0.4 3.9 ± 0.4 2.5 ± 0.3 3.0 ± 0.3 2.5 ± 0.2 3.6 ± 0.3 2.3 ± 0.3 3.3 ± 0.4 5.1 ± 0.4 (+122)t 4.0 ± 0.3 4.3 ± 0.3 (+87)t 3.9 ± 0.2 2.6 ± 0.5 4.0 ± 0.3 2.6 ± 0.3 3.9 ± 0.2 2.3 ± 0.1 5.5 ± 0.7 (+49)* 2.2 ± 0.3 4.3 ± 0.6 2.1 ± 0.2 4.9 ± 0.8 2.0 ± 0.3 4.5 ± 0.5 4.7 ± 0.2 (+27)* 5.3 ± 0.5 (+43)¶ 4.3 ± 0.7 4.1 ± 0.5

CFU-GEMM 3.1 ± 0.2 3.2 ± 0.1 2.7 ± 0.2 3.7 ± 2.8 ± 2.7 ± 2.9 ± 2.5 ± 2.9 ± 3.0 ± 3.1 ± 2.8 ± 2.7 ± 2.5 ± 4.2 ± 3.0 ± 3.2 ± 2.8 ±

0.6 0.2 0.2 0.2 0.4 0.2 0.3 0.3 0.1 0.2 0.3 0.3 (+35)§ 0.2 0.1 0.2 3.0 ± 0.3 3.0 ± 0.3 2.3 ± 0.2 2.6 ± 0.3

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Table 3. Lowest concentrations of CSFs having activity for marrow hematopoietic progenitor cell cycling and numbers when CSFs are administered alone or in combination to mice pretreated with LF CFU-GM I3FU-E-2 BFU-E-1 CFU-GEMM CSF CSF with CSF CSF with CSF CSF with CSF CSP with alone,* other CSF,t Fold alone,* other CSF,t Fold alone,* other CSF,t Fold alone,t other CSF,t Fold units units change: units units change units units changet units units change* Cycling status, % progenitors in S phase CSF-1 2,000 50 40 10,000 50 200 10,000 50 200 10,000 50 200 IL-3 500 10 50 100 10 10 iOo-200 10 10-20 100-200 10 10-20 GM-CSF 2,000 50 40 >20,000 50 >400 >20,000 50 >400 5,000 50 100 Numbers, progenitors per femur 20,000 500 40 20,000 100 200 20,000 500 40 20,000 500 40 ?§ IL-3 2,000 50 40 2,000 25 80 500 25 20 250 >50 ?§ >20,000 GM-CSF >20,000 500 >40 >20,000 >500 100 >200 >20,000 500 >40 *Lowest concentrations of CSFs having activity when administered alone are derived from studies reported in this paper and elsewhere (12). tLowest concentration of CSF having no activity alone, but having activity when administered with a dosage of another type of CSF, that also has no activity when used alone. tFold change refers to differences between lowered CSF concentrations that are active when CSFs are given together compared to when CSFs are administered to mice in the absence of other exogenous forms of CSFs. §Available data do not allow us to determine whether or not these combinations result in greater than additive effects.

CSF-1

cycling rates of BFU-E-1 and CFU-GEMM but had no effect on the cycling of CFU-GM (Table 1). Influences were also assessed on the numbers of nucleated cells and progenitors per femur in these mice (Table 2). None of the CSFs alone or in combination had a significant effect on nucleated cellularity and none of the CSFs administered to mice as individual molecules influenced progenitor cell numbers. However, 500 units of CSF-1 with 50 units of IL-3 significantly increased numbers of CFU-GM and BFU-E-2, 100 units of CSF-1 with 25 units of IL-3 significantly increased numbers of BFU-E-2, 500 units of CSF-1 with 500 units of GM-CSF significantly increased numbers of CFU-GM, BFU-E-1, and CFUGEMM, 50 units of IL-3 with 500 units of GM-CSF significantly increased numbers of CFU-GM and BFU-E-1, and 25 units of IL-3 With 100 units of GM-CSF significantly increased numbers of BFU-E-1. A comparison of the concentrations of CSFs having activity when administered alone or in combination to LFpretreated mice is shown in Table 3. The values for CSFs alone are based on those reported here and in a previous paper (12). It is apparent that much smaller quantities of CSFs can be used to elicit a stimulating effect in vivo on progenitor cell cycling and numbers when combinations of CSFs are used in comparison to the administration to the mice of single forms of CSF. Thus, much less CSF-1, IL-3, and GM-CSF (concentrations were reduced by factors of 40-200, 10-50, and 40->400, respectively) can be used to increase progenitor cell cycling in vivo when these CSFs are administered with another type of CSF. Moreover, less

CSF-1, IL-3, and GM-CSF (concentrations were reduced by factors of 40-500, 20-80, arid >40-200, respectively) can be used to increase marrow progenitor cell numbers in vivo when these CSFs are administered with a different type of CSF.

DISCUSSION Administration of individual CSFs to animals has demonstrated that CSFs can function in vivo (9-16), and animal models, using mice pretreated with LF, have been useful for demonstrating the short-term effects of low dosages of CSFs on enhancement of progenitor cell cycling and numbers (12). The present results demonstrate that CSFs, when used at low concentrations, which themselves have no myeloid-stimulating effects in vivo, can stimulate the cycling rates and numbers of hematopoietic progenitor cells in the marrows of mice pretreated with LF when the CSFs are combined with

low dosages of a different CSF. These results are consistent with the definition of synergy in action between molecules (33) and the studies in vitro by others (4-7) and ourselves (8) demonstrating synergistic stimulation of myeloid colony formation with combinations of CSFs. These in vivo studies do not allow us to conclude how the synergistic effects of the CSFs are being mediated, but they may reflect direct and indirect effects on the progenitor cells. The molecules may bind directly to specific receptors on the same progenitor cells and/or they may bind to accessory cells and enhance myelopoiesis by inducing the release of other CSFs (34-36) or of other molecules, such as interleukin 1 (37-39), y-interferon (40-42), and/or tumor necrosis factor (43-45), which could subsequently trigger the release of CSFs or other intermediary molecules from other accessory cells. Regardless of the mechanisms involved, synergy in vivo with combinations of CSFs is most likely of significance in the physiological regulation of myeloid blood cell production in vivo. We thank Dr. David Urdal and Dr. Abdul Waheed for their help with the preparations of IL-3 and CSF-1, respectively, and Mrs. Shirley Duke for typing the manuscript. These studies were supported by Public Health Service Grants CA 36464 and CA 36740 (to H.E.B.) and CA 15237 (to R.K.S.) from the National Cancer Institute. D.E.W. was supported by National Ihstitutes of Health Training Program IT32 AM 07519. 1. Metcalf, D. (1985) Science 229, 16-22. 2. Broxmeyer, H. E. (1983) C)?C Crit. Rev. Oncol. Hematol. 1,

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