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Effects of Bacillus subtilis spores on interferon production
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Pharmacological Research, Vol . 26, Supplement 2, 1992
EFFECTS OF BACILLUS SUBTILIS SPORES ON INTERFERON PRODUCTION M . Muscettola*, G. Grasso**, Z. Blach-Olszewski°, P . Migliaccio**, C . BorghesiNicoletti**, M. Giarratana**, V .C. Gallo§ . Institutes of *General Physiology and **Human Anatomy, Siena (Italy) . °Institute of Immunology and Experimental Therapy, Wroclaw (Poland) and §Sanofi-Winthrop SpA (Milan) Key words : Interferon - B. subtilis - Mucosal immunity - Spleen cells - Peritoneal cells
Introduction It is well established that Bacillus subtilis spores have immunomodulatory and immunostimulating activities in experimental animals and man . However the mechanism responsible for their immunomodulatory properties is not clear and their effects on interferon (IFN) production are little unsterstood . In a recent study (1) we showed that oral administration of B. subtilis spores in rabbits induces high levels of plasma IFN-like activity which persist for eight days after suspension of treatment . Unfortunately, the lack of monoclonal antibodies to rabbit IFNs prevented IFN characterization . The aim of the present study was to evaluate the effect of B. subtilis spores on IFN production ex vivo by murine peritoneal and spleen cells after stimulation, respectively, with LPS and SEA or ConA mitogens . Materials and Methods Balb/c female mice were randomly allocated to two different groups and kept under conventional conditions for one week before the experiment . Lyophilized spores* were suspended in saline and administered orally at a concentration of 2.5x 106 spores per day per animal for 14 days . Two groups of animals were sacrificed by cervical dislocation, then peritoneal and spleen cells were suspended in RPMI 1640 . After washing, the splenocytes were stimulated for 72 h with ConA (5 .tg/ml at 37°C) and SEA (0 .01 µg/ml at 37°C) . Peritoneal cells were stimulated for 24 h with LPS (2 pg/ml at 26°C) . The microplaque reduction assay, described elsewere (2), was used throughout for the titration of IFN activity using L 929 cells as indicator cells and vesicular stomatitis virus as challenge virus . Each assay included the international standards for murine IFN-a/3 and IFN-y to check the sensitivity and reproducibility of our test system . Samples were tested at least twice in quadruplicate . IFN activity was expressed in IU/ml . Typing of IFN species was carried out using specific anti-IFN monoclonal antibodies . The data were processed by ANOVA . Differences were considered statistically significant for P < 0 .05 . Results Table 1 shows that oral administration of B. subtilis spores for 14 days induced increased IFN production by stimulated peritoneal and spleen cells from treated mice compared to controls . Typing of IFN species with specific murine anti-IFN monoclonal antibodies showed that LPS-stimulated peritoneal cells and Con A- or SEA-stimulated spleen cells released, respectively, IFN-3 and IFN-y . Light microscopy showed increased lymphocyte traffic in Peyer's patches of treated animals, with the perifollicular lymphatic sinuses expanded and full of lymphocytes . *Enterogermina, Sanofi-Winthrop SpA, Milan .
