Immunology 1990 71 560-565
Suppression of host resistance against Listeria monocytogenes infection by 15-deoxyspergualin in mice A. NAKANE, A. NUMATA & T. MINAGAWA Department of Microbiology, Hokkaido University School of Medicine, Kita Nishi, Kita-ku, Sapporo, Japan
Acceptedfor publication 24 July 1990
SUMMARY The effects of 15-deoxyspergualin (DSG), an immunosuppressive agent, on host resistance against Listeria monocytogenes were studied in mice. Administration of DSG in the early phase of infection resulted in fatal listeriosis by preventing acquired anti-listerial resistance, even though the infectious dose was sublethal for the untreated controls. In contrast, DSG treatment started after development of the acquired immunity was ineffective. Endogenous production of interferon-gamma (IFN-y) and tumour necrosis factor (TNF) in the bloodstreams induced by the infection was normal in DSGtreated mice. Nevertheless, augmentation of macrophage functions such as expression of major histocompatibility complex (MHC) class II antigens, phagocytic activity and listericidal activity induced by the infection was abrogated by DSG treatment. These results suggest that the inhibitory effect of DSG on antilisterial resistance might be different from cyclosporine A (CsA).
INTRODUCTION (l-amino-19-guanidino- I1,1 5-dihydroxy-4,9,12Spergualin triazanonadecan-10, 13-dione), which is a metabolite of Bacillus laterosporus, has immunosuppressive activities as well as antitumour activity (Umezawa et al., 1981, 1985). The study of analogues revealed that 15-deoxyspergualin (DSG), which lacks the hydroxy group in position 15, is one of the most active compounds (Iwasawa et al., 1982). DSG has been reported to prolong allogeneic graft survival in experimental transplantation models (Ochiai et al., 1987; Suzuki, Kanashiro & Amemiya, 1987; Walter et al., 1987). DSG suppresses T-celldependent immune responses involving delayed-type hypersensitivity and induction of alloreactive secondary cytotoxic T lymphocytes (CTL) (Nemoto et al., 1987b; Nishimura & Tokunaga, 1989). Moreover, Dickneite et al. (1987) reported that DSG interferes with the macrophage (MO)/monocyte system involving generation of °2 radicals, secretion of hydrolytic enzymes, and expression of major histocompatibility complex (MHC) class II antigens. On the other hand, Nemoto et al. (1987a, 1988) demonstrated that the drug failed to inhibit
production of interleukin (IL)-1, IL-2 and IL-3, different from cyclosporine A (CsA) (Scebach, 1985). However, immunosuppressive mechanisms of DSG have been poorly understood. Complete elimination of Listeria monocytogenes, a facultative intracellular bacterium, from the tissues of infected animals is performed by macrophages activated by T-cell-dependent mechanisms (Mackaness, 1969; Kaufmann, 1987). Production of endogenous cytokines involving interferon-gamma (IFN-y) and tumour necrosis factor (TNF) and the essential role of these cytokines in anti-listerial resistance by in vivo injections of antibodies against these factors has been demonstrated (Buchmeier & Schreiber, 1985; Havell, 1987; Nakane, Minagawa & Kato, 1988a; Nakane et al., 1989). We studied the effects of DSG on host resistance against L. monocytogenes infection in mice. In this report, we provide evidence that DSG suppresses anti-listerial resistance, and that the effect might be caused by the decrease of MOi functions, but not by the suppression of production of endogenous IFN-y or TNF.
MATERIALS AND METHODS
Abbreviations: ABC, avidin-biotin complex; CsA, cyclosporine A; CTL, cytotoxic T lymphocytes; DSG, 15-deoxyspergualin; ELISA, enzyme-linked immunosorbent assay; IFN-y, interferon-gamma; IL, interleukin; i.p., intraperitoneally; i.v., intravenously; LD50, 50% lethal dose; LPS, lipopolysaccharide; mAb, monoclonal antibody; MO, macrophages; PBS, phosphate-buffered saline; PEC, peritoneal exudate cells; TdR, thymidine; TNF, tumour necrosis factor. Correspondence: Dr A. Nakane, Dept. Microbiology, Hokkaido University School of Medicine, Kita 15 Nishi 7, Kita-ku, Sapporo, 060 Japan.
Drug
15-deoxyspergualin (1 -amino- 19-guanidino- 1 -hydroxy-4,9,12triazanonadecan-10, 1 3-dione; DSG) was supplied by Nippon Kayaku Co. Ltd, Tokyo, Japan. The drug was dissolved in pyrogen-free physiological saline, sterilized by passing through a 0-22 gm filter membrane and stored at - 80° until use. DSG was injected intraperitoneally (i.p.) into mice at various doses.
560
Effect of DSG on listeriosis Mice Female ddY mice (obtained from SLC, Hamamatsu, Shizuoka, Japan), 5-7 weeks old, were used. In some experiments, female BALB/c mice (SLC), 6-8 weeks old, were used. Bacteria L. monocytogenes lb 1684 cells were prepared as described previously (Nakane et al., 1988b). The concentration of the washed cells was adjusted spectrophotometrically at 550 nm. Mice were infected intravenously (i.v.) with 0-2 ml of a solution containing 2 x 104 colony-forming units (CFU) of viable L. monocytogenes cells in saline. In some experiments, they were reinfected i.v. with 5 x 106 CFU [25 50% lethal doses (LD50)] of the bacterium 10 days after the primary infection.
Determination of numbers of viable L. monocytogenes cells in organs The numbers of viable L. monocytogenes cells in the spleens and livers of the infected animals were established by plating serial 10-fold dilutions of organ homogenates in 0-01 M phosphatebuffered saline (PBS, pH 74) on tryptic soy agar (Difco Laboratories, Detroit, MI). Colonies were routinely counted 18-24 hr later.
Induction of cytokines IFN-y in the early phase of L. monocytogenes infection was determined by using the serum specimens obtained from mice 48 hr after infection (Nakane et al., 1990). IFN-y was also induced by i.v. injection of 25 LD50 of viable L. monocytogenes cells into mice on Day 10 of infection, and the sera were collected 6 hr later (Nakane et al., 1989). TNF was induced by injecting mice i.v. with 25 pg of lipopolysaccharide (LPS) on Day 2 after L. monocytogenes infection, and the sera were obtained 2 hr later. Salmonella typhimurium LPS, purified by the method of Westphal & Jann (1965), was purchased from Difco Laboratories. IFN-y assay The IFN-y assay was carried out by a double-sandwich enzymelinked immunosorbent assay (ELISA) developed by Curry et al. (1987). Purified rat anti-mouse IFN-y monoclonal antibody (mAb) and rabbit anti-recombinant mouse IFN-y serum were used as a primary and a secondary antibodies, respectively (Nakane et al., 1990). All ELISAs were run with recombinant mouse IFN-y, produced and purified by Genentech, Inc., San Francisco, CA. TNF assay TNF titres were determined by the cytolytic activity against TNF-sensitive L-929 cells, as described previously (Nakane et
al., 1988a). Bactericidal assay This assay was adapted from that described by Davies (1982) and modified by Cheers et al. (1989). Briefly, mice that had been infected i.v. with 2 x 104 CFU of L. monocytogenes cells were injected with DSG at a dose of 5 mg/kg/day or with saline only from Days 0-3 of infection. The uninfected controls were treated with DSG or saline only in the same manner. Peritoneal exudate cells (PEC) were collected on Day 7 of infection. L. monocytogenes cells were radiolabelled by 16 hr of incubation at
561
26° of 107 organisms with 370 kBq of [3H]thymidine ([3H]TdR; 1-85 TBq/mmol; American Radiolabeled Chemicals Inc., St Louis, MO) in 10 ml of tryptic soy broth (Difco). Labelled bacteria were washed free of unincorporated [3H]TdR by centrifuging at 4000 g for 15 min at 40 and suspending in RPMI1640 medium (Gibco Laboratories, Grand Island, NY) four times. Bacterial numbers were adjusted spectrophotometrically to 2 x 108 CFU/ml. To a 1-5-ml Ependorf centrifuge tube was added 0-25 ml each ofcells (4 x 106/ml) and bacteria, and 50,ul of the fresh homologous serum. After the mixture was incubated for 30 min in a CO2 incubator at 370 to allow phagocytosis of the bacteria, cells were washed free of non-associated bacteria by centrifuging at 400 g for 5 min at 4°. They were resuspended in RPMI-1640 medium supplemented with 10% homologous serum and sampled for the amount of cell-associated radioactivity. After 0 and 120 min of incubation at 370 in a CO2 incubator, the tubes were removed to ice, and 0.5 ml of trichloroacetic acid was added. After equilibration for at least 1 hr at 40, the tubes were centrifuged in a Microcentrifuge (Hitachi Koki Co. Ltd, Tokyo, Japan) at I 0,000g for 5 min. Soluble label release by digestion of the bacteria following phagocytosis was measured.
Determination of MHC class II antigens MHC class II antigens on peritoneal macrophages were determined by the avidin-biotin complex (ABC) method (Guesdon, Tenynck & Aurameas, 1979) using mAb 1 E-4 (rat IgG2b), which recognizes at least I-A"" and I-Ek/b d k molecules (Ogasawara et al., 1987). mAb 1E-4 was provided by K. Ogasawara of Institute of Immunological Science, Hokkaido University, Sapporo, Japan. Briefly, BALB/c mice (H-2d) that had been infected i.p. with 5 x 104 CFU of L. monocytogenes cells were injected with DSG (3 3 mg/kg/day or 5 mg/kg/day) on Days 0-3 of infection. The number of PEC collected on Day 7 of infection was adjusted to 1 x 107 cells in 1 ml of RPMI-1640 medium supplemented with foetal calf serum (10%; Gibco), penicillin G (100 U/ml) and streptomycin (100 Mg/ml) and cultured in LabTek chamber slides (type 4804; Nunc, Roskilde, Denmark) for 1 hr in a CO2 incubator at 37°. After the non-adherent cells were taken out, the adherent cells were fixed with cold acetone. The slides were incubated successively with normal rabit serum for 15 min, an optimal dilution of mAb 1 E-4 or normal rat IgG for 60 min, biotinylated rabbit anti-rat IgG (Vector Laboratories, Burlingame, CA) for 30 min, and then avidin-biotinylated peroxidase complex (Vector Laboratories) for 30 min. The endogenous peroxidase activity was blocked by incubating the slides in 3% hydrogen peroxide in PBS for 15 min before the avidin-biotin peroxidase step. The slides were stained by incubation in a solution of 3-amino-9-ethyl-carbazole (Aldrich Chemicals Co. Inc., Milwaukee, WI), counterstained with haematoxylin, and mounted in PBS-glycerol. The specificity of the immunohistochemical procedure was confirmed by the following findings: (i) cells performed by ABC procedure without mAb IE-4 were never stained; (ii) no positive reaction was seen when normal rat IgG was used as a control. Statistical evaluation of the data Data were expressed as mean + SD, and the Wilcoxon rank sum test was used to determine the significance of the differences between control and experimental groups.
A. Nakane, A. Numata & T. Minagawa
562
Table 2. Effect of administration of DSG on production of IFN-y and TNF in the bloodstreams of mice in the early phase of L. monocytogenes infection
Titre (U/ml)t t
Infection*
0
+
A
+
0
2
8 6 Days after infection
4
10
12
Figure 1. Effect of administration of different doses of DSG on mortality of L. monocytogenes-infected mice. ddY mice received 1 mg (-), 3.3 mg (O) or 10 mg (0) of DSG per kg per day from Day 0 to 3 of infection. The controls received saline (A). Each result represents a group of 12 mice from two experiments.
Table 1. Growth of L. monocytogenes cells in the spleens and livers of mice that received DSG Log bacteria/organ§
Infection* Day 2
Day 5
Spleen
Treatment
Salinet DSG (3-3 mg/kg/day)t DSG (10 mg/kg/day)t
Saline$ DSG (3 3 mg/kg/day)J DSG (10 mg/kg/day)J
5-86+0 23 5-94+0 20 609+026 2 42+0 45 5l10+0 33¶ 6 84+0-45T
Liver 4 65+0 35 4 99 +0 27 528+022 2 02+0 20 6-22 +044T 6 91 +027¶
ddY mice were infected i.v. with 2 x 104 CFU of L. monocytogenes Day 0. t DSG or saline were administered i.p. on Days -2-1 of infection. t DSG or saline were administered i.p. on Days 0-3 of infection. § Each result represents the mean + SD for a group of five mice. Significantly different from the value for saline-treated controls at P 0-05). Since our previous study (Nakane et al., 1988a) demonstrated that production of endogenous TNF in L. monocytogenes-infected mice might occur in the early phase of infection but that the titre in the serum was under the detectable level by a bioassay with L-929 cells, TNF production in vivo was amplified by stimulation of LPS. Mice which had been treated with DSG or saline only or non-infected controls were injected i.v. with LPS (25 yg) on Day 2 of infection, and the serum samples for TNF titrations were taken 2 hr later. Enhancement of TNF production was observed in L. monocytogenes-infected mice (Table 2). The titres of TNF in serum obtained from DSG-treated mice were comparable to those from the drug-free animals. Effect of administration of DSG on expression of MHC class II antigens on the surface of peritoneal MO Expression of MHC class II antigens on the surface of peritoneal MO obtained from mice that had been administered
Effect of DSG Table 3. Effect of administration of DSG on expression of MHC class II antigens on the surface of peritoneal macrophages obtained from L. monocytogenes-infected mice
DSG treatmentt
Infection*
(mg/kg/day)
563
listeriosis
Table 4. Effect of administration of DSG on phagocytic activity and bactericidal activity of peritoneal macrophages against L. monocytogenes cells
MHC class II antigenpositive cells (%)t
0 0 33 5
+ + +
on
Infection*
9+2 81+6
55±6§ 35+10§
* BALB/c mice were infected i.p. with 5 x 104 CFU of L. monocytogenes cells. t DSG or saline were administered on Days 0-3 of infection. I Each result represents the mean+ SD for a group of five mice. § Significantly different from the value for the infected and saline-treated controls at P < 0-01.
DSG (3 3 mg/kg/day or 5 mg/kg/day) or saline only on Days 0-3, were determined on Day 7 of the i.p. infection with 5 x 104 CFU of L. monocytogenes (Table 3). The infection enhanced class II antigen (Table 3) expression of peritoneal MO (81 + 6%) over uninfected controls (9+2%). However, augmentation of expression of the antigens induced by the infection was significantly suppressed when mice were treated with DSG (P
Suppression of host resistance against Listeria monocytogenes infection by 15-deoxyspergualin in mice A. NAKANE, A. NUMATA & T. MINAGAWA Department of Microbiology, Hokkaido University School of Medicine, Kita Nishi, Kita-ku, Sapporo, Japan
Acceptedfor publication 24 July 1990
SUMMARY The effects of 15-deoxyspergualin (DSG), an immunosuppressive agent, on host resistance against Listeria monocytogenes were studied in mice. Administration of DSG in the early phase of infection resulted in fatal listeriosis by preventing acquired anti-listerial resistance, even though the infectious dose was sublethal for the untreated controls. In contrast, DSG treatment started after development of the acquired immunity was ineffective. Endogenous production of interferon-gamma (IFN-y) and tumour necrosis factor (TNF) in the bloodstreams induced by the infection was normal in DSGtreated mice. Nevertheless, augmentation of macrophage functions such as expression of major histocompatibility complex (MHC) class II antigens, phagocytic activity and listericidal activity induced by the infection was abrogated by DSG treatment. These results suggest that the inhibitory effect of DSG on antilisterial resistance might be different from cyclosporine A (CsA).
INTRODUCTION (l-amino-19-guanidino- I1,1 5-dihydroxy-4,9,12Spergualin triazanonadecan-10, 13-dione), which is a metabolite of Bacillus laterosporus, has immunosuppressive activities as well as antitumour activity (Umezawa et al., 1981, 1985). The study of analogues revealed that 15-deoxyspergualin (DSG), which lacks the hydroxy group in position 15, is one of the most active compounds (Iwasawa et al., 1982). DSG has been reported to prolong allogeneic graft survival in experimental transplantation models (Ochiai et al., 1987; Suzuki, Kanashiro & Amemiya, 1987; Walter et al., 1987). DSG suppresses T-celldependent immune responses involving delayed-type hypersensitivity and induction of alloreactive secondary cytotoxic T lymphocytes (CTL) (Nemoto et al., 1987b; Nishimura & Tokunaga, 1989). Moreover, Dickneite et al. (1987) reported that DSG interferes with the macrophage (MO)/monocyte system involving generation of °2 radicals, secretion of hydrolytic enzymes, and expression of major histocompatibility complex (MHC) class II antigens. On the other hand, Nemoto et al. (1987a, 1988) demonstrated that the drug failed to inhibit
production of interleukin (IL)-1, IL-2 and IL-3, different from cyclosporine A (CsA) (Scebach, 1985). However, immunosuppressive mechanisms of DSG have been poorly understood. Complete elimination of Listeria monocytogenes, a facultative intracellular bacterium, from the tissues of infected animals is performed by macrophages activated by T-cell-dependent mechanisms (Mackaness, 1969; Kaufmann, 1987). Production of endogenous cytokines involving interferon-gamma (IFN-y) and tumour necrosis factor (TNF) and the essential role of these cytokines in anti-listerial resistance by in vivo injections of antibodies against these factors has been demonstrated (Buchmeier & Schreiber, 1985; Havell, 1987; Nakane, Minagawa & Kato, 1988a; Nakane et al., 1989). We studied the effects of DSG on host resistance against L. monocytogenes infection in mice. In this report, we provide evidence that DSG suppresses anti-listerial resistance, and that the effect might be caused by the decrease of MOi functions, but not by the suppression of production of endogenous IFN-y or TNF.
MATERIALS AND METHODS
Abbreviations: ABC, avidin-biotin complex; CsA, cyclosporine A; CTL, cytotoxic T lymphocytes; DSG, 15-deoxyspergualin; ELISA, enzyme-linked immunosorbent assay; IFN-y, interferon-gamma; IL, interleukin; i.p., intraperitoneally; i.v., intravenously; LD50, 50% lethal dose; LPS, lipopolysaccharide; mAb, monoclonal antibody; MO, macrophages; PBS, phosphate-buffered saline; PEC, peritoneal exudate cells; TdR, thymidine; TNF, tumour necrosis factor. Correspondence: Dr A. Nakane, Dept. Microbiology, Hokkaido University School of Medicine, Kita 15 Nishi 7, Kita-ku, Sapporo, 060 Japan.
Drug
15-deoxyspergualin (1 -amino- 19-guanidino- 1 -hydroxy-4,9,12triazanonadecan-10, 1 3-dione; DSG) was supplied by Nippon Kayaku Co. Ltd, Tokyo, Japan. The drug was dissolved in pyrogen-free physiological saline, sterilized by passing through a 0-22 gm filter membrane and stored at - 80° until use. DSG was injected intraperitoneally (i.p.) into mice at various doses.
560
Effect of DSG on listeriosis Mice Female ddY mice (obtained from SLC, Hamamatsu, Shizuoka, Japan), 5-7 weeks old, were used. In some experiments, female BALB/c mice (SLC), 6-8 weeks old, were used. Bacteria L. monocytogenes lb 1684 cells were prepared as described previously (Nakane et al., 1988b). The concentration of the washed cells was adjusted spectrophotometrically at 550 nm. Mice were infected intravenously (i.v.) with 0-2 ml of a solution containing 2 x 104 colony-forming units (CFU) of viable L. monocytogenes cells in saline. In some experiments, they were reinfected i.v. with 5 x 106 CFU [25 50% lethal doses (LD50)] of the bacterium 10 days after the primary infection.
Determination of numbers of viable L. monocytogenes cells in organs The numbers of viable L. monocytogenes cells in the spleens and livers of the infected animals were established by plating serial 10-fold dilutions of organ homogenates in 0-01 M phosphatebuffered saline (PBS, pH 74) on tryptic soy agar (Difco Laboratories, Detroit, MI). Colonies were routinely counted 18-24 hr later.
Induction of cytokines IFN-y in the early phase of L. monocytogenes infection was determined by using the serum specimens obtained from mice 48 hr after infection (Nakane et al., 1990). IFN-y was also induced by i.v. injection of 25 LD50 of viable L. monocytogenes cells into mice on Day 10 of infection, and the sera were collected 6 hr later (Nakane et al., 1989). TNF was induced by injecting mice i.v. with 25 pg of lipopolysaccharide (LPS) on Day 2 after L. monocytogenes infection, and the sera were obtained 2 hr later. Salmonella typhimurium LPS, purified by the method of Westphal & Jann (1965), was purchased from Difco Laboratories. IFN-y assay The IFN-y assay was carried out by a double-sandwich enzymelinked immunosorbent assay (ELISA) developed by Curry et al. (1987). Purified rat anti-mouse IFN-y monoclonal antibody (mAb) and rabbit anti-recombinant mouse IFN-y serum were used as a primary and a secondary antibodies, respectively (Nakane et al., 1990). All ELISAs were run with recombinant mouse IFN-y, produced and purified by Genentech, Inc., San Francisco, CA. TNF assay TNF titres were determined by the cytolytic activity against TNF-sensitive L-929 cells, as described previously (Nakane et
al., 1988a). Bactericidal assay This assay was adapted from that described by Davies (1982) and modified by Cheers et al. (1989). Briefly, mice that had been infected i.v. with 2 x 104 CFU of L. monocytogenes cells were injected with DSG at a dose of 5 mg/kg/day or with saline only from Days 0-3 of infection. The uninfected controls were treated with DSG or saline only in the same manner. Peritoneal exudate cells (PEC) were collected on Day 7 of infection. L. monocytogenes cells were radiolabelled by 16 hr of incubation at
561
26° of 107 organisms with 370 kBq of [3H]thymidine ([3H]TdR; 1-85 TBq/mmol; American Radiolabeled Chemicals Inc., St Louis, MO) in 10 ml of tryptic soy broth (Difco). Labelled bacteria were washed free of unincorporated [3H]TdR by centrifuging at 4000 g for 15 min at 40 and suspending in RPMI1640 medium (Gibco Laboratories, Grand Island, NY) four times. Bacterial numbers were adjusted spectrophotometrically to 2 x 108 CFU/ml. To a 1-5-ml Ependorf centrifuge tube was added 0-25 ml each ofcells (4 x 106/ml) and bacteria, and 50,ul of the fresh homologous serum. After the mixture was incubated for 30 min in a CO2 incubator at 370 to allow phagocytosis of the bacteria, cells were washed free of non-associated bacteria by centrifuging at 400 g for 5 min at 4°. They were resuspended in RPMI-1640 medium supplemented with 10% homologous serum and sampled for the amount of cell-associated radioactivity. After 0 and 120 min of incubation at 370 in a CO2 incubator, the tubes were removed to ice, and 0.5 ml of trichloroacetic acid was added. After equilibration for at least 1 hr at 40, the tubes were centrifuged in a Microcentrifuge (Hitachi Koki Co. Ltd, Tokyo, Japan) at I 0,000g for 5 min. Soluble label release by digestion of the bacteria following phagocytosis was measured.
Determination of MHC class II antigens MHC class II antigens on peritoneal macrophages were determined by the avidin-biotin complex (ABC) method (Guesdon, Tenynck & Aurameas, 1979) using mAb 1 E-4 (rat IgG2b), which recognizes at least I-A"" and I-Ek/b d k molecules (Ogasawara et al., 1987). mAb 1E-4 was provided by K. Ogasawara of Institute of Immunological Science, Hokkaido University, Sapporo, Japan. Briefly, BALB/c mice (H-2d) that had been infected i.p. with 5 x 104 CFU of L. monocytogenes cells were injected with DSG (3 3 mg/kg/day or 5 mg/kg/day) on Days 0-3 of infection. The number of PEC collected on Day 7 of infection was adjusted to 1 x 107 cells in 1 ml of RPMI-1640 medium supplemented with foetal calf serum (10%; Gibco), penicillin G (100 U/ml) and streptomycin (100 Mg/ml) and cultured in LabTek chamber slides (type 4804; Nunc, Roskilde, Denmark) for 1 hr in a CO2 incubator at 37°. After the non-adherent cells were taken out, the adherent cells were fixed with cold acetone. The slides were incubated successively with normal rabit serum for 15 min, an optimal dilution of mAb 1 E-4 or normal rat IgG for 60 min, biotinylated rabbit anti-rat IgG (Vector Laboratories, Burlingame, CA) for 30 min, and then avidin-biotinylated peroxidase complex (Vector Laboratories) for 30 min. The endogenous peroxidase activity was blocked by incubating the slides in 3% hydrogen peroxide in PBS for 15 min before the avidin-biotin peroxidase step. The slides were stained by incubation in a solution of 3-amino-9-ethyl-carbazole (Aldrich Chemicals Co. Inc., Milwaukee, WI), counterstained with haematoxylin, and mounted in PBS-glycerol. The specificity of the immunohistochemical procedure was confirmed by the following findings: (i) cells performed by ABC procedure without mAb IE-4 were never stained; (ii) no positive reaction was seen when normal rat IgG was used as a control. Statistical evaluation of the data Data were expressed as mean + SD, and the Wilcoxon rank sum test was used to determine the significance of the differences between control and experimental groups.
A. Nakane, A. Numata & T. Minagawa
562
Table 2. Effect of administration of DSG on production of IFN-y and TNF in the bloodstreams of mice in the early phase of L. monocytogenes infection
Titre (U/ml)t t
Infection*
0
+
A
+
0
2
8 6 Days after infection
4
10
12
Figure 1. Effect of administration of different doses of DSG on mortality of L. monocytogenes-infected mice. ddY mice received 1 mg (-), 3.3 mg (O) or 10 mg (0) of DSG per kg per day from Day 0 to 3 of infection. The controls received saline (A). Each result represents a group of 12 mice from two experiments.
Table 1. Growth of L. monocytogenes cells in the spleens and livers of mice that received DSG Log bacteria/organ§
Infection* Day 2
Day 5
Spleen
Treatment
Salinet DSG (3-3 mg/kg/day)t DSG (10 mg/kg/day)t
Saline$ DSG (3 3 mg/kg/day)J DSG (10 mg/kg/day)J
5-86+0 23 5-94+0 20 609+026 2 42+0 45 5l10+0 33¶ 6 84+0-45T
Liver 4 65+0 35 4 99 +0 27 528+022 2 02+0 20 6-22 +044T 6 91 +027¶
ddY mice were infected i.v. with 2 x 104 CFU of L. monocytogenes Day 0. t DSG or saline were administered i.p. on Days -2-1 of infection. t DSG or saline were administered i.p. on Days 0-3 of infection. § Each result represents the mean + SD for a group of five mice. Significantly different from the value for saline-treated controls at P 0-05). Since our previous study (Nakane et al., 1988a) demonstrated that production of endogenous TNF in L. monocytogenes-infected mice might occur in the early phase of infection but that the titre in the serum was under the detectable level by a bioassay with L-929 cells, TNF production in vivo was amplified by stimulation of LPS. Mice which had been treated with DSG or saline only or non-infected controls were injected i.v. with LPS (25 yg) on Day 2 of infection, and the serum samples for TNF titrations were taken 2 hr later. Enhancement of TNF production was observed in L. monocytogenes-infected mice (Table 2). The titres of TNF in serum obtained from DSG-treated mice were comparable to those from the drug-free animals. Effect of administration of DSG on expression of MHC class II antigens on the surface of peritoneal MO Expression of MHC class II antigens on the surface of peritoneal MO obtained from mice that had been administered
Effect of DSG Table 3. Effect of administration of DSG on expression of MHC class II antigens on the surface of peritoneal macrophages obtained from L. monocytogenes-infected mice
DSG treatmentt
Infection*
(mg/kg/day)
563
listeriosis
Table 4. Effect of administration of DSG on phagocytic activity and bactericidal activity of peritoneal macrophages against L. monocytogenes cells
MHC class II antigenpositive cells (%)t
0 0 33 5
+ + +
on
Infection*
9+2 81+6
55±6§ 35+10§
* BALB/c mice were infected i.p. with 5 x 104 CFU of L. monocytogenes cells. t DSG or saline were administered on Days 0-3 of infection. I Each result represents the mean+ SD for a group of five mice. § Significantly different from the value for the infected and saline-treated controls at P < 0-01.
DSG (3 3 mg/kg/day or 5 mg/kg/day) or saline only on Days 0-3, were determined on Day 7 of the i.p. infection with 5 x 104 CFU of L. monocytogenes (Table 3). The infection enhanced class II antigen (Table 3) expression of peritoneal MO (81 + 6%) over uninfected controls (9+2%). However, augmentation of expression of the antigens induced by the infection was significantly suppressed when mice were treated with DSG (P
