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A Novel in Vivo Screening Method for Immunomodulating Substances: Development of an Assay System a
ab
Ryohei F. Tsuji , Junji Magae , Kazuo Nagai
ab
a
& Makari Yamasaki
a
Department of Agricultural Chemistry, The University of Tokyo, Yayoi 1–1–1, Bunkyo-ku, Tokyo 113, Japan b
Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 227, Japan Published online: 12 Jun 2014.
To cite this article: Ryohei F. Tsuji, Junji Magae, Kazuo Nagai & Makari Yamasaki (1992) A Novel in Vivo Screening Method for Immunomodulating Substances: Development of an Assay System, Bioscience, Biotechnology, and Biochemistry, 56:9, 1497-1498, DOI: 10.1271/bbb.56.1497 To link to this article: http://dx.doi.org/10.1271/bbb.56.1497
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Biosci. Biotech. Biochem., 56 (9), 1497-1498, 1992
Note
A Novel in Vivo Screening Method for Immunomodulating Substances: Development of an Assay System Ryohei F. TSUJI,t Junji MAGAE,* Kazuo NAGAI,* and Makari YAMASAKI Department of Agricultural Chemistry, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113, Japan Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 227, Japan Received March 19, 1992
Downloaded by [Thomas Jefferson University] at 16:43 23 December 2014
* Department of Bioengineering,
A screening method for selective immunomodulators effective in vivo was developed using sheep red blood cells (SRBC) as a thymus-dependent (TD) antigen and heat-killed Brucella abortus cells as a thymus-independent (TI) antigen 1) for antibody production, and trinitrobenzene sulfonic acid (TNBS) for delayed-type hypersensitivity (DTH) response. Por optimal antibody production against TD antigens, B cells require stimulation derived from helper T cells in addition to stimulation of surface immunoglobulin by antigens. 2) On the other hand, TI antigens do not require helper T cell function; B cells that recognize antigen can proliferate and differentiate by themselves to produce antibody.3) Antibody production against TD antigen requires type 2 helper T cells (Th 2), while DTH requires type 1 helper T cells (Th 1). Thl activate inflammatory cells such as macrophages and neutrophiles in the efferent phase of DTH response. 4) We believe that by using these antigens in our system, it is possible to separately evaluate functions of B cells, Th l , Th 2, and inflammatory cells in vivo. Several groups have found interesting immunomodulating substances among microbial metabolites in in vitro screening systems. 5 -7) Because compounds that are effective in vitro are not necessarily effective in vivo, we developed an in vivo mouse screening system. Pemale 6-wk-old CDP 1 mice (SPP grade) were immunized with TD and TI antigens as described previously. 8) CDP 1 mice were simultaneously injected iv with 1 x 10 8 SRBC and 1 x 10 9 Brucella abortus cells. Six days later, 1 x 109 Brucella abortus cells were used for secondary immunization. Twelve days thereafter, sera and spleens were obtained, and used to measure antibody titers and the extent of splenomegaly. The sera were incubated with or without 0.1 M 2-mercaptoethanol (2-ME) for 60 min at 3rC to inactivate IgM antibody, which is sensitive to 2-ME. They were serially diluted with phosphatebuffered saline (PBS; 0.29% Na2HP04, 0.02% KH 2P04, 0.8% NaCI, 0.02% KCI, pH 7.4) and incubated with 1.5 x 10 7 SRBC in round bottomed micro titer wells or with 2.5 x 10 8 Brucella abortus cells in V-shape-bottomed micro titer wells for 20 hr at 37°C. Antibody titer, defined