Effect of Glucan on Granulopoiesis and Macrophage Genesis in Mice1

[CANCER RESEARCH 37, 1739-1742, June 1977] Effect of Glucan on Granulopoiesis and Macrophage Genesis in Mice1

Carmen Burgaleta and David W. GoIde2

Division of Hematology-Oncology, Department of Medicine, UCLA School of Medicine, Los Angeles, California 90024

SUMMARY reticuboendothelial system (6, 31). Glucan consists of a chain of glucopynanose units joined by 1-fJ glucoside link Glucan, a potent meticuloendothelial stimulant, is a gluco ages. When glucan is injected into rodents, it causes a pymanose polysacchanide derived from zymosan. Because marked increase in size and weight of the spleen, liver, and of glucan's potential as an immunotherapeutic agent, we lungs, and appears to increase macnophage activity in these performed studies in order to determine its effect on granu organs (7-9). Glucan administration has been reported to bopoiesis and macrophage production in mice. One week cause transient tumor regression in rats with Shay chboro afterthe i.p. injection of 4 mg of glucan, there was a tenfold leukemia (8) as well as inhibition of tumor growth in synge increase in colony-forming cells in the spleen and approxi neic systems such as B16 mouse melanoma and BW 10232 mately a twofold increment of cells in the bone marrow and adenocancinoma (24). There is also,.some limited expeni the penitoneal cavity capable of colony formation in vitro. ence with glucan immunotherapy in man (24, 25). Themewas a relative and absolute increase in the number of We undertook a series of experiments designed to assess pure macrophage colonies from bone marrow and spleen. the effect of glucan on granubocyte-monocyte progenitor The total macnophage content in spleen, penitoneal cavity, cells and macnophage genesis in the mouse. and bone marrow was also increased in the treated mice. Serum fromglucan-injectedmicehad highcobony-stimubat ing activity levels, and the peritoneal macrophages elabo MATERIALS AND METHODS rated increased colony-stimulating activity in vitro as com Glucan was prepared in the laboratory of Dr. N. Di Luzio pared to controls. Peripheral white blood cell counts were by techniques previously described (19). The material was two times greater than those of control in the glucan-tneated provided in a stock solution of 37 mg/mb and diluted to 2 mice.These studiesindicatethatglucanadministrationme mg/mb in 5% dextrose. The preparation contained no endo suIts in increased granubocyte and macnophage production. toxin as determined by the limulus assay. The enhanced Ieukopoiesis is probably mediated in part by Female DBA/2 mice, 8 to 10 weeks old, were used in all augmented release of colony-stimulating activity from mac experiments. The mice received i.p. injections of 2 mg nophages. These observations suggest that the use of glu glucan (approximately 200 mg/kg) on Days 0 and 1 and can as an immunotherapeutic agent can result in an in were sacrificed by cervical dislocation 6, 10, 15, and 20 days creased number of available effecton cells. after the 1st injection. Control mice received equal volumes of 0.9% NaCI solution or 5% dextrose. A minimum of 3 INTRODUCTION mice/group were used in each experiment. The spleens were removed aseptically, and single-cell suspensions were A number of agents referred to as neticuboendothelial prepared in a medium (Flow Laboratories, Inglewood, Calif.) stimulants on macmophage activators have been considered with 15% fetal calf serum and antibiotics. Bone marrow was useful in immunotherapy (1, 13, 15, 20, 27, 28). These flushed from the femona, and the cells were suspended in agents are frequently of plant on microbial origin and have a the same medium. Penitoneal cells were obtained by wash multitude of biological effects. The immunophammacobogy ing the penitoneal cavity with Hanks' balanced salt solution of the macrophage activators has recently come under containing 10 units of preservative-free hepanin per ml, 15% close scrutiny because of their potential robe in cancer fetal calf serum, and antibiotics. The cells were washed therapy. twice and resuspended in a medium. Total nucleated cell Pilbememand Ecken (30) applied the name zymosan to a counts in the spleen, femur, and penitoneal cavity were yeast cell wall fraction of Saccharomyces cerevisiae which determined by using a hemocytometem. The number of mac produced prominent hyperplasia and hypenfunction of the rophages present in bone marrow, spleen, and the penito neticuboendothelial system when administered to expemi neal cavity was determined by differential counts performed mental animals. Glucan, a polysacchanide of approximately on cytocentrifuge preparations stained with Giemsa and for 6500 daltons, has been isolated from zymosan and shown to a-naphthyl butyrase (32). be responsible for most of the stimulatory effects on the Cells from the 3 sources were assayed for colony-forming capacity in agam.A single-layer culture method was used 1 Supported by USPHS Grants CA 15688 and CA 15619.