1043-6618/92/26110176-02/$03 .00/0
© 1992 The Italian Pharmacological Society
Pharmacological Research, Vol . 26, Supplement 2, 1992
Table 1 . IFN-4 and -'y production by peritoneal and spleen cells from mice fed for 14 days with B. subtilis spores compared to controls ----------------------------------------------------------------------------------------------------------IFN-0 (IU/ml) IFN1y (IU/ml) ----------------------------------------------------------------------------------------------------------------Controls : - LPS-stimulated peritoneal cells 29.24+20 .3 - SEA-stimulated spleen cells 16 .10+6.9 - ConA-stimulated spleen cells 21 .50+4.5 Treated : - LPS-stimulated peritoneal cells 475 .4+ 143 .9 - SEA-stimulated spleen cells 77.6+ 17.8 - ConA-stimulated spleen cells 90 ± 9 .4 --------------------------------------------------------------------------------------------------------------Discussion In accordance with previous data (2,3), the present findings demonstrate that ConA and SEA-stimulated spleen cells from mice fed with B . subtilis spores produce significantly higher IFN-y levels than those of controls . LPS-stimulated peritoneal cells from treated animals also produce significantly higher IFN-P levels than those of controls . This increased IFN-y production is in line with other effects of B. subtilis such as enhanced macrophage-mediated cytotoxicity, lymphocyte proliferation in response to PHA and ConA, NK cell function (4) and increased percentage of HLADR+ T lymphocytes (5) and justifies at least in part the interesting therapeutic results . Moreover the increased lymphocyte traffic in Peyer's patches and the enhanced levels of IFN-y and IFN-13 in treated mice confirm that B . subtilis spores have local and systemic immunostimulating activities and suggest that such activities can be partially modulated by increased secretion of these cytokines . References 1) Muscettola M, Grasso G, Migliaccio P, Gallo VC . Plasma interferon-like activity in rabbits after oral administration of Bacillus subtilis spores . J . Chemother. 1991 ; 3 (Suppl . 3) : 130-2. 2) Muscettola M, Grasso G . Somatostatin and vasoactive intestinal peptide reduce interferon gamma production by human peripheral blood mononuclear cells . Immunobiol . 1990 ; 180 :419-30. 3) Nozaki-Renard J . Induction d'interferon par Bacillus subtilis. Ann. Microbiol . (Inst . Pasteur) 1978 ; 129A: 525-43 . 4) Spreafico F, Polentarutti N, Vecchi A, Filippeschi S, Tagliabue A, Sironi M, Moras ML. L'effetto immunostimolatore delle spore di Bacillus subtilis aspetti sperimentali . Chemioter . Antimicrob . 1980 ; 3 : 259-66 . 5) Gonzales R, Pirali F, Ravizzola G, Pinsi G, Zanoni L, De Francesco M, Foresti I, Caruso A . Stimolazione selettiva dell'espressione di antigen HLA-DR su linfociti T in vivo in seguito a somministrazione di "vaccini" orali . Boll . Microbiol . Indag. Lab. 1989 ; 9 : 5-9.
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Pharmacological Research, Vol . 26, Supplement 2, 1992
EFFECTS OF BACILLUS SUBTILIS SPORES ON INTERFERON PRODUCTION M . Muscettola*, G. Grasso**, Z. Blach-Olszewski°, P . Migliaccio**, C . BorghesiNicoletti**, M. Giarratana**, V .C. Gallo§ . Institutes of *General Physiology and **Human Anatomy, Siena (Italy) . °Institute of Immunology and Experimental Therapy, Wroclaw (Poland) and §Sanofi-Winthrop SpA (Milan) Key words : Interferon - B. subtilis - Mucosal immunity - Spleen cells - Peritoneal cells
Introduction It is well established that Bacillus subtilis spores have immunomodulatory and immunostimulating activities in experimental animals and man . However the mechanism responsible for their immunomodulatory properties is not clear and their effects on interferon (IFN) production are little unsterstood . In a recent study (1) we showed that oral administration of B. subtilis spores in rabbits induces high levels of plasma IFN-like activity which persist for eight days after suspension of treatment . Unfortunately, the lack of monoclonal antibodies to rabbit IFNs prevented IFN characterization . The aim of the present study was to evaluate the effect of B. subtilis spores on IFN production ex vivo by murine peritoneal and spleen cells after stimulation, respectively, with LPS and SEA or ConA mitogens . Materials and Methods Balb/c female mice were randomly allocated to two different groups and kept under conventional conditions for one week before the experiment . Lyophilized spores* were suspended in saline and administered orally at a concentration of 2.5x 106 spores per day per animal for 14 days . Two groups of animals were sacrificed by cervical dislocation, then peritoneal and spleen cells were suspended in RPMI 1640 . After washing, the splenocytes were stimulated for 72 h with ConA (5 .tg/ml at 37°C) and SEA (0 .01 µg/ml at 37°C) . Peritoneal cells were stimulated for 24 h with LPS (2 pg/ml at 26°C) . The microplaque reduction assay, described elsewere (2), was used throughout for the titration of IFN activity using L 929 cells as indicator cells and vesicular stomatitis virus as challenge virus . Each assay included the international standards for murine IFN-a/3 and IFN-y to check the sensitivity and reproducibility of our test system . Samples were tested at least twice in quadruplicate . IFN activity was expressed in IU/ml . Typing of IFN species was carried out using specific anti-IFN monoclonal antibodies . The data were processed by ANOVA . Differences were considered statistically significant for P < 0 .05 . Results Table 1 shows that oral administration of B. subtilis spores for 14 days induced increased IFN production by stimulated peritoneal and spleen cells from treated mice compared to controls . Typing of IFN species with specific murine anti-IFN monoclonal antibodies showed that LPS-stimulated peritoneal cells and Con A- or SEA-stimulated spleen cells released, respectively, IFN-3 and IFN-y . Light microscopy showed increased lymphocyte traffic in Peyer's patches of treated animals, with the perifollicular lymphatic sinuses expanded and full of lymphocytes . *Enterogermina, Sanofi-Winthrop SpA, Milan .