as half maximum agglutinating ability, was expressed as log2' CDP 1 mice were also injected subcutaneously with 200 ft! of 10mM TNBS on day 3. On day 11, 40,t! of 10 mM TNBS was challenged by subcutaneous injection into the left hind footpad. Increase in footpad thickness was measured 24 hr after the elicitation with a dial-caliper (Ozaki, Mfg Co., Ltd., Japan). The right footpad was also measured as a control. Two hundred microliter samples from culture broth or known compounds were dissolved in PBS containing 0.2% Tween 80 and administered intraperitoneally on days 1, 5, and 8, except for FK506 which was administered orally. When SRBC as a TD antigen and Brucella abortus as a TI antigen were simultaneously injected into a mouse, 2-ME resistant antibody production, mainly of the IgO isotype, was detected only in the case of TD antigen (Table I). This is probably because isotype class switching requires helper T lymphocytes,9) which produce class-switching factors such as IL-4, IL-5 and IPN-y, or cognately interact with B cells. 10) This suggests that the system permits a selective evaluation of effects of T cells and B cells. We investigated the effects of several active compounds whose targets have already been identified (Table I). As shown in our previous report,8) PK506,11) an immunosuppressant specific for helper T lymphocytes, selectively inhibited antibody production against TD antigen, while antibody production against TI antigen was not affected. The most striking inhibition by PK506 was shown in 2-ME resistant antibody production against TD antigen. The DTH response was also significantly suppressed by the same dose that inhibited antibody production. Prodigiosin 25-C
Table I. Effects of Active Compounds on Antibody Production and Delayed-type Hypersensitivity (DTH) in Vivo Dose Anti-SRBC Anti-Brucella DTH Increase (mg/ in footpad kg) - 2ME + 2ME - 2ME + 2ME thickne~s, cm x 10 2 Exp. 1 Vehicle PK506 Exp. 2 Vehicle Prodigiosin 25-C Exp. 3 Vehicle Mitomycin C Exp. 4 Vehicle Cyclophosphamide Concanavalin A Lipopolysaccharide Exp. 5 Vehicle Actinomycin D Exp. 6 Vehicle Cycloheximide Antimycin Exp. 7 Vehicle Lentinan Zymosan Na~Alginate
5.2 5.2 4.2
4.2 3.8 o.oa
4.0 4.7 4.3
0.0 0.0 0.0
7.5 ± 1.0 8.0±4.5 2.3±2.0a
0.5 1.0
5.2 5.0 5.2
4.5 4.7 4.3
4.7 5.0 4.8
0.0 0.0 0.0
8.2±2.4 3.5±2.6 0.7 ±0.6a
3.0 4.0
5.2 4.0 O.oa
4.2 2.0a O.oa
4.0 4.3 O.oa
0.0 0.0 0.0
7.5 ± 1.0 7.8 ±2.0 3.5±0.oa
20 40 3 10 0.1 1.0
5.0 5.0 3.8 5.2 5.7 5.5 4.5
3.0 1.8 0.0" 3.7 4.3 4.2 2.8
3.7 4.3 0.0" 3.5 4.3 4.2 3.7
0.0 0.0 0.0 0.0 1.8" 1.5 a 1.3 a
7.3±2.9 7.1 ±0.8 3.7±1.0 4.0±0.5 3.8±0.6 5.5 ± 1.3 2.3±0.7a
6.0 6.0 5.8
5.0 3.8" 4.7
3.0 3.0 2.2
0.0 0.0 0.0
9.8 ± 1.0 9.2±2.2 5.7±3.7
10 50 0.3 1.0
6.2 5.3 5.0 5.3 5.3
5.0 2.7" 1.2a 3.8 2.8"
4.3 4.3 4.0 3.7 4.3
0.0 0.0 0.0 0.0 0.0
7.7 ±2.8 9.2±2.4 12.5±2.3a 5.8± 1.8 7.7
1 3 10 30 10 30
5.3 6.0 5.7 6.7 6.0 5.7 6.0
4.0 5.7 5.5 5.8 4.7 5.2 5.3
5.0 6.0 6.0 6.0 6.3 6.0 6.3
0.0 1.5 a 1.8 a 0.7" 1.7 a 3.0a 2.2a
100 300
0.03 0.1
N.Db N.D N.D N.D N.D N.D N.D
Each active compounds were injected ip into CDP 1 mice except PKS06, which was administered po as described in the text. Number of mice in each experimental groups was more than three. a Statistically significant (two tailed Student's I-test, p < 0.05). b Not determined.
t Present address: Noda Institute for Scientific Research, 399 Noda, Noda-shi, Chiba 278, Japan. Abbreviations: MMC, mitomycin C; CY, cyclophosphamide; AcD, actinomycin D; CX, cycloheximide; con A, concanavalin A; LPS, lipopolysaccharide; PrO 25-C, prodigiosin 25-C.
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(PrG 25_C),12.13) an immunosuppressant that preferentially suppresses function of cytotoxic T lymphocytes, affected neither the response to TD antigen nor to TI antigen even at a dose of 1 mg/kg where the compound completely suppresses induction of cytotoxic T lymphocytes. S) However, PrG 25-C significantly suppressed DTH response and this effect was shown to occur through suppression of the efferent phase of DTH (unpublished data). Antibody production against both TD and TI antigens and DTH response was inhibited by the inhibitors of DNA synthesis, mitomycin C (MMC) and cyclophosphamide (CY). Strong inhibition was seen at 4mg/kg ofMMC and at 40mg/kg ofCY, respectively. The dose ofMMC (4 mg/kg) was extremely toxic and 4 mice out of 6 tested died during the experiment. Data from the two mice are shown in Table I, indicating that antibody production against both antigens was completely suppressed. 2-ME resistant antibody production against TD antigen was more strongly inhibited than 2-ME untreated antibody production, suggesting that these agents preferentially suppressed class switching. More selective inhibition of 2-ME resistant antibody production against TD antigen was observed after treatment with cycloheximide (CX), an inhibitor of protein synthesis, at a doses greater than 10 mg/kg. CX only negligibly inhibited 2-ME sensitive antibody production against TD antigen and antibody production against TI antigen. By contrast, CX enhanced footpad swelling at doses greater than 10 mg/kg. This result is consistent with those of Zubiaga, who reported that CX increased lymphokine production by inhibiting synthesis of trans-acting DNA binding proteins, which regulated expression of lymphokine genes. 14) When mice were treated with actinomycin D (AcD), an inhibitor of RNA synthesis, antibody production was still observed in spite of great decreases of body weight. Antimycin did not have a significant effect in this system although 2-ME resistant antibody production against TD antigen was slightly suppressed at the dose of 1.0 mg/kg. Con A and LPS are polyclonal mitogens specific for T cells and B cells, respectively. When these immunostimulants were used in our system, 2-ME resistant antibody against TI antigens was produced. Whole antibody production against TD antigen and TI antigen slightly increased. The results indicated that isotype class switching ofTI antigen was induced by both Con A and LPS. To confirm this result, lentinan,15) zymosan,16) and sodium alginate, 1 7) potent immunostimulants that are effective in vivo, were used in our system. They also showed similar effects as the mitogens described above. Thus, it seems likely that macromolecular immunostimulants commonly induce isotype class switching of antibodies against TI antigens. Both Con A and LPS suppressed DTH responses. It is possible that Con A exerted its suppressive ability through activation of suppressor T cells. A suppressive effect ofLPS on DTH was also reported by Molendijk et al. 1S ) The tested antigens produced a remarkable splenomegaly (data not shown). The increase in spleen size was two or three-fold over controls. MMC and AcD, the inhibitors of DNA and RNA synthesis respectively, suppressed the splenomegaly. In the case of the mice treated with 4 mg/kg of MMC or 0.1 mg/kg of AcD, the spleen was even smaller than that of non-immunized controls. FK506 and prodigiosin 25~C did not affect the responses, which suggests that splenomegaly was not induced by T cell activation. Immunostimulants such as Con A, LPS, lentinan, zymosan, and sodium alginate greatly enhanced splenomegaly. Therefore, splenomegaly seems to be a useful indicator of macromolecules that induce isotype class switching. This screening system was used with fourteen hundred culture broth filtrates of actinomycetes isolated from soil samples (Table II). There was no T cell specific immunosuppressing activity but non-specific immunosuppression was seen with one extract, class switch induction of antibody isotype with 13, and DTH-specific immunosuppression with 18 extracts.
et al.
Table II.
Classification of the Culture Broths by Selective Effects in Vivo
Anti-SRBC
Anti-Brucella
-2ME +2ME
-2ME +2ME
DTH Known drugs
t
1
t
-+
-+
-+
-+
-+
-+
-+
-+
-+
-+
i, enhanced;
-+,
-+
t -+(1)
not affected;
! !
MMC, CY Mitogen PrG 25-C FK506
~o. of. actIve strams
13 18 0
L suppressed.
Isolation and characterization of these active compounds will be published elsewhere. Acknowledgment. the manuscript.
We thank M. Schaechter for the help in preparing
References 1) 1. Scher, Adv. Immunol., 33, 1--71 (1982). 2) R. J. Noelle, E. C. Snow, J. W. Uhr, and E. S. Vitetta, Proc. Natl. Acad. Sci. U.S.A., 80, 6628-6631 (1983). 3) M. A. Barton and E. Diener, Transplant. Rev., 23, 5-22 (1975). 4) J. L. Platt, B. W. Grant, A. A. Eddy, and A. F. Michael, J. Exp. Med., 158, 1227-1242 (1983). 5) A. Nakamura, K. Nagai, S. Suzuki, K. Ando, and G. Tamura, J. Antibiotics, 39, 1148-1154 (1986). 6) T. Kino, H. Hatanaka, M. Hashimoto, M. Nishiyama, T. Goto, M. Okuhara, M. Kohsaka, H. Aoki, and H. Imanaka, J. Antibiotics, 40, 1249-1255 (1987). 7) H. Umezawa, M. Ishizuka, T. Takeuchi, F. Abe, K. Nemoto, K. Shibuya, and T. Nakamura, J. Antibiotics, 38, 283-284 (1985). 8) R. F. Tsuji, M. Yamamoto, A. Nakamura, T. Kataoka, J. Magae, K. Nagai, and M. Yamasaki, J. Antibiotics, 43, 1293-1301 (1990). 9) R. L. Coffman, B. W. P. Seymour, D. A. Lebman, D. D. Hiraki, J. A. Christiansen, B. Shrader, H. M. Cherwinski, H. F. J. Savelkoul, F. D. Finkelman, M. W. Bond, and T. R. Mosmann, Immunol. Rev., 102, 5-28 (1988). 10) V. M. Sanders, J. M. Snyder, J. W. Uhr, and E. S. Vitetta, J. Immunol., 137, 2395-2404 (1986). 11) M. J. Tocci, D. M. Matkovich, K. A. Collier, P. Kwok, F. Dumont, S. Lin, S. Degubicibus, J. J. Siekietka, J. Chin, and N. 1. Hutchinson, J. Immunol. 143, 718-726 (1989). 12) A. Nakamura, K. Nagai, K. Ando, and G. Tamura, J. Antibiotics, 39, 1155-1159 (1986). 13) A. Nakamura, J. Magae, R. F. Tsuji, M. Yamasaki, and K. Nagai, Transplantation, 47, lO13-1016 (1989). 14) A. M. Zubiaga, E. Munoz, and B. T. Huber, J. Immunol., 146, 3857-3863 (1991). 15) D. W. Dresser andJ. M. Phillips, Immunology, 27, 895-902 (1974). 16) A. Nicholson, V. Brade, G. D. Lee, H. S. Shin, and M. M. Mayer, J. Immunol., 112, 1115-1123 (1974). 17) M. Fujihara, K. Komiyama, 1. Umezawa, and T. Nagumo, Chemotharapy, 32, 1004---1009 (1984). 18) A. Molendijk, H. Bril, L. M. Hussaarts-Odijk, R. J. H. L. M. van Gurp,.and R. Benner, Immunobiology, 176,255-271 (1988).
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A Novel in Vivo Screening Method for Immunomodulating Substances: Development of an Assay System a
ab
Ryohei F. Tsuji , Junji Magae , Kazuo Nagai
ab
a
& Makari Yamasaki
a
Department of Agricultural Chemistry, The University of Tokyo, Yayoi 1–1–1, Bunkyo-ku, Tokyo 113, Japan b
Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 227, Japan Published online: 12 Jun 2014.
To cite this article: Ryohei F. Tsuji, Junji Magae, Kazuo Nagai & Makari Yamasaki (1992) A Novel in Vivo Screening Method for Immunomodulating Substances: Development of an Assay System, Bioscience, Biotechnology, and Biochemistry, 56:9, 1497-1498, DOI: 10.1271/bbb.56.1497 To link to this article: http://dx.doi.org/10.1271/bbb.56.1497
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Biosci. Biotech. Biochem., 56 (9), 1497-1498, 1992
Note
A Novel in Vivo Screening Method for Immunomodulating Substances: Development of an Assay System Ryohei F. TSUJI,t Junji MAGAE,* Kazuo NAGAI,* and Makari YAMASAKI Department of Agricultural Chemistry, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113, Japan Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 227, Japan Received March 19, 1992
Downloaded by [Thomas Jefferson University] at 16:43 23 December 2014
* Department of Bioengineering,
A screening method for selective immunomodulators effective in vivo was developed using sheep red blood cells (SRBC) as a thymus-dependent (TD) antigen and heat-killed Brucella abortus cells as a thymus-independent (TI) antigen 1) for antibody production, and trinitrobenzene sulfonic acid (TNBS) for delayed-type hypersensitivity (DTH) response. Por optimal antibody production against TD antigens, B cells require stimulation derived from helper T cells in addition to stimulation of surface immunoglobulin by antigens. 2) On the other hand, TI antigens do not require helper T cell function; B cells that recognize antigen can proliferate and differentiate by themselves to produce antibody.3) Antibody production against TD antigen requires type 2 helper T cells (Th 2), while DTH requires type 1 helper T cells (Th 1). Thl activate inflammatory cells such as macrophages and neutrophiles in the efferent phase of DTH response. 4) We believe that by using these antigens in our system, it is possible to separately evaluate functions of B cells, Th l , Th 2, and inflammatory cells in vivo. Several groups have found interesting immunomodulating substances among microbial metabolites in in vitro screening systems. 5 -7) Because compounds that are effective in vitro are not necessarily effective in vivo, we developed an in vivo mouse screening system. Pemale 6-wk-old CDP 1 mice (SPP grade) were immunized with TD and TI antigens as described previously. 8) CDP 1 mice were simultaneously injected iv with 1 x 10 8 SRBC and 1 x 10 9 Brucella abortus cells. Six days later, 1 x 109 Brucella abortus cells were used for secondary immunization. Twelve days thereafter, sera and spleens were obtained, and used to measure antibody titers and the extent of splenomegaly. The sera were incubated with or without 0.1 M 2-mercaptoethanol (2-ME) for 60 min at 3rC to inactivate IgM antibody, which is sensitive to 2-ME. They were serially diluted with phosphatebuffered saline (PBS; 0.29% Na2HP04, 0.02% KH 2P04, 0.8% NaCI, 0.02% KCI, pH 7.4) and incubated with 1.5 x 10 7 SRBC in round bottomed micro titer wells or with 2.5 x 10 8 Brucella abortus cells in V-shape-bottomed micro titer wells for 20 hr at 37°C. Antibody titer, defined as half maximum agglutinating ability, was expressed as log2' CDP 1 mice were also injected subcutaneously with 200 ft! of 10mM TNBS on day 3. On day 11, 40,t! of 10 mM TNBS was challenged by subcutaneous injection into the left hind footpad. Increase in footpad thickness was measured 24 hr after the elicitation with a dial-caliper (Ozaki, Mfg Co., Ltd., Japan). The right footpad was also measured as a control. Two hundred microliter samples from culture broth or known compounds were dissolved in PBS containing 0.2% Tween 80 and administered intraperitoneally on days 1, 5, and 8, except for FK506 which was administered orally. When SRBC as a TD antigen and Brucella abortus as a TI antigen were simultaneously injected into a mouse, 2-ME resistant antibody production, mainly of the IgO isotype, was detected only in the case of TD antigen (Table I). This is probably because isotype class switching requires helper T lymphocytes,9) which produce class-switching factors such as IL-4, IL-5 and IPN-y, or cognately interact with B cells. 10) This suggests that the system permits a selective evaluation of effects of T cells and B cells. We investigated the effects of several active compounds whose targets have already been identified (Table I). As shown in our previous report,8) PK506,11) an immunosuppressant specific for helper T lymphocytes, selectively inhibited antibody production against TD antigen, while antibody production against TI antigen was not affected. The most striking inhibition by PK506 was shown in 2-ME resistant antibody production against TD antigen. The DTH response was also significantly suppressed by the same dose that inhibited antibody production. Prodigiosin 25-C
Table I. Effects of Active Compounds on Antibody Production and Delayed-type Hypersensitivity (DTH) in Vivo Dose Anti-SRBC Anti-Brucella DTH Increase (mg/ in footpad kg) - 2ME + 2ME - 2ME + 2ME thickne~s, cm x 10 2 Exp. 1 Vehicle PK506 Exp. 2 Vehicle Prodigiosin 25-C Exp. 3 Vehicle Mitomycin C Exp. 4 Vehicle Cyclophosphamide Concanavalin A Lipopolysaccharide Exp. 5 Vehicle Actinomycin D Exp. 6 Vehicle Cycloheximide Antimycin Exp. 7 Vehicle Lentinan Zymosan Na~Alginate
5.2 5.2 4.2
4.2 3.8 o.oa
4.0 4.7 4.3
0.0 0.0 0.0
7.5 ± 1.0 8.0±4.5 2.3±2.0a
0.5 1.0
5.2 5.0 5.2
4.5 4.7 4.3
4.7 5.0 4.8
0.0 0.0 0.0
8.2±2.4 3.5±2.6 0.7 ±0.6a
3.0 4.0
5.2 4.0 O.oa
4.2 2.0a O.oa
4.0 4.3 O.oa
0.0 0.0 0.0
7.5 ± 1.0 7.8 ±2.0 3.5±0.oa
20 40 3 10 0.1 1.0
5.0 5.0 3.8 5.2 5.7 5.5 4.5
3.0 1.8 0.0" 3.7 4.3 4.2 2.8
3.7 4.3 0.0" 3.5 4.3 4.2 3.7
0.0 0.0 0.0 0.0 1.8" 1.5 a 1.3 a
7.3±2.9 7.1 ±0.8 3.7±1.0 4.0±0.5 3.8±0.6 5.5 ± 1.3 2.3±0.7a
6.0 6.0 5.8
5.0 3.8" 4.7
3.0 3.0 2.2
0.0 0.0 0.0
9.8 ± 1.0 9.2±2.2 5.7±3.7
10 50 0.3 1.0
6.2 5.3 5.0 5.3 5.3
5.0 2.7" 1.2a 3.8 2.8"
4.3 4.3 4.0 3.7 4.3
0.0 0.0 0.0 0.0 0.0
7.7 ±2.8 9.2±2.4 12.5±2.3a 5.8± 1.8 7.7
1 3 10 30 10 30
5.3 6.0 5.7 6.7 6.0 5.7 6.0
4.0 5.7 5.5 5.8 4.7 5.2 5.3
5.0 6.0 6.0 6.0 6.3 6.0 6.3
0.0 1.5 a 1.8 a 0.7" 1.7 a 3.0a 2.2a
100 300
0.03 0.1
N.Db N.D N.D N.D N.D N.D N.D
Each active compounds were injected ip into CDP 1 mice except PKS06, which was administered po as described in the text. Number of mice in each experimental groups was more than three. a Statistically significant (two tailed Student's I-test, p < 0.05). b Not determined.
t Present address: Noda Institute for Scientific Research, 399 Noda, Noda-shi, Chiba 278, Japan. Abbreviations: MMC, mitomycin C; CY, cyclophosphamide; AcD, actinomycin D; CX, cycloheximide; con A, concanavalin A; LPS, lipopolysaccharide; PrO 25-C, prodigiosin 25-C.
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R. F.
TSUJI
(PrG 25_C),12.13) an immunosuppressant that preferentially suppresses function of cytotoxic T lymphocytes, affected neither the response to TD antigen nor to TI antigen even at a dose of 1 mg/kg where the compound completely suppresses induction of cytotoxic T lymphocytes. S) However, PrG 25-C significantly suppressed DTH response and this effect was shown to occur through suppression of the efferent phase of DTH (unpublished data). Antibody production against both TD and TI antigens and DTH response was inhibited by the inhibitors of DNA synthesis, mitomycin C (MMC) and cyclophosphamide (CY). Strong inhibition was seen at 4mg/kg ofMMC and at 40mg/kg ofCY, respectively. The dose ofMMC (4 mg/kg) was extremely toxic and 4 mice out of 6 tested died during the experiment. Data from the two mice are shown in Table I, indicating that antibody production against both antigens was completely suppressed. 2-ME resistant antibody production against TD antigen was more strongly inhibited than 2-ME untreated antibody production, suggesting that these agents preferentially suppressed class switching. More selective inhibition of 2-ME resistant antibody production against TD antigen was observed after treatment with cycloheximide (CX), an inhibitor of protein synthesis, at a doses greater than 10 mg/kg. CX only negligibly inhibited 2-ME sensitive antibody production against TD antigen and antibody production against TI antigen. By contrast, CX enhanced footpad swelling at doses greater than 10 mg/kg. This result is consistent with those of Zubiaga, who reported that CX increased lymphokine production by inhibiting synthesis of trans-acting DNA binding proteins, which regulated expression of lymphokine genes. 14) When mice were treated with actinomycin D (AcD), an inhibitor of RNA synthesis, antibody production was still observed in spite of great decreases of body weight. Antimycin did not have a significant effect in this system although 2-ME resistant antibody production against TD antigen was slightly suppressed at the dose of 1.0 mg/kg. Con A and LPS are polyclonal mitogens specific for T cells and B cells, respectively. When these immunostimulants were used in our system, 2-ME resistant antibody against TI antigens was produced. Whole antibody production against TD antigen and TI antigen slightly increased. The results indicated that isotype class switching ofTI antigen was induced by both Con A and LPS. To confirm this result, lentinan,15) zymosan,16) and sodium alginate, 1 7) potent immunostimulants that are effective in vivo, were used in our system. They also showed similar effects as the mitogens described above. Thus, it seems likely that macromolecular immunostimulants commonly induce isotype class switching of antibodies against TI antigens. Both Con A and LPS suppressed DTH responses. It is possible that Con A exerted its suppressive ability through activation of suppressor T cells. A suppressive effect ofLPS on DTH was also reported by Molendijk et al. 1S ) The tested antigens produced a remarkable splenomegaly (data not shown). The increase in spleen size was two or three-fold over controls. MMC and AcD, the inhibitors of DNA and RNA synthesis respectively, suppressed the splenomegaly. In the case of the mice treated with 4 mg/kg of MMC or 0.1 mg/kg of AcD, the spleen was even smaller than that of non-immunized controls. FK506 and prodigiosin 25~C did not affect the responses, which suggests that splenomegaly was not induced by T cell activation. Immunostimulants such as Con A, LPS, lentinan, zymosan, and sodium alginate greatly enhanced splenomegaly. Therefore, splenomegaly seems to be a useful indicator of macromolecules that induce isotype class switching. This screening system was used with fourteen hundred culture broth filtrates of actinomycetes isolated from soil samples (Table II). There was no T cell specific immunosuppressing activity but non-specific immunosuppression was seen with one extract, class switch induction of antibody isotype with 13, and DTH-specific immunosuppression with 18 extracts.
et al.
Table II.
Classification of the Culture Broths by Selective Effects in Vivo
Anti-SRBC
Anti-Brucella
-2ME +2ME
-2ME +2ME
DTH Known drugs
t
1
t
-+
-+
-+
-+
-+
-+
-+
-+
-+
-+
i, enhanced;
-+,
-+
t -+(1)
not affected;
! !
MMC, CY Mitogen PrG 25-C FK506
~o. of. actIve strams
13 18 0
L suppressed.
Isolation and characterization of these active compounds will be published elsewhere. Acknowledgment. the manuscript.
We thank M. Schaechter for the help in preparing
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