2 To whom requests for reprints should be addressed. with 0.3% agar, a medium, and 15% fetal calf serum using Received December 28, 1976; accepted March 10, 1977. techniques previously described (16, 18). An extract of

JUNE 1977 1739

Table 1 EffectcellsSpleenâ€•Bone of glucan on cell counts and in viâ€˜trocolony-forming marrowâ€•Viable

10@)Daycell countb (x 108) Total CFU@Cb (x 10@)Viable cell countb (x 108) Total CFU@Cb (x

20Glucan1.3 7 Day 20 Day 7 Day 20Day 7 Day 20 Day 7 Day Â±1 2.1 Â±0.3 9.3 Â±2 42 Â±2 Â±2 4.1 Â±0.3 9.3 Â±1 20 Â±0.6 0.05)â€•Control1.0 (p 0.01)â€•(p 0.01)c1.6 (p 0.05)c (p

Â±2 1.4 Â±10.4Penitoneum 0.9 Â±0.3 1.1 Â±0.31.4 Â±3 2.6 Â±2 5.6 Â±1 7.1 Â± a blood leukocytesa Differentialcountsâ€•Granubocyte Total Peritoneal cellsâ€• Total macrophagesb macrophage Lymphocyte Monocyte (%)Glucan (x 10@) (x 10â€•) colonies (x 102)PeripheralWBC/mlâ€• (%) (%) Â±0.7 4.4 Â±0.4 2 Â±0.2 Â±500 26 Â±4.9 67 Â±4.9 7 Â±1.2 @ (p 0.01)â€• (p 0.01)â€• (p 0.01)â€• Control7.3 2.4 Â±0.3 1.8 Â±0.2 1 Â±0.26662 3025 Â±275 18 Â±3 78 Â±7 4 Â±1

a Seven days after glucan injection. b Mean of 7 experiments Â± S.E.

C Student's t test (1-tailed). d Mean of 9 experiments Â± S.E.

pregnant mouse uterus was used as the source of CSA3(3). (Table 1). Bone marrow CFU-C were increased approxi This preparation had an activity of 3000 to 6000 cobonies/ mately 75% in the treated group (P 0.05; t test) (Table 1). mg protein. Fifty pi were added to each culture, providing a These studies on spleen and bone marrow were repeated CSA concentration known to stimulate maximal colony for 10, 15, and 20 days after injection. By 20 days themewas a mation. Plates were incubated in a humidified environment further increase in spleen and bone marrow CFU-C (Table of 7.5% CO2 in air at 37Â°,and colonies were enumerated 1). after 7 to 10 days for bone marrow and spleen cultures and Glucan injection produced a consistent increase in the 30 days for pemitoneal cell cultures. The minimum colony total number of cells recovered from the penitoneal cavity. size was taken as 50 cells. The ability of spleen cells to clone The number of macnophages and macnophage colony-form without added GSA (â€œautostimulation'â€˜)wasstudied by plat ing cells in the penitoneal cavity were 2 times higher in the ing high concentrations of cells (1 x 10@to 4 x 106/plate), treated group (Table 1). and the colonies and clusters were counted at Day 7. Cob Spleen cells from glucan-treated animals fonmed colonies nies were examined morphobogicabby at high power with without added CSA more readily than did spleen cells from an inverted microscope, and selected colonies were picked controls (Table 2). At a concentration of 4 x 106cells/plate, with a finely drawn pipet, washed in phosphate-buffered themewere 4 times more autostimulated CFU-C, suggesting saline (No. 420; Grand Island Biological Co. , Grand Island, increased numbers and/or activity of colony-stimulating N.Y.), and deposited on glass slides with a cytocentnifuge cells. Normal spleen and bone marrow CFU-C were of gran for morphological and cytochemical analysis. ubocytic, monocyte-macmophage, and mixed types. In cub In order to assay serum CSA activity, 0.1 ml of serum was tunes from glucan-treated animals, 40% of the colonies were added to normal mouse bone marrow cultures in the dou exclusively macnophage (attaching to and spread on the ble-bayer agan culture system as previously described (16, 17). Glucan-stimulated and control macmophages (1 and 2 x Table 2 105/plate) were also incorporated as feeder layers in 0.5% Effect of glucan on elaboration of CSA agan undenlayems to test for the elaboration of CSA in the Splenic CFU-C with marrowMacrophagesSerumx 10@bon out added GSA double-layer system. cellsa.e Blood was obtained by cardiac puncture in lightly ether (autostimulation) ized mice, and WBC and differential counts were pen Cell no. formed. plated io5Glucan1(x 10) Coloniesâ€•Clusters/2@b)1x 10â€• 2 x (100 61Â±195Â±15Control1 4Â±2 RESULTS 2 8Â±2.3 4 16Â±3.322Â±4.5 Glucan administration had relatively little effect on total nucleated cell counts in spleen and femur, but the CFU-C 0.6 Â±0.3 Â±0.6 14 Â±758 Â±18 pen spleen was increased tenfold in the gbucan-tmeatedani 2 3.5Â±0.3 4 4.3Â±0.86.3 mals 7 days after injection when compared to controls a Studies performed 7 days after glucan injection.

3 The abbreviations used are: CSA, colony-stimulating activity; CFU-C, b Mean of 4 experiments Â± SE. colony-forming unit, culture. C Mean of 3 experiments Â± S.E.

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1977 American Association for Cancer Research. Glucan Effect on Macrophages and Granulopoiesis bottom of the plate), whereas these constituted only 2 to 3% augmented production of macrophages in vitro was found of the colonies in control cultures. The colonies formed to coincide with in vivo events. Total macrophage counts in from peritoneal cells were all of the macrophage type. Total spleen, peritoneal cavity, and bone marrow were substan macnophage counts in spleen, peritoneal cavity, and bone tially increased in mice receiving glucan. marrow were increased 2 to 3 times in the treated mice. Studies of GSA production in vivo and in vitro were con Serum from glucan-treated mice had increased CSA in ducted in an effort to define the mechanism of increased comparison with control (Table 2). Glucan-activated macro leukopoiesis. Colony and cluster formation occurred in phage underlayers also exhibited markedly increased GSA agan cultures of spleen cells from treated animals plated at (Table2). high density without an exogenous source of CSA. This Peripheral WBC counts were 2 times greater than control phenomenon of autostimulation is due to increased num values in glucan-treated mice (Table 1). There was no signif bens or activity of colony-stimulating cells (monocytes and icant difference in the differential counts. macmophages) capable of releasing GSA in the culture dish (18, 26). Furthermore, glucan-activated macmophages elab orated considerably more GSA than did control cells when DISCUSSION tested as a feeder layer in the agar culture system. The serum from glucan-treated mice also had increased GSA. A variety of chemicals and biological materials are me Supporting evidence that the increased GSA resulted in ferred to as macrophage activators (1, 4, 15, 27, 28). These augmented leukopoiesis in vivo was obtained with the ob substances cause a characteristic general increase in mac servation of twofold higher peripheral WBC counts in the rophage function (5). More importantly, some are reported treated mice. to induce the capacity for syngeneic tumor cell cytotoxicity Our data indicate that glucan administration increased (1 , 15, 21 , 29). However, all â€œactivatedâ€•macrophages may granubocyte and monocyte-macnophage production in not exhibit cytotoxicity. There is now a large literature on mice. The augmented leukopoiesis is likely mediated by macmophage activation and the role of macmophages in con increased release of GSA from macrophages occurring con taming and combatting tumor growth (11-13, 20, 28). A comitantly with the activation process. These observations lymphocyte product (macmophage-activating factor), me suggest that when glucan is used as an immunotherapeutic leased during mitogen or antigen stimulation, is also a agent it can increase the number of available effector cells. prominent macmophageactivator (5, 12). Nonspecific immu notherapy is believed to be mediated in animals by the direct and indirect activation of macnophages. These con REFERENCES siderations constitute much of the rationale for trials of 1. Alexander, P., and Evans, R. Endotoxin and Double Stranded RNA nonspecific immunotherapy in man. Render Macrophages Cytotoxic. Nature New Biol., 232: 76-78, 1971. Macrophage tumor cell destruction may rebate to the 2. Baum, M., and Fisher, B. Macrophage Production by the Bone Marrow numbers of effector cells present at the site of tumor growth of Tumor-bearing Mice. Cancer Res., 32: 2813-2817, 1972. 3. Bradley, T. R., Stanley, E. R., and Sumner, M. A. 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Carmen Burgaleta and David W. Golde

Cancer Res 1977;37:1739-1742.

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