Int. J. lmmunopharmac., Vol. 14, No. 1, pp. 11-17, 1992. Printed in Great Britain.

0192-0561/92 $5.00 + .00 Pergamon Press plc.

International Society for Immunopharmacology.

PROTECTIVE EFFECT OF SPR-901 (RBS) ON THE DECREASE OF PERIPHERAL LEUKOCYTE NUMBER IN 5-FLUOROURACIL-TREATED

MICE

HIROYUKI MIYAZAKI, *t YASUNOBU YOSHIKAI, ¢§ MAKOTO TANAKA, t YASUYUKI TAKEDA, t SUGURU TAKEO t and KIKUO NOMOTO*

*Department of Immunology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812; ~Pharmaceutical Research Laboratories, Sapporo Breweries Ltd, 10 Okatohme, Yaizu, Shizuoka 425; and

*Laboratory of Germfree Life, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, 65, Tsurumai-cho, Showa-ku, Nagoya 466, Japan

(Received 19 April 1991 and in final form 26 July 1991)

A b s t r a c t - - 5-Fluorouracil (5-FU) induces a decrease in the number of peripheral leukocytes (leukopenia), which is one of the major obstacles in the chemotherapy of cancer. The number of peripheral leukocytes decreased by day 4 in mice injected i.p. with 130 mg/kg of 5-FU and recovered to the normal level by day 8. Such a decrease by 5-FU was prevented to some extent by the oral administration of 30 mg/kg/day of SPR-901. Proliferative responses of bone marrow cells to granulocyte/macrophage colony stimulating factor (GM-CSF) or granulocyte colony stimulating factor (G-CSF) were suppressed by 5-FU treatment and their recoveries were enhanced by SPR-901. The serum level of IL-6 in 5-FU-treated mice was increased by SPR-901. All of the mice treated with 300 mg/kg of 5-FU in combination with SPR-901 survived over 15 days, however, only 4 of 10 mice treated only with 300 mg/kg of 5-FU survived. These results suggest that SPR-901 acts on macrophages directly or indirectly, giving rise to the enhanced production of IL-1, IL-6, and other factors. Some of the factors derived from SPR-901 activated macrophages, perhaps mainly IL-6, act on the early stage of development of multipotent bone marrow progenitors synergistically with GM-CSF.

Since chemotherapeutic agents give rise to a profound and prolonged leukopenia, bacterial infection is a major cause of death of cancer patients during chemotherapy (Kregar, Craven, Carling & McCabe, 1980; Tancrede & Andremont, 1985; Wells, Ferrieri, Weisdorf & Rhame, 1987). So the doses of chemotherapeutic agents are limited by this side-effect. Therefore, the reduction of the side- effect during chemotherapy is important. Several attempts have been made using mice to prevent the leukopenia and subsequent bacterial infections induced by chemotherapeutic agents (Matsumoto, Matsubara, Matsuno, Tamura, Hattori, Nomura, Ono & Yokota, 1987; Konishi, Tanaka, Kumamoto, Hasegawa, Okuda, Yoshikai & Nomoto, 1990; Nomoto, Yokokura, Mitsuyama, Yoshikai & Nomoto, in press). For example, the effectiveness of human granulocyte colony stimulating factor was well examined (Matsumoto et al., 1987).

~Author to whom correspondence should be addressed.

We have previously reported that SPR-901 (RBS), an a-glucan, exhibited antitumor activities against syngeneic mouse tumors when administered to mice not only intraperitoneally (i.p.) but also orally (p.o.) (Takeo, Kado, Yamamoto, Kamimura, Watanabe, Uchida & Mori, 1988; Kado, Yoneta, Takeo, Mitsui & Watanabe, 1991). Most of the polysaccharides exhibiting antitumor effects such as lentinan, schizophyllan and PS-K are fl-glucans (Chihara, Hamuro, Maeda, Arai & Fukuoka, 1970; Tsukagoshi, & Ohashi, 1974; Sugawara, Leek & Wong, 1984). In this respect SPR-901 has an interesting structure. And we have reported that oral administration of SPR-901 (RBS) enhanced protection against listerial infection through activation of macrophages acting as accessory cells for immune response (Takeda, Yoshikai, Ohga & Nomoto, 1990; Takeda, Yoshikai, Ohga & Nomoto, in press).

12 H. MIYAZAKI el al.

...... ---[- ot-o -Glc- (1----~ 6)-]-~m C~-D -GIc- (1----~ 6) ....... 3

T 1

...... -aL- Ct.-D -GIc- (1---~ 6 ) - ~ Ot-O -GIc

Cell counts

Peripheral blood was obtained from the retro- orbital plexus. Numbers of total leukocytes were counted with a MICROCELLCOUNTER (Sysmex, Japan). Differential counts were done on Giemsa- stained smears of leukocytes.

Fig. 1. A probable structure of SPR-901 (RBS). Glc; glucopyranose, m + n = 24 (m, n; 0-24).

The results of experiments to measure antitumor activity of SPR-901 suggested that antitumor effects of this agent were enhanced in combination with various chemotherapeutic agents such as 5-fluo- rouracil (5-FU) (unpublished data). In this report, therefore, we examined whether SPR-901 had a protective effect on the leukopenia induced by a chemotherapeutic agent, 5-FU.

E X P E R I M E N T A L P R O C E D U R E S

Mice

Female mice of an inbred BALB/c strain were obtained from Charles River Co., Ltd (Atsugi, Japan) and used at 6 - 8 weeks of age. Each experimental group consisted of five to ten mice.

Proliferative responses o f bone marrow cells to GM-CSF or G-CSF

Bone marrow cells were prepared, as described previously (Watoson, 1983), from femurs and tibias of mice treated with 5-FU and SPR-901. Bone marrow cells were resuspended in RPMI 1640 medium, supplemented with 10070 FCS, 5 x 10 5 M 2-mercaptoethanol, 100 U/ml pen- icillin, and 100/ag/ml streptomycin. One hundred microliters of the bone marrow cell suspension containing 1 x 10 6 cells/ml were seeded in 96 well plates with 100 tal of reciprocal concentration of either recombinant mouse granulocyte/macrophage colony stimulating factor (Cosmo Bio, Japan: GM-CSF) or recombinant human granulocyte colony stimulating factor (Kyowa Hakko Kogyo, Japan: G-CSF), incubated at 37°C in an atmosphere of 5070 CO: for 3 days, and added with 18.5 kBq/well of 3H-thymidine (Amersham, Japan) at the final 20 h of culture (Watoson, 1983). Then radioactivity was measured by a liquid scintillation counter.

Preparation o f SPR-901

SPR-901 (RBS) was synthesized from sucrose using dextran sucrase prepared from Leuconostoc mesenteroides subsp, dextranicum (Kado et al., 1991). We used the name of SPR-901 (serial development number of RBS) in this report. A probable structure of SPR-901 is presented in Fig. 1 (Takeo et al., 1988). Its molecular weight is over 1,000,000 (Takeo et al., 1988) and the contamination of LPS is less than 1 ng/mg. The level of LPS was determined by Toxicolor system (Seikagaku Corp., Japan).

Measurement o f cytokines

Spleen and serum were obtained from mice on day 4. Serum was inactivated by incubation at 56°C for 30 min, and 1 × l 0 6 cells/ml of spleen cells were cultured with 5 ~g/ml of concanavalin A (Sigma, America; Con A) at 37°C in an atmosphere of 507o CO2 for 24 h. IL-2, IL-3, and IL-6 were measured by cytokine dependent cell lines, CTLL-2, FDCP2 and MH60-BSF2, respectively, as described previously (Prestige, Watson, Urdal, Mochizuki, Conlon & Gillis, 1984; Nakajima, Martinez-Maza, Hirano, Breen, Nishanian, Salazar-Gonzalez, Fahey & Kishimoto, 1989). One unit of each cytokine was determined by the half of the maximal growth.

Treatment o f mice with SPR-901 and 5-FU

Mice (five mice per group) were administered p.o. 30 mg/kg/day of SPR-901 from day - 3 to day 4, by gastric gavage and injected i.p. with 130 mg/kg 5-fluorouracil (Kyowa Hakkou Kogyo, Tokyo; 5-FU) on day 0. Observation was done on days 2, 4, 6 ,8 .

Survival from high dose o f 5-FU injection

Mice (10 mice per group) were injected i.p. with 5-FU (400 and 300 mg/kg) on day 3 and administ- rated p.o. SPR-901 (30 mg/kg/day) from day 0 to day 9. Then mice were observed for survival for 14 days after the administration of 5-FU.

% 6t

% ~ 40

E == 20

o

Effect of SPR-901 on 5-FU-Induced Leukopenia

"°°%% ~ ....... 6

• . . . . . . . . . . . . ..

Days after 5-FU administration

Fig. 2. Effect of SPR-901 on the reduction in peripheral blood leukocytes in 5-FU-treated mice. Female BALB/c mice were treated i.p. with 130 mg/kg of 5-FU on day 0. The mice were divided into two groups and administered p.o. with 30 mg/kg/day SPR-901 (open circle), or water (control, open square) in 0.3 ml from day - 3 to day 4. Peripheral blood was obtained from the retro-orbital plexus on the indicated days. Each point represents the mean _+ standard deviation for five mice. *P<0.05 compared with

control by the Student's t-test.

Statistics

The statistical significance of the data was determined by Student's t-test or the generalized Wilcoxon test (Gehan, 1965).

RESULTS

Effect o f SPR-901 on leukocyte number

5-Fluorouracil damages bone marrow cells and induces a decrease of leukocyte number (Nomoto et al., in press). Therefore, we examined the protective effect of SPR-901 on the 5-FU-induced decrease of leukocyte number. Mice injected with 5-FU were examined on the number of peripheral leukocytes on days 2, 4, 6, and 8 (Fig. 2). The peripheral leukocyte number of mice injected only with 5-FU decreased from a normal level of 8610_+ 710/mm 3 to 2770 + 690/mm 3 on day 4, although the number recovered rapidly to the normal level thereafter (Fig. 2 open square). On the other hand, mice given SPR-901 exhibited a significantly smaller decrease of peripheral leukocyte number (the peripheral leuko- cyte number was 4760 _+ 510/mm 3 after 4 days) after an injection of 5-FU (Fig. 2 open circle, P<0.05). Similar results were obtained on days 2 and 6 (P<0.05). The peripheral leukocyte number of mice administered p.o. with only 30mg/kg /day of

13

SPR-901 showed much the same level as untreated control mice at any stage (data not shown). The proportion of polymorphonuclear leukocytes was not significantly changed in both cases of mice injected only with 5-FU and mice administered SPR- 901 in combination with 5-FU (data not shown).

Proliferative responses o f bone marrow cells to GM-CSF or G-CSF

Although the proliferative response of control mouse bone marrow cells to GM-CSF exhibited a dose-dependent curve, that to G-CSF showed a saturated curve (Fig. 3 open triangle). The pattern was dramatically changed when mice were injected i.p. with 130 mg/kg of 5-FU. In 5-FU-treated mice, the proliferative response of bone marrow cells to GM-CSF was depressed on day 2. And the response was recovered gradually to the normal level until day 6 (Fig. 3 open square). When SPR-901 was administered in combination with 5-FU, the decrease in the response was prevented to some degree and 50% of the control level was exhibited on day 2. And until day 6 the response recovered gradually to the normal level (Fig. 3 open circle). The same tendency was found in the case of spleen cells (data not shown). At the same time, the CFU-c assay was done and the number of hemopoietic progenitor cells also increased in the 5-FU+SPR-901 group compared with the 5-FU group (data not shown).

The response to G-CSF was damaged by 5-FU on day 2, and gradually recovered to the normal level by day 6. SPR-901 showed a slight effect on the proliferative response to G-CSF. 5-FU damaged the proliferative response of bone marrow cells to both GM-CSF and G-CSF. In 5-FU-treated mice, SPR-901 enhanced the recovery of the response to GM-CSF more effectively than that to G-CSF. Thereafter SPR-901 was thought to promote the recovery of the earlier stage of hematopoietic cells.

Cytokines level o f serum and culture supernatant

Serum and spleen cells were prepared from mice 4 days after 5-FU injection. SPR-901 administration was continued from 3 days before 5-FU injection to 4 days after 5-FU injection. Cytokines examined in this experiment were IL-2, IL-3, and IL-6 (shown in Table 1). Cytokine levels in Con A-stimulated culture supernatant were decreased by the injection of 5-FU. And the decreased level was unchanged by the administration of SPR-901. However, the IL-6 level of serum was increased by the administration of SPR-901 in combination with 5-FU. It was supposed that this increase of serum IL-6 and/or other soluble

14 H. MIYAZAKI et al.

E

%

[ -

60

40

20

a) s S

ss~ SS

s ~

c ~ ~ [] [3---

0 3 6 12 25

10

t

0 3 6 12 25

b)

0 3 6 12 25

GM-CSF concentration (U/ml)

e)

/

L i i ~ T

0 3 6 12 25

G-CSF concentration (U/ml)

t c)

0 3 6 12 25

E) .,.~

0 3 6 12 25

Fig. 3. Effect of SPR-901 on the CSF-response of bone marrow cells prepared from mice treated with 5-FU. Female BALB/c mice were treated in the same manner as in Fig. 2. Mice were treated i.p. with 130 mg/kg of 5-FU, and divided into two groups and administered p.o. with 30 mg/kg/day of SPR-901 (open circle), or water (open square). Control mice were treated i.p. with saline and p.o. with water (open triangle). Bone marrow cells were prepared at day 2 (a, d), day 4 (b, e), and day 6 (c, f), and cultured with reciprocal concentration of either GM-CSF (a, b, c) or G-CSF (d, e, f). Each point

represents the mean _+ standard deviation for five mice.

factors contributed to the protective effect of SPR- 901 on 5-FU-induced leukopenia. Other experiments revealed the high expression of I L - l a and IL-6 m R N A of peritoneal exudate cells f rom mice administered p.o. with SPR-901 (Takeda et al., 1990, and unpublished data).

Protective effect o f SPR-901 on 5-FU-induced endogenous infection

We showed the protective effect of SPR-901 on 5-FU-induced leukopenia, then examined whether SPR-901 exhibited the protective effect on 5-FU- induced endogenous infection. All mice given 400 m g / k g of 5-FU died until day 11, and 60°7o of mice given 300 mg /kg of 5-FU died until day 15 (Fig. 4). When mice were administered p.o. with SPR-901 for 10 consecutive days (5-FU i.p. injection was done at day 4), the survival t ime of mice given a lethal dose of 5-FU (400 mg/kg) was prolonged (P<0.05), and 100% o f mice given a sublethal dose of 5-FU (300 mg/kg) survived (P<0.05).

DISCUSSION

The effects of SPR-901 on CSF responses of bone marrow cells suggested some mechanisms of its preventive effect on the decrease of leukocyte number induced by 5-FU. The GM-CSF response of bone marrow cells prepared f rom mice treated with SPR-901 and 5-FU exhibited 50°7o of the level of normal mice on days 2 and 4, whereas bone marrow cells f rom mice treated only with 5-FU exhibited no or little response. Therefore, the protective effect of SPR-901 on the decrease of peripheral leukocyte number induced by 5-FU was due to the recovery of the CSF response f rom the damage by 5-FU, Moreover IL-6 was raised in serum when 5-FU- treated mice were administered p.o. with SPR-901, Then IL-6 was also related to the SPR-901 effects, And the effect of SPR-901 on 5-FU-induced leukopenia was reflected by the in vivo experiment to observe the survival of 5-FU-treated mice.

Effect of SPR-901 on 5-FU-Induced Leukopenia

Table 1. Cytokines in serum and culture supernatant of spleen cells

IL2*

Concentration ( U / m l )

Con A sup.* Serum t

Control 105.6 N.D. ~ 5-FU 45.9 N.D2 5-FU + SPR-901 41.4 N.D.§

IL3* Control 452.5 29.3 5-FU 113.1 34.8 5-FU + SPR-901 91.9 27.3

IL6* Control 203.9 12.3 5-FU 55.8 29.3 5-FU + SPR-901 74.6 56.6

*Spleen cells were cultured at 1 × l06 cells/ml with 5/~g/ml Con A for 24 h. tSerum was inactivated by incubation at 56°C for 30 min. *IL2, IL3, and IL6 were determined by growth of CTLL-2, FDCP2 and MH60-BSF2, respectively. ~Not detected.

15

"7.

100

50

~--.!

!

! ~ - " 1

I i i i i I I

0 5 10

I * I I

! l ! i I I I L . ! L . . . . :

! I 1 5

Survival Time ( d a y )

Fig. 4. Effect of SPR-901 on the survival time of 5-FU- treated mice. Female BALB/c mice were treated i.p. with 300 mg/kg of 5-FU on day 3, and treated p.o. with water (control, ----), or 30 mg/kg/day of SPR-901 ( ) from day 0 to day 9. And mice were treated i.p. with 400 mg/kg of 5-FU, and treated p.o. with water (control, - - . . . . ), or 30 mg/kg/day of SPR-901 ( - - - - - ) . *P<0.05 com- pared with each control by the generalized Wilcoxon test.

The effects of cytokines on the differentiat ion of hemopoiet ic progenitors were well discussed. The effects of IL-3 and GM-CSF on hematopoiesis were well characterized (Koike, Ogawa, Ihle, Miyake, Shimizu, Miyaj ima, Yokota & Arai, 1987; Hil ton, Nicola, Gough & Metcalf , 1988; Sonoda, Yang, Wong, Clark & Ogawa, 1988a,b). IL-3 was considered to exert its effect on the early stage of development of mult ipotent progenitors (Sonoda et

al., 1988b). The effects of GM-CSF on multipotent progenitors were considered to be the same as those of IL-3 (Koike et al., 1987; Sonoda et al., 1988a, b). But with respect to colony format ion on methylcellulose, the activity of GM-CSF was less than that of IL-3. Thereby it was suggested that the primary targets of IL-3 are primitive progenitors and the targets of GM-CSF are intermediate progenitors (Sonoda et al., 1988a, b).

IL-6 and G-CSF acted on hematopoiet ic progenitors synergistically with IL-3 (Ikebuchi, Wong, Clark, Ihle, Hirai & Ogawa, 1987; Ikebuchi, Clark, Ihle, Souza & Ogawa, 1988; Wong, Witek- Gianotti , Temple, Kriz, Ferenz, Hewick, Clark, Ikebuchi & Ogawa, 1988). Blast cells f rom spleen cells of 5-FU-treated mice could grow in the presence of IL-3. The starting time of the growth of blast cells was shortened by combinat ion with IL-6 or G-CSF when compared with only IL-3. But the growth rate was not significantly different between a treatment with IL-3 only and that with IL-3+ IL-6/G-CSF. Thus IL-3 and IL-6 (or G-CSF) act synergistically to support the proliferation of hematopoiet ic progenitors and at least some part of the effect results f rom a decrease in the Go period of individual stem cells (Ikebuchi et al., 1987, 1988).

Previous reports suggested that SPR-901 acted on macrophages and the activated macrophages were enhanced in the production of IL-1 and IL-6 (Takeda et al., 1990, in press). In this report SPR-901 exhibited the protective effects on the reduction of peripheral blood leukocytes and on the damage of

16

the CSF response of bone marrow cells in mice treated with 5-FU. It was suggested that macrophages are activated directly or indirectly by SPR-901, and the activated macrophages produce preferentially IL-1 and IL-6. And IL-6 a n d / o r other factors act synergistically to support the prolifer- ation of hematopoiet ic progenitors at the early stage of development in the process of recovery f rom 5-FU-induced damage. In fact, the serum level of IL-6 was higher in mice treated with 5-FU and SPR-901 than in non- t reated controls or 5-FU- treated controls. But, since other factors such as GM-CSF or G-CSF were not examined, the contr ibut ion of factors other than IL-6 could not be

ruled out.

H. MIYAZAKI et al.

In conclusion, the reduct ion of peripheral blood leukocytes was slighter in 5-FU-treated (i.p.) and SPR-901 administered (p.o.) mice than in 5-FU- treated control mice. Mice administered SPR-901 survived even after t reatment with lethal doses of 5-FU. Therefore , we suggest that SPR-901 prevents 5-FU-induced leukopenia and acts synergistically with 5-FU in the ant i tumor effect.

Acknowledgements - - We thank Professor Tadamitsu Kishimoto and co-workers for the IL-6 dependent cell line, MH60-BSF2. And we thank Professor Katsuo Kumagai and co-workers for the IL-3 dependent cell line, FDCP2.

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