1043-6618/92/26110176-02/$03 .00/0
© 1992 The Italian Pharmacological Society
Pharmacological Research, Vol . 26, Supplement 2, 1992
Table 1 . IFN-4 and -'y production by peritoneal and spleen cells from mice fed for 14 days with B. subtilis spores compared to controls ----------------------------------------------------------------------------------------------------------IFN-0 (IU/ml) IFN1y (IU/ml) ----------------------------------------------------------------------------------------------------------------Controls : - LPS-stimulated peritoneal cells 29.24+20 .3 - SEA-stimulated spleen cells 16 .10+6.9 - ConA-stimulated spleen cells 21 .50+4.5 Treated : - LPS-stimulated peritoneal cells 475 .4+ 143 .9 - SEA-stimulated spleen cells 77.6+ 17.8 - ConA-stimulated spleen cells 90 ± 9 .4 --------------------------------------------------------------------------------------------------------------Discussion In accordance with previous data (2,3), the present findings demonstrate that ConA and SEA-stimulated spleen cells from mice fed with B . subtilis spores produce significantly higher IFN-y levels than those of controls . LPS-stimulated peritoneal cells from treated animals also produce significantly higher IFN-P levels than those of controls . This increased IFN-y production is in line with other effects of B. subtilis such as enhanced macrophage-mediated cytotoxicity, lymphocyte proliferation in response to PHA and ConA, NK cell function (4) and increased percentage of HLADR+ T lymphocytes (5) and justifies at least in part the interesting therapeutic results . Moreover the increased lymphocyte traffic in Peyer's patches and the enhanced levels of IFN-y and IFN-13 in treated mice confirm that B . subtilis spores have local and systemic immunostimulating activities and suggest that such activities can be partially modulated by increased secretion of these cytokines . References 1) Muscettola M, Grasso G, Migliaccio P, Gallo VC . Plasma interferon-like activity in rabbits after oral administration of Bacillus subtilis spores . J . Chemother. 1991 ; 3 (Suppl . 3) : 130-2. 2) Muscettola M, Grasso G . Somatostatin and vasoactive intestinal peptide reduce interferon gamma production by human peripheral blood mononuclear cells . Immunobiol . 1990 ; 180 :419-30. 3) Nozaki-Renard J . Induction d'interferon par Bacillus subtilis. Ann. Microbiol . (Inst . Pasteur) 1978 ; 129A: 525-43 . 4) Spreafico F, Polentarutti N, Vecchi A, Filippeschi S, Tagliabue A, Sironi M, Moras ML. L'effetto immunostimolatore delle spore di Bacillus subtilis aspetti sperimentali . Chemioter . Antimicrob . 1980 ; 3 : 259-66 . 5) Gonzales R, Pirali F, Ravizzola G, Pinsi G, Zanoni L, De Francesco M, Foresti I, Caruso A . Stimolazione selettiva dell'espressione di antigen HLA-DR su linfociti T in vivo in seguito a somministrazione di "vaccini" orali . Boll . Microbiol . Indag. Lab. 1989 ; 9 : 5-9.
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