Folia Microbiol. 37 (2), 153-156 (1992)
Functional and Metabolic Changes of Guinea Pig Peritoneal Macrophages Induced by Bacterial Vaccines J. TAKe,OVAa, D. KOTULOVAb, A. KA~I(ZKOVAand M. FEREN~[Kc aHospital with Policlinic, Department of Clinical Biochemistry, 054 11 Levoda, Czechoslovakia bDepartment of Medical Microbiology and bmmmology, Comenius Unh,ersity, 811 08 Bratislava, Czechoslovakia Clnstitute of lmmttnology, Comenius University 811 08 Bratislava, Czechoslovakia Received May 29, 1991
ABSTRACT. Phagocytic activity of peritoneal macrophages of guinea pigs injected subcutaneously with bacterial vaccines was found to be increased. The phagocytic index remained unchanged or was decreased. In addition, a decreased candidacidal activity was observed. Metabolic activation of macrophages, measured by the INT test, was inhibited in unstimulated cells while cells stimulated with zymosan or opsonized zymosan exhibited higher values of the INT test as compared with control animals. After injection of vaccines the number of peritoneal macrophages was increased, but the spleen mass decreased.
Vaccinotherapy belongs to the oldest methods used in the therapy of bacterial allergic diseases and chronic microbial infections that are resistant to therapy by means of antibiotics and chemotherapeutics. Thus, bacterial vaccines are supposed to possess immunizing and hyposensitizing effects. The immunizing effect results in enhanced specific immune resistance and the hyposensitizing effect is manifested by decrease intensity of the hypersensitivity reaction to the given allergen. It seems that the decisive role is played by immunostimulatory effects that are manifested by activation of macrophages and NK cells, enhancement of specific cell-mediated immunity reactions, B-lymphocyte stimulation and increased biosynthesis and secretion of immunoglobulins, particularly secretory IgA (Girard and Fleury 1979; Milatovic 1982; Kr~l et al. 1984; Maestroni and Losa 1984; Rosenthal 1986). Stock vaccines are indicated primarily for hyposensitization reasons, but obviously they possess also an immunopreventive capacity in terms of immunostimulation (Beneg 1986). Also, such vaccines stimulate TH lymphocytes ((~ip and Buransk~ 1986). The Staphylococctts attretts vaccine induces, after a short-term administration to guinea pigs, an increased number of peritoneal macrophages as well as enhancement of their phagocytic index. The candidacidal and phagocytic activity of these macrophages was significantly decreased. As regards b~ vitro experiments, after a short-term contact of human granulocytes with the Staphylococcus vaccine, stimulation of INT reduction, superoxide production and liberation of extracellular enzymes such as myeloperoxidase, lysozyme and fl-D-glucuronidase was observed (Kotulov~i et al. 1988). The Propionibacterium ache vaccines possess a stimulatory effect for phagocytes and potentiate the secretory function of murine peritoneal macrophages releasing elastase, glucuronidase and hydrogen peroxide (Drews 1985). The staphylococcal lyzate STAVA contains important antigenic components of staphylococci and living phage particles. When injected subcