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Oral administration of a streptococcal agent OK-432 activates alveolar macrophages in mice
Int. J. Immunopharmac., Vol. 14, No. 2, pp. 205-211, 1992. Printed in Great Britain.
0192-0561/92 $5.00 + .00 Pergamon Press plc. International Society for lmmunopharmacology.
O R A L A D M I N I S T R A T I O N OF A S T R E P T O C O C C A L A G E N T OK-432 A C T I V A T E S A L V E O L A R M A C R O P H A G E S IN MICE AKIRA KINOSHITA, IWAO SUZUKI, TERUAKI SAKURAIand TOSHIRO YADOMAE* Laboratory of Immunopharmacology of Microbial Products, Tokyo College of Pharmacy, Horinouchi 1432-1, Hachioji, Tokyo 192-03, Japan (Received 6 March 1991 and in final form 5 August 1991)
Abstract -- The effect of orally administered OK-432, a streptococcal preparation, on the functions of
alveolar macrophages in mice was examined. The oral administration of OK-432 (1, 2 or 4 KE, four times every 3 days) augmented phagocytic activity, lysosomal enzyme activity and interleukin 1 (IL-I) production of murine alveolar macrophages recovered 5 days after the final administration while it did not augment H202 production. The number of alveolar macrophages was not affected by the same treatment. These results suggested that oral administration of OK-432 activates alveolar macrophages qualitatively to protect the lung from the metastasis of cancer cells and infectious diseases by pathogenic micro-organisms.
The streptococcal preparation, OK-432, is a lyophilized powder of avirulent Streptococcus pyogenes Su strain treated with penicillin. OK-432 is a biological response modifier (BRM) which has been widely used for cancer patients in Japan. In recent years, oral administration of OK-432 has been reported to exhibit an immunomodulatory effect and antitumor activity in animals and humans (Akimoto, Abe & Kasai, 1985; Nakajima, Akimoto, Kimura, Iwasaki, Matano, Hirakawa & Mori, 1987). This administration method has the advantage of easy use for patients accompanied by no pain by injection and no significant side-effects (Nio, Ohgaki, Henmi, Kan, Inamoto, Tsuchitani, Kodama & Tobe, 1982). A clinical trial of oral administration of OK-432 has been performed for more than a thousand gastric cancer patients in Japan and resulted in an accumulative survival rate of the oral administration of OK-432 after curative surgery was significantly higher than those of the placebo and intradermally administered groups (Nio & Tobe, 1989). In a previous paper, we showed that OK-432 augmented peritoneal macrophage functions including lysosomal enzyme activity, phagocytic activity and IL-1 production when it was administered by the oral route into mice (Suzuki, Kinoshita, Tanaka & Yadomae, 1990). In this paper, we studied the effect of orally administered OK-432 *Author to whom correspondence should be addressed. 205
on alveolar macrophage functions in mice. Alveolar macrophages are located on the mucosal surfaces of alveoli. The alveolar macrophages are the first effector cells to antigens which invaded the host by respiration, and are considered to affect the mucosal immunity on respiratory surfaces. There are few reports on the activation of alveolar macrophages by an orally administered agent (Radermecker, Rommain, Maldague, Bury & Smets, 1988). The activation of alveolar macrophages by OK-432 administered orally is of value for investigation from the point of view of the clinical usefulness of this agent and interest in the immunological phenomenon at respiratory mucosal surfaces. EXPERIMENTAL PROCEDURES
Animals Specific pathogen-free male mice of CDFt (BALB/c × DBA/2) and C 3 H / H e J mice were purchased from Japan SLC, Inc. (Shizuoka) and used at 6 - 8 weeks of age. The animals were bred under specific pathogen-free conditions. 0K-432 OK-432 (Picibanil, a group A streptococcal preparation) was kindly provided by Chugai
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A. KINOSH1TAet al.
Pharmaceutical Co., Ltd, Tokyo. One KE (Klinishe Einheit) of OK-432 is equivalent to 0.1 mg lyophilized powder of S. pyogenes.
Macrophages Alveolar macrophages were collected from normal or OK-432 administered mice by the bronchoalveolar lavage method (Akagawa, Kamoshita & Tokunaga, 1988) with a slight modification. Briefly, mice were anesthetized by intraperitoneal injection of 10 mg/0.2 ml pentobarbital sodium solution (Dainippon Pharmaceutical Co., Osaka). Bronchoalveolar lavage was performed with cooled Ca 2', Mg2+-free phosphate-buffered saline containing 0.05% EDTA (Dojindo Laboratories, Kumamoto). The cells were resuspended in RPMI 1640 medium (Nissui Seiyaku Co., Ltd, Tokyo) supplemented with 10070 heat-inactivated fetal calf serum (FCS, Boehringer Manhein GmbH), 5 mM HEPES, penicillin (100 U/ml) and streptomycin (100/~g/ml). The cell number was counted with a hemocytometer, and cells were differentiated by using a Diff-Quik Stain Kit (Kokusai Shiyaku Co., Ltd, Hyogo). Aliquots (0.25 ml) of the cell suspension containing an adequate number of macrophages were plated in a flat-bottomed 96- or 24-well tissue culture plate (Sumitomo Bakelite Co., Ltd, Tokyo). After incubation for 2 h at 37°C in a CO2 incubator, nonadherent cells were removed by washing twice with warmed (37°C) RPMI 1640 medium. The resultant cells were used as alveolar macrophages.
Zymosan A; Sigma Chemical Co., MO) opsonized with complement in 96-well tissue culture plates (Sumitomo) for 20 min at 37°C in a CO2 incubator. Then the macrophages were washed twice with warmed (37°C) RPMI 1640 medium to remove any nonphagocytosed zymosan particles, and solubilized by the addition of 0.1 ml of 50 mM sodium cholate. The fluorescence intensity was measured with a fluorescence spectrophotometer (Corona Electric Co., Ltd, Ibaragi) with an excitation wavelength of 490 nm and an emission wavelength of 530 nm.
Assay for cellular lysosomal enzyme activity Macrophage monolayers in tissue culture plates (Sumitomo) (1 × 105/well) were solubilized by the addition of 0.01 ml of 0.1% Triton X-100. Thirtyseven millimolar p-nitrophenyl phosphate (Sigma) solution was added as a substrate for acid phosphatase. Then, 0.02 ml of 0.1 M citrate buffer (pH 5.0) were added to each well. After incubation for 30 min at 37°C, 0.2 ml of 0.2 M borate buffer (pH 9.8) were added to the reaction mixture, and the optimal density at 405 nm was measured.
Measurement of H202 production Macrophage monolayers in 96-well tissue culture plates (2 × 105/well) were overlaid with 50/A of Krebs - Ringer phosphate buffer (pH 7.4) containing 6 mM scopoletin, 0.1 U horseradish peroxidase (Sigma), 6 mM glucose and 0.016 nM phorbol myristate acetate (PMA, Sigma) for 2 h at 37°C in a CO2 incubator. Then the HzO2 release into the buffer was measured.
Experimental protocols OK-432 was suspended in physiological saline, and aliquots (0.2 ml) of the suspension were used for the administration into mice. For the intragastric (oral) administration, the suspension was delivered to the stomachs of mice by using an intubation needle. OK-432 was administered orally every 3 days for two to eight times (four times, days 0, 3, 6 and 9 in most experiments). Individual alveolar macrophages were collected 5 days after the final administration, and their functions were assayed.
Assay for phagocytic activity Phagocytic activity was assayed by a method described previously (Suzuki, Tanaka, Adachi & Yadomae, 1988). Briefly, macrophage monolayers (2 × 105/well) were incubated with fluoresceinconjugated zymosan particles (prepared from
Measurement of IL-1 production Macrophage monolayers in 24-well tissue culture plates (Sumitomo) (1 × 105/well) were incubated for 72 h at 37°C under 5°70 CO2 in 0.4 ml of RPMI 1640 medium containing 5°7o FCS. Supernatants of the cultures were collected, filtered through 0.45/~m syringe filters (Coming Laboratory Sciences Company, NY), and stored at - 8 0 ° C . IL-1 activity in the culture supernatants was measured in terms of the thymocyte proliferation response (Meltzer, Oppenheim & Rosenstreich, 1978). Briefly, a single cell suspension of thymocytes (1.5 × 106/well)taken from C3H/HeJ mice was placed in a flat-bottomed 96-well tissue culture plate (Sumitomo). To the cell suspension, 50/A of macrophage culture supernatant was added. The culture was then incubated in the presence of a submitogenic concentration of Con A (1 ~g/ml) for 72 h at 37°C in a CO2 incubator. These
Oral Administration of OK-432 ~
115,
cases assayed [Fig. 2(B)]. In a time course assay, a maximum activity was observed 3 weeks after start of administration of OK-432 (2 KE, four times), and thereafter the activity decreased [Fig. 2(C)].
o tO 0
207
10
N
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d Z Nil
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2 K~
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OK-43E p.o. x 4 Fig. 1. Effect of OK-432 on the number of alveolar macrophages. Groups of CDF~ mice were administered OK-432 orally (p.o.) four times (days 0, 3, 6 and 9). Alveolar macrophages were taken from these mice by washing with 10 ml of cooled Ca~'+ and Mg2+-free PBS containing 0.05°70 EDTA, and counted with a hemocytometer. cultures were pulse labelled with 3H-TdR (New England Nuclear, Boston, MA) (0.5 ~Ci/well) for the final 12 h of the incubation periods. At the end of the incubation periods the cells were harvested and radioactivity of the cells was measured in a liquid scintillation counter.
Statistics All results are expressed as the mean + standard deviation (S.D). Statistical evaluations in all experiments were performed by Student's t-test. A value of P<0.05 was considered significant.
RESULTS
Effect on number o f macrophages Alveolar macrophages occupied 98% of total cells recovered from each mouse orally administered OK-432 by the lavage method. The administration of OK-432 (1, 2 or 4 KE, four times every 3 days) did not affect the numbers of alveolar macrophages obtained on day 14 (Fig. 1). Effect on phagocytic activity Figure 2 shows the phagocytic activity of alveolar macrophages from mice administered OK-432 orally. Multiple oral administrations of OK-432 (1, 2 or 4 KE, four times) significantly enhanced the phagocytic activity, and the enhancing effects seemed to be dose dependent [Fig. 2(A)]. Furthermore, the administration of OK-432 (2 KE) from two to eight times enhanced the activities in all
Effect on lysosomal enzyme activity The effect of OK-432 administration on the lysosomal enzyme activity of alveolar macrophages was examined in a system of four times oral administrations at 2 day intervals. The acid phosphatase activities were enhanced slightly in all groups of mice administered OK-432 orally (Fig. 3). Effect on H202 production The ability of macrophages to produce H2O 2 was examined (Fig. 4). The peritoneal macrophages obtained from mice that received a single intraperitoneal administration of OK-432 showed elevated H202 production in a PMA-triggered system. On the other hand, the production by alveolar macrophages was not augmented by oral administration of OK-432 (1,2 or 4 KE, four times). Effect on IL-1 production The effect of OK-432 administration on IL-1 production by alveolar macrophages was examined. As shown in Fig. 5, significant enhancements of IL-1 production (3 or 4-fold) were observed in the alveolar macrophages from mice received OK-432 (1, 2 or 4 KE, four times) orally.
DISCUSSION In this study, we examined the effect of orally administered OK-432 on several functions of alveolar macrophages including cell number, phagocytic activity, lysosomal enzyme activity, IL-1 production and H202 production. Oral administration of OK-432 did not affect the number of alveolar macrophages in mice. It was reported that oral administration of RU 41.740 (BiostimTM), an extract of Klebsiella pneumoniae, increased the number of alveolar macrophages in guinea-pigs and augmented their phagocytic activity (Radermecker et al., 1988). These findings suggest that the effects of immunomodulators administered orally on alveolar macrophages are different from each other depending on their physico-chemical properties and/or their absorption mechanism from the intestine. The alveolar macrophages are considered to originate from monocytes in blood
208
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Days a f t e r t h e f i r s t a d m i n i s t r a t i o n
p.o. ( 2KE )
Fig. 2.(A) Effect of OK-432 on phagocytic activity of alveolar macrophages. Groups of CDF~ mice were administered OK-432 orally (p.o.) four times (days 0, 3, 6 and 9). The phagocytic activity of alveolar macrophages taken from these mice were determined 5 days after the last administration. *P<0.05, **P<0.01. (B) Effect of multiple administration of OK-432 on phagocytic activity of alveolar macrophages. Groups of CDF~ mice were administered OK-432 orally (p.o.) every 3 days for two, four, six or eight times. The phagocytic activity of alveolar macrophages taken from these mice were determined 5 days after the last administration. *P<0.01. (C) Time course assay of phagocytic activity of alveolar macrophages from mice given OK-432. Groups of CDF, mice were administered OK-432 (2 KE) orally (p.o.) four times (days, 0, 3, 6 and 9) and phagocytic activities were determined 14, 21 or 28 days after the first administration (day 0). *P<0.05, **P<0.01, ***P<0.001. 1000m
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4 KE p.o. x 4
Fig. 3. Effect of OK-432 on lysosomal enzyme activity of alveolar macrophages. Groups of CDF~ mice were administered OK-432 (1, 2 or 4 KE, four times) orally, and acid phosphatase activities were determined 14 days after the first administration (day 0). *P<0.05.
Nil
1KE 0K-432
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Fig. 4. Effect of OK-432 o n H 2 0 : production of alveolar macrophages. Groups of CDF, mice were administered OK-432 (1, 2 or 4 KE, four times) orally. One group of CDF~ mice were administered OK-432 (1 KE) i.p. and the H202 production from their peritoneal macrophages harvested 5 days after the administration was comparatively determined.
Oral Administration of OK-432
0 ~-4
Nil
IKE
2KE
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0K-432 p.o. x 4 Fig. 5. Effect of OK-432 on IL-1 production of macrophages. Groups of CDF~ mice were administered OK-432 orally (1, 2 or 4 KE, four times). Five days after the last administration, their alveolar macrophages were harvested and cultured for 3 days. The culture supernatants were collected and added to a C3H/HeJ thymocyte culture (1.5 x 10~) at a final dilution of 1:4 with Con A (1 ~g/ml), and then cultured for 72 h. *P<0.05, **P<0.01. (Thomas, Ramberg, Sale, Sparkes & Golde, 1976) or be due to the growth of alveolar macrophages at alveoli (Golde, Byers & Finley, 1974). The differentiation and growth of alveolar macrophages are reported to be regulated by some kind of cytokines including colony stimulating factors (Chen, Muller & Chou, 1988). These findings suggest that OK-432 administered orally did not affect the growth or differentiation of alveolar macrophages at alveoli or promotion of the inflow of macrophages into the alveoli. Oral administration of OK-432 increased slightly the phagocytic and lysosomal enzyme activities of alveolar macrophages in mice, whereas the same treatment did not augment the H202 production. This phenomenon was also observed in the peritoneal macrophages taken from mice given OK-432 orally (Suzuki et al., 1990). It was reported that the H202 production (Hoidal, Fox, LeMarbre, Takiff & Repine, 1980; Ando, Suga, Sugimoto & Tokumi, 1979; Sugimoto, Ando, Senba & Tokumi, 1980) and the lysosomal enzyme activities (Cohn & Wiener, 1963) of alveolar macrophages were modulated by several environmental factors, such as air pressure, antigenic substances and surfactants. Our finding using OK-432 indicates that the mechanisms of oxygen-independent antigen digestion by alveolar macrophages can be modulated by an immunomodulating material. On the other hand, Loesberg, Henricks & Nijkamp (1989) reported that the feeding of some dietary fatty acids to mice improved the lung functions possibly via
209
modulation of the production of radicals from alveolar macrophages. These phenomena, including our results, suggest the presence of some mechanisms to regulate the functions of alveolar macrophages by orally administered agents. Oxygen metabolites are known to act as effector molecules to neoplasms (Nathan, Silverstein, Brukres & Cohn, 1979) and micro-organisms (Rosen & Klebanoff, 1979; Sasada & Johnston, 1980). On the other hand, these molecules are also known to injure normal blood vessels or lung tissues (Deneke & Fanburg, 1980; Frank, Summerville & Massaro, 1980) and promote pulmonary emphysema (McCormick, Harkin, Johnson & Ward, 1981) or fibrosis (Johnson & Ward, 1981). No effect of OK-432 administered orally on oxygen metabolism of both alveolar and peritoneal macrophages was considered to confer an advantage to the use OK-432 for patients. Oral administration of OK-432 into mice augmented the production of IL-1 from alveolar macrophages, and the effect was similar to that on peritoneal macrophages in mice (Suzuki et al., 1990). Therefore, it is considered that OK-432 administered orally affects both the nonspecific and specific immune responses at alveoli of mice. In this study, we indicated that oral administration of OK-432 augmented several functions of alveolar macrophages in mice. However, the regulatory mechanism of alveolar macrophages in vivo by this agent is not known. An existence of a common mucosal immune system on mucosal surfaces of animals and humans was proposed from several laboratories (Mfiller-Schoop & Good, 1975; Theppen, van Rooijen & Kraal, 1989). According to this concept, the major natural pathway to stimulate the immune system of animals is through gutassociated lymphoreticular tissues (GALT) and bronchus-associated lymphoreticular tissue (BALT) where introduced antigens penetrate through pinocytotic and phagocytotic microfold cells (M cells) covering GALT and BALT and interact with accessory and lymphoid cells. A recent finding that T-cells bearing y, d T-cell receptor preferentially localize to epithelial organs such as the gut (Pardoll et al., 1987; Stingle et al., 1987) and epidermis (Goodman & Lefrancois, 1988; Bonneville, Janeway, Ito, Haser, Nakanishi & Tonegawa, 1988) attracts great interest. The function of y, 6 T-cells is still uncertain. However, it is suggested that they play a role in the elimination of infected epithelial cells by micro-organisms. Thus, it is considered that there is a close relationship among mucosal organs of animals in respect to immunological defense
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mechanisms. Recently, it was r e p o r t e d that heattreated S. pyogenes induced proliferation o f h u m a n lymphocytes in vitro, and 10-60°70 o f these lymphoblasts were ),, d T-cells (Abo, Seki, Rikiishi & Kumagai, 1989). The reasons why OK-432 administered orally could affect alveolar m a c r o p h a g e functions may be explained by the effect o f
S. pyogenes on mucosal immunity. It may be possible to use OK-432 as a tool for elucidation. Acknowledgements - - This work was supported by a grantin-aid from the Uehara Memorial Foundation. We thank Miss M. Tohmiya for her technical assistance and Chugai Pharmaceutical Co. for generous provision of OK-432.
REFERENCES
ADO, T., SEKI, S., R1KIISHI, H. & KUMAGAI,K. (1989). Induction of human TcR y, 6 cells by bacterial stimulation. Proc. Jpn Soc. Immun., 19, 130 (in Japanese). AKAGAWA,K. S., KAMOSHITA,K. & TOKUNAGA,T. (1988). Effects of granulocyte - macrophage colony-stimulating factor and colony-stimulating factor-1 on the proliferation and differentiation of murine alveolar macrophages. J. Immun., 141, 3383 - 3390. AKIMOTO, M., ABE, R. & KASAI,M. (1985). Augmentation of natural killer activity of the gut associated lymphoid tissue by oral administration of streptococcal preparation, OK-432. Clin. Immun., 17, 855 - 857 (in Japanese). ANDO, M., SUGA, M., SUGIMOTO, M. & TOKUMI, H. (1979). Superoxide production in pulmonary alveolar macrophages and killing of BCG by the superoxide-generating system with or without catalase. Infect. Immun., 24, 404-410. BONNEVILLE, M., JANEWAY, C. A. JR, ITO, K., HASER, W., NAKANISH1, N. & TONEGAWA, S. (1988). Intestinal intraepithelial lymphocytes are a distinct subset of gamma/delta T cells. Naure, 336, 479-481. CHEN, B, D,, MULLER,M. & CHOU, T. H. (1988). Role of granulocyte/macrophage colony-stimulating factor in regulation of murine alveolar macrophage proliferation and differentiation. J. Immun., 141, 139- 144. COHN, Z. A. & WIENER,E. (1963). The particulate hydrolases of macrophages. I. Comparative enzymology, isolation, and properties. J. exp. Med., 118, 991 - 1008. DENEKE, S. M. & FANBURG, B. L. (1980). Normobaric oxygen toxicity of the lung. New Engl. J. Med., 303, 76-86. FRANK, L. & MASSARO, D. (1980). Oxygen toxicity. Am. J. Med., 69, 117-126. FRANK, L., SUMMERVILLE,J. & MASSARO, D. (1980). Protection from oxygen toxicity with endotoxin. Role of the endogenous antioxidant enzyme of the lung. J. clin. Invest., 65, 1104-1110. GOLDE, D. W., DYERS,L. A. & FINLEY, T. N. (1974). Proliferation capacity of human alveolar macrophage. Nature, 247, 373-375. GOODMAN, T. & LEFRANCOJS, L. (1988). Expression of the T-cell receptor on intestinal CD8 + intraepithelial lymphocytes. Nature, 333, 855- 858. HOIDAL, J. R., FOX, R. B., LEMARBRE, P. A., TAK1FF, H. E. & REPINE, J. E. (1980). Oxidant metabolism of alveolar macrophages from young asymptomatic cigarette smokers. Increased superoxide anion release and its potential consequences. Chest, 77, 270-272. JOHNSON, K. J. & WARD, P. A. (1981). Role of oxygen metabolites in immune complex injury of lung. J. Immun., 126, 2365 - 2369. LOESBERG, C., HENRICKS, P. A. J. & NIJKAMP,F. P. (1989). Inverse relationship of guinea pig alveolar macrophages and tracheal-adrenergic receptor function; influence of dietary polyunsaturated fatty acids. Int. J. bnmunopharmac., l 1, 165- 171. McCoRMICK, J. R., HARKIN, M. M., JOHNSON, K. J. & WARD, P. A. (1981). Suppression by superoxide dismutase of immune complex induced pulmonary alveolitis and dermal inflammation. Am. J. Path., 102, 5 5 - 61. MELTZER, S. B., OPPENHEIM,J. J. & ROSENSTREICH, D. L. (1978). Characterization of lymphocyte-activating factor (LAF) produced by the macrophage cell line, P388D~. I. Enhancement of LAF production by activated T lymphocytes. J. Immun., 120, 1497-1503. MOLLER-SCHOOP, J. W. & GOOD, R. A. (1975). Functional studies of Peyer's patches: evidence for their participation in intestinal immune response. J. Immun., 114, 1757- 1760. NAKAJ1MA, Y., AKIMOTO, M., KIMURA, M., IWASAKI, H., MATANO, S., HIRAKAWA, H. & MORI, S. (1987). Metastatic behavior of footpad-inoculated tumor in mice and the impact of OK-432 per os administration. Jpn J. Cancer Chemother., 14, 1229-1233 (in Japanese). NATHAN, C. F., Sn_VERSTEIN, S. C., BRUKRES, L. H. & COHN, Z. A. (1979). Extracellular cytolysis by activated macrophages and granulocytes. II. Hydrogen peroxide as a mediator of cytotoxicity. J. exp. Med., 149, 100- 113.
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NlO, Y., OHGAKI,K., HENMI, K., KAN, N., INAMOTO,T., TSUCH1TANI,T., KODAMA,H. & TOBE, T. (1982). Clinical trial of oral immunotherapy with Streptococcal preparation OK-432 on gastric cancer. J. Jpn Soc. Cancer Ther., 17, 728- 734 (in Japanese). NIO, Y. & TOBE, T. (1989). Antitumor immunity of gut and oral immunotherapy. Bifidobacteria and Microflora, 8, 59-73. PARDOLL, D. M., FOWLKES,B. J., BLUESTONE,J. A., KRUISBEEK,A., MALOY,W. L., COLIGAN, J. E. & SCHWARTZ,R. H. (1987). Differential expression of two distinct T-cell receptors during thymocyte development. Nature, 326, 79-81. RADERMECKER, M., ROMMAIN, M., MALDAGUE,M. P., BURY, T. & SMETS, P. (1988). Increase in the number and the phagocytic function of guinea pig pulmonary and peritoneal and macrophages following oral administration of RU 41740, a glycoprotein extract from Klebsiella pneumoniae. Int. J. Immunopharmac., 10, 913- 917. ROSEN, H. & KLEBANOFF,S. J. (1979). Bactericidal activity of a superoxide anion-generating system. A model for the polymorphonuclear leukocyte. J. exp. Med., 149, 27-39. SASADA,M. & JOHNSTON,R. B. JR (1980). Macrophage microbicidal activity. Correlation between phagocytosis associated oxidative metabolism and the killing of Candida by macrophages. J. exp. Med., 152, 85-98. STINGLE, G., KONING, F., YAMADA,H., YOKOYAMA,W. M., TSCHACHLER,E., BLUESTONE,J, A., STEINER,G., SAMELSON, L. E., LEW, A. M., COLmAN, J. E. & SHEVACH,E. M. (1987). Thy-I + dendritic epidermal cells express T3 antigen and the T-cell receptor y chain. Proc. natn. Acad. Sci. U.S.A., 84, 4586-4590. SUGIMOTO, M., ANDO, M., SENNA, H. & TOKUML H. (1980). Lung defence in neonates: effects of bronchial lavage fluids from adult and neonatal rabbits on superoxide production by their alveolar macrophages. J. Reticuloendothel. Soc., 27, 595 - 606. SUZUKI, [., KINOSHITA, A,, TANAKA, H. & YADOMAE, T. (1990). Oral administration of a streptococcal agent OK-432 activates peritoneal macrophages in mice. Int. J. Immunopharmac., 13, 147-154. SUZUKI, I., TANAKA,H., ADACH1,S. & YADOMAE,T. (1988). Rapid measurement of phagocytosis by macrophages. Chem. Pharmac. Bull., 36, 4871 - 4875. THEPPEN, T., ROOIJEN, N. VAN& KRAAL,G. (1989). Alveolar macrophage elimination in vivo is associated with an increase in pulmonary immune response in mice. J. exp. Med., 170, 499-509. THOMAS, E. D., RAMBERG,R. E., SALE, G. E., SPARKES,R. S. & GOLDE, D. W. (1976). Direct evidence for a bone marrow origin of the alveolar macrophage in man. Science, 192, 1016- 1018.
0192-0561/92 $5.00 + .00 Pergamon Press plc. International Society for lmmunopharmacology.
O R A L A D M I N I S T R A T I O N OF A S T R E P T O C O C C A L A G E N T OK-432 A C T I V A T E S A L V E O L A R M A C R O P H A G E S IN MICE AKIRA KINOSHITA, IWAO SUZUKI, TERUAKI SAKURAIand TOSHIRO YADOMAE* Laboratory of Immunopharmacology of Microbial Products, Tokyo College of Pharmacy, Horinouchi 1432-1, Hachioji, Tokyo 192-03, Japan (Received 6 March 1991 and in final form 5 August 1991)
Abstract -- The effect of orally administered OK-432, a streptococcal preparation, on the functions of
alveolar macrophages in mice was examined. The oral administration of OK-432 (1, 2 or 4 KE, four times every 3 days) augmented phagocytic activity, lysosomal enzyme activity and interleukin 1 (IL-I) production of murine alveolar macrophages recovered 5 days after the final administration while it did not augment H202 production. The number of alveolar macrophages was not affected by the same treatment. These results suggested that oral administration of OK-432 activates alveolar macrophages qualitatively to protect the lung from the metastasis of cancer cells and infectious diseases by pathogenic micro-organisms.
The streptococcal preparation, OK-432, is a lyophilized powder of avirulent Streptococcus pyogenes Su strain treated with penicillin. OK-432 is a biological response modifier (BRM) which has been widely used for cancer patients in Japan. In recent years, oral administration of OK-432 has been reported to exhibit an immunomodulatory effect and antitumor activity in animals and humans (Akimoto, Abe & Kasai, 1985; Nakajima, Akimoto, Kimura, Iwasaki, Matano, Hirakawa & Mori, 1987). This administration method has the advantage of easy use for patients accompanied by no pain by injection and no significant side-effects (Nio, Ohgaki, Henmi, Kan, Inamoto, Tsuchitani, Kodama & Tobe, 1982). A clinical trial of oral administration of OK-432 has been performed for more than a thousand gastric cancer patients in Japan and resulted in an accumulative survival rate of the oral administration of OK-432 after curative surgery was significantly higher than those of the placebo and intradermally administered groups (Nio & Tobe, 1989). In a previous paper, we showed that OK-432 augmented peritoneal macrophage functions including lysosomal enzyme activity, phagocytic activity and IL-1 production when it was administered by the oral route into mice (Suzuki, Kinoshita, Tanaka & Yadomae, 1990). In this paper, we studied the effect of orally administered OK-432 *Author to whom correspondence should be addressed. 205
on alveolar macrophage functions in mice. Alveolar macrophages are located on the mucosal surfaces of alveoli. The alveolar macrophages are the first effector cells to antigens which invaded the host by respiration, and are considered to affect the mucosal immunity on respiratory surfaces. There are few reports on the activation of alveolar macrophages by an orally administered agent (Radermecker, Rommain, Maldague, Bury & Smets, 1988). The activation of alveolar macrophages by OK-432 administered orally is of value for investigation from the point of view of the clinical usefulness of this agent and interest in the immunological phenomenon at respiratory mucosal surfaces. EXPERIMENTAL PROCEDURES
Animals Specific pathogen-free male mice of CDFt (BALB/c × DBA/2) and C 3 H / H e J mice were purchased from Japan SLC, Inc. (Shizuoka) and used at 6 - 8 weeks of age. The animals were bred under specific pathogen-free conditions. 0K-432 OK-432 (Picibanil, a group A streptococcal preparation) was kindly provided by Chugai
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A. KINOSH1TAet al.
Pharmaceutical Co., Ltd, Tokyo. One KE (Klinishe Einheit) of OK-432 is equivalent to 0.1 mg lyophilized powder of S. pyogenes.
Macrophages Alveolar macrophages were collected from normal or OK-432 administered mice by the bronchoalveolar lavage method (Akagawa, Kamoshita & Tokunaga, 1988) with a slight modification. Briefly, mice were anesthetized by intraperitoneal injection of 10 mg/0.2 ml pentobarbital sodium solution (Dainippon Pharmaceutical Co., Osaka). Bronchoalveolar lavage was performed with cooled Ca 2', Mg2+-free phosphate-buffered saline containing 0.05% EDTA (Dojindo Laboratories, Kumamoto). The cells were resuspended in RPMI 1640 medium (Nissui Seiyaku Co., Ltd, Tokyo) supplemented with 10070 heat-inactivated fetal calf serum (FCS, Boehringer Manhein GmbH), 5 mM HEPES, penicillin (100 U/ml) and streptomycin (100/~g/ml). The cell number was counted with a hemocytometer, and cells were differentiated by using a Diff-Quik Stain Kit (Kokusai Shiyaku Co., Ltd, Hyogo). Aliquots (0.25 ml) of the cell suspension containing an adequate number of macrophages were plated in a flat-bottomed 96- or 24-well tissue culture plate (Sumitomo Bakelite Co., Ltd, Tokyo). After incubation for 2 h at 37°C in a CO2 incubator, nonadherent cells were removed by washing twice with warmed (37°C) RPMI 1640 medium. The resultant cells were used as alveolar macrophages.
Zymosan A; Sigma Chemical Co., MO) opsonized with complement in 96-well tissue culture plates (Sumitomo) for 20 min at 37°C in a CO2 incubator. Then the macrophages were washed twice with warmed (37°C) RPMI 1640 medium to remove any nonphagocytosed zymosan particles, and solubilized by the addition of 0.1 ml of 50 mM sodium cholate. The fluorescence intensity was measured with a fluorescence spectrophotometer (Corona Electric Co., Ltd, Ibaragi) with an excitation wavelength of 490 nm and an emission wavelength of 530 nm.
Assay for cellular lysosomal enzyme activity Macrophage monolayers in tissue culture plates (Sumitomo) (1 × 105/well) were solubilized by the addition of 0.01 ml of 0.1% Triton X-100. Thirtyseven millimolar p-nitrophenyl phosphate (Sigma) solution was added as a substrate for acid phosphatase. Then, 0.02 ml of 0.1 M citrate buffer (pH 5.0) were added to each well. After incubation for 30 min at 37°C, 0.2 ml of 0.2 M borate buffer (pH 9.8) were added to the reaction mixture, and the optimal density at 405 nm was measured.
Measurement of H202 production Macrophage monolayers in 96-well tissue culture plates (2 × 105/well) were overlaid with 50/A of Krebs - Ringer phosphate buffer (pH 7.4) containing 6 mM scopoletin, 0.1 U horseradish peroxidase (Sigma), 6 mM glucose and 0.016 nM phorbol myristate acetate (PMA, Sigma) for 2 h at 37°C in a CO2 incubator. Then the HzO2 release into the buffer was measured.
Experimental protocols OK-432 was suspended in physiological saline, and aliquots (0.2 ml) of the suspension were used for the administration into mice. For the intragastric (oral) administration, the suspension was delivered to the stomachs of mice by using an intubation needle. OK-432 was administered orally every 3 days for two to eight times (four times, days 0, 3, 6 and 9 in most experiments). Individual alveolar macrophages were collected 5 days after the final administration, and their functions were assayed.
Assay for phagocytic activity Phagocytic activity was assayed by a method described previously (Suzuki, Tanaka, Adachi & Yadomae, 1988). Briefly, macrophage monolayers (2 × 105/well) were incubated with fluoresceinconjugated zymosan particles (prepared from
Measurement of IL-1 production Macrophage monolayers in 24-well tissue culture plates (Sumitomo) (1 × 105/well) were incubated for 72 h at 37°C under 5°70 CO2 in 0.4 ml of RPMI 1640 medium containing 5°7o FCS. Supernatants of the cultures were collected, filtered through 0.45/~m syringe filters (Coming Laboratory Sciences Company, NY), and stored at - 8 0 ° C . IL-1 activity in the culture supernatants was measured in terms of the thymocyte proliferation response (Meltzer, Oppenheim & Rosenstreich, 1978). Briefly, a single cell suspension of thymocytes (1.5 × 106/well)taken from C3H/HeJ mice was placed in a flat-bottomed 96-well tissue culture plate (Sumitomo). To the cell suspension, 50/A of macrophage culture supernatant was added. The culture was then incubated in the presence of a submitogenic concentration of Con A (1 ~g/ml) for 72 h at 37°C in a CO2 incubator. These
Oral Administration of OK-432 ~
115,
cases assayed [Fig. 2(B)]. In a time course assay, a maximum activity was observed 3 weeks after start of administration of OK-432 (2 KE, four times), and thereafter the activity decreased [Fig. 2(C)].
o tO 0
207
10
N
5
d Z Nil
1 KE
2 K~
4 KE
OK-43E p.o. x 4 Fig. 1. Effect of OK-432 on the number of alveolar macrophages. Groups of CDF~ mice were administered OK-432 orally (p.o.) four times (days 0, 3, 6 and 9). Alveolar macrophages were taken from these mice by washing with 10 ml of cooled Ca~'+ and Mg2+-free PBS containing 0.05°70 EDTA, and counted with a hemocytometer. cultures were pulse labelled with 3H-TdR (New England Nuclear, Boston, MA) (0.5 ~Ci/well) for the final 12 h of the incubation periods. At the end of the incubation periods the cells were harvested and radioactivity of the cells was measured in a liquid scintillation counter.
Statistics All results are expressed as the mean + standard deviation (S.D). Statistical evaluations in all experiments were performed by Student's t-test. A value of P<0.05 was considered significant.
RESULTS
Effect on number o f macrophages Alveolar macrophages occupied 98% of total cells recovered from each mouse orally administered OK-432 by the lavage method. The administration of OK-432 (1, 2 or 4 KE, four times every 3 days) did not affect the numbers of alveolar macrophages obtained on day 14 (Fig. 1). Effect on phagocytic activity Figure 2 shows the phagocytic activity of alveolar macrophages from mice administered OK-432 orally. Multiple oral administrations of OK-432 (1, 2 or 4 KE, four times) significantly enhanced the phagocytic activity, and the enhancing effects seemed to be dose dependent [Fig. 2(A)]. Furthermore, the administration of OK-432 (2 KE) from two to eight times enhanced the activities in all
Effect on lysosomal enzyme activity The effect of OK-432 administration on the lysosomal enzyme activity of alveolar macrophages was examined in a system of four times oral administrations at 2 day intervals. The acid phosphatase activities were enhanced slightly in all groups of mice administered OK-432 orally (Fig. 3). Effect on H202 production The ability of macrophages to produce H2O 2 was examined (Fig. 4). The peritoneal macrophages obtained from mice that received a single intraperitoneal administration of OK-432 showed elevated H202 production in a PMA-triggered system. On the other hand, the production by alveolar macrophages was not augmented by oral administration of OK-432 (1,2 or 4 KE, four times). Effect on IL-1 production The effect of OK-432 administration on IL-1 production by alveolar macrophages was examined. As shown in Fig. 5, significant enhancements of IL-1 production (3 or 4-fold) were observed in the alveolar macrophages from mice received OK-432 (1, 2 or 4 KE, four times) orally.
DISCUSSION In this study, we examined the effect of orally administered OK-432 on several functions of alveolar macrophages including cell number, phagocytic activity, lysosomal enzyme activity, IL-1 production and H202 production. Oral administration of OK-432 did not affect the number of alveolar macrophages in mice. It was reported that oral administration of RU 41.740 (BiostimTM), an extract of Klebsiella pneumoniae, increased the number of alveolar macrophages in guinea-pigs and augmented their phagocytic activity (Radermecker et al., 1988). These findings suggest that the effects of immunomodulators administered orally on alveolar macrophages are different from each other depending on their physico-chemical properties and/or their absorption mechanism from the intestine. The alveolar macrophages are considered to originate from monocytes in blood
208
A . KINOSHITA et al.
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(A)
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p.o. x 4
(C)
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2
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40
30
20
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0
Nil
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X8
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21
28
Days a f t e r t h e f i r s t a d m i n i s t r a t i o n
p.o. ( 2KE )
Fig. 2.(A) Effect of OK-432 on phagocytic activity of alveolar macrophages. Groups of CDF~ mice were administered OK-432 orally (p.o.) four times (days 0, 3, 6 and 9). The phagocytic activity of alveolar macrophages taken from these mice were determined 5 days after the last administration. *P<0.05, **P<0.01. (B) Effect of multiple administration of OK-432 on phagocytic activity of alveolar macrophages. Groups of CDF~ mice were administered OK-432 orally (p.o.) every 3 days for two, four, six or eight times. The phagocytic activity of alveolar macrophages taken from these mice were determined 5 days after the last administration. *P<0.01. (C) Time course assay of phagocytic activity of alveolar macrophages from mice given OK-432. Groups of CDF, mice were administered OK-432 (2 KE) orally (p.o.) four times (days, 0, 3, 6 and 9) and phagocytic activities were determined 14, 21 or 28 days after the first administration (day 0). *P<0.05, **P<0.01, ***P<0.001. 1000m
~o
% 4
600-
PM
600¢g
%
400_= 2
200.
t
0
0
Nil
i KE
2 KE 0K-432
4 KE p.o. x 4
Fig. 3. Effect of OK-432 on lysosomal enzyme activity of alveolar macrophages. Groups of CDF~ mice were administered OK-432 (1, 2 or 4 KE, four times) orally, and acid phosphatase activities were determined 14 days after the first administration (day 0). *P<0.05.
Nil
1KE 0K-432
2KE
4KE
p.o.x4
IKE i.p.xl
Fig. 4. Effect of OK-432 o n H 2 0 : production of alveolar macrophages. Groups of CDF, mice were administered OK-432 (1, 2 or 4 KE, four times) orally. One group of CDF~ mice were administered OK-432 (1 KE) i.p. and the H202 production from their peritoneal macrophages harvested 5 days after the administration was comparatively determined.
Oral Administration of OK-432
0 ~-4
Nil
IKE
2KE
4KE
0K-432 p.o. x 4 Fig. 5. Effect of OK-432 on IL-1 production of macrophages. Groups of CDF~ mice were administered OK-432 orally (1, 2 or 4 KE, four times). Five days after the last administration, their alveolar macrophages were harvested and cultured for 3 days. The culture supernatants were collected and added to a C3H/HeJ thymocyte culture (1.5 x 10~) at a final dilution of 1:4 with Con A (1 ~g/ml), and then cultured for 72 h. *P<0.05, **P<0.01. (Thomas, Ramberg, Sale, Sparkes & Golde, 1976) or be due to the growth of alveolar macrophages at alveoli (Golde, Byers & Finley, 1974). The differentiation and growth of alveolar macrophages are reported to be regulated by some kind of cytokines including colony stimulating factors (Chen, Muller & Chou, 1988). These findings suggest that OK-432 administered orally did not affect the growth or differentiation of alveolar macrophages at alveoli or promotion of the inflow of macrophages into the alveoli. Oral administration of OK-432 increased slightly the phagocytic and lysosomal enzyme activities of alveolar macrophages in mice, whereas the same treatment did not augment the H202 production. This phenomenon was also observed in the peritoneal macrophages taken from mice given OK-432 orally (Suzuki et al., 1990). It was reported that the H202 production (Hoidal, Fox, LeMarbre, Takiff & Repine, 1980; Ando, Suga, Sugimoto & Tokumi, 1979; Sugimoto, Ando, Senba & Tokumi, 1980) and the lysosomal enzyme activities (Cohn & Wiener, 1963) of alveolar macrophages were modulated by several environmental factors, such as air pressure, antigenic substances and surfactants. Our finding using OK-432 indicates that the mechanisms of oxygen-independent antigen digestion by alveolar macrophages can be modulated by an immunomodulating material. On the other hand, Loesberg, Henricks & Nijkamp (1989) reported that the feeding of some dietary fatty acids to mice improved the lung functions possibly via
209
modulation of the production of radicals from alveolar macrophages. These phenomena, including our results, suggest the presence of some mechanisms to regulate the functions of alveolar macrophages by orally administered agents. Oxygen metabolites are known to act as effector molecules to neoplasms (Nathan, Silverstein, Brukres & Cohn, 1979) and micro-organisms (Rosen & Klebanoff, 1979; Sasada & Johnston, 1980). On the other hand, these molecules are also known to injure normal blood vessels or lung tissues (Deneke & Fanburg, 1980; Frank, Summerville & Massaro, 1980) and promote pulmonary emphysema (McCormick, Harkin, Johnson & Ward, 1981) or fibrosis (Johnson & Ward, 1981). No effect of OK-432 administered orally on oxygen metabolism of both alveolar and peritoneal macrophages was considered to confer an advantage to the use OK-432 for patients. Oral administration of OK-432 into mice augmented the production of IL-1 from alveolar macrophages, and the effect was similar to that on peritoneal macrophages in mice (Suzuki et al., 1990). Therefore, it is considered that OK-432 administered orally affects both the nonspecific and specific immune responses at alveoli of mice. In this study, we indicated that oral administration of OK-432 augmented several functions of alveolar macrophages in mice. However, the regulatory mechanism of alveolar macrophages in vivo by this agent is not known. An existence of a common mucosal immune system on mucosal surfaces of animals and humans was proposed from several laboratories (Mfiller-Schoop & Good, 1975; Theppen, van Rooijen & Kraal, 1989). According to this concept, the major natural pathway to stimulate the immune system of animals is through gutassociated lymphoreticular tissues (GALT) and bronchus-associated lymphoreticular tissue (BALT) where introduced antigens penetrate through pinocytotic and phagocytotic microfold cells (M cells) covering GALT and BALT and interact with accessory and lymphoid cells. A recent finding that T-cells bearing y, d T-cell receptor preferentially localize to epithelial organs such as the gut (Pardoll et al., 1987; Stingle et al., 1987) and epidermis (Goodman & Lefrancois, 1988; Bonneville, Janeway, Ito, Haser, Nakanishi & Tonegawa, 1988) attracts great interest. The function of y, 6 T-cells is still uncertain. However, it is suggested that they play a role in the elimination of infected epithelial cells by micro-organisms. Thus, it is considered that there is a close relationship among mucosal organs of animals in respect to immunological defense
210
A. KINOSHITAet al.
mechanisms. Recently, it was r e p o r t e d that heattreated S. pyogenes induced proliferation o f h u m a n lymphocytes in vitro, and 10-60°70 o f these lymphoblasts were ),, d T-cells (Abo, Seki, Rikiishi & Kumagai, 1989). The reasons why OK-432 administered orally could affect alveolar m a c r o p h a g e functions may be explained by the effect o f
S. pyogenes on mucosal immunity. It may be possible to use OK-432 as a tool for elucidation. Acknowledgements - - This work was supported by a grantin-aid from the Uehara Memorial Foundation. We thank Miss M. Tohmiya for her technical assistance and Chugai Pharmaceutical Co. for generous provision of OK-432.
REFERENCES
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NlO, Y., OHGAKI,K., HENMI, K., KAN, N., INAMOTO,T., TSUCH1TANI,T., KODAMA,H. & TOBE, T. (1982). Clinical trial of oral immunotherapy with Streptococcal preparation OK-432 on gastric cancer. J. Jpn Soc. Cancer Ther., 17, 728- 734 (in Japanese). NIO, Y. & TOBE, T. (1989). Antitumor immunity of gut and oral immunotherapy. Bifidobacteria and Microflora, 8, 59-73. PARDOLL, D. M., FOWLKES,B. J., BLUESTONE,J. A., KRUISBEEK,A., MALOY,W. L., COLIGAN, J. E. & SCHWARTZ,R. H. (1987). Differential expression of two distinct T-cell receptors during thymocyte development. Nature, 326, 79-81. RADERMECKER, M., ROMMAIN, M., MALDAGUE,M. P., BURY, T. & SMETS, P. (1988). Increase in the number and the phagocytic function of guinea pig pulmonary and peritoneal and macrophages following oral administration of RU 41740, a glycoprotein extract from Klebsiella pneumoniae. Int. J. Immunopharmac., 10, 913- 917. ROSEN, H. & KLEBANOFF,S. J. (1979). Bactericidal activity of a superoxide anion-generating system. A model for the polymorphonuclear leukocyte. J. exp. Med., 149, 27-39. SASADA,M. & JOHNSTON,R. B. JR (1980). Macrophage microbicidal activity. Correlation between phagocytosis associated oxidative metabolism and the killing of Candida by macrophages. J. exp. Med., 152, 85-98. STINGLE, G., KONING, F., YAMADA,H., YOKOYAMA,W. M., TSCHACHLER,E., BLUESTONE,J, A., STEINER,G., SAMELSON, L. E., LEW, A. M., COLmAN, J. E. & SHEVACH,E. M. (1987). Thy-I + dendritic epidermal cells express T3 antigen and the T-cell receptor y chain. Proc. natn. Acad. Sci. U.S.A., 84, 4586-4590. SUGIMOTO, M., ANDO, M., SENNA, H. & TOKUML H. (1980). Lung defence in neonates: effects of bronchial lavage fluids from adult and neonatal rabbits on superoxide production by their alveolar macrophages. J. Reticuloendothel. Soc., 27, 595 - 606. SUZUKI, [., KINOSHITA, A,, TANAKA, H. & YADOMAE, T. (1990). Oral administration of a streptococcal agent OK-432 activates peritoneal macrophages in mice. Int. J. Immunopharmac., 13, 147-154. SUZUKI, I., TANAKA,H., ADACH1,S. & YADOMAE,T. (1988). Rapid measurement of phagocytosis by macrophages. Chem. Pharmac. Bull., 36, 4871 - 4875. THEPPEN, T., ROOIJEN, N. VAN& KRAAL,G. (1989). Alveolar macrophage elimination in vivo is associated with an increase in pulmonary immune response in mice. J. exp. Med., 170, 499-509. THOMAS, E. D., RAMBERG,R. E., SALE, G. E., SPARKES,R. S. & GOLDE, D. W. (1976). Direct evidence for a bone marrow origin of the alveolar macrophage in man. Science, 192, 1016- 1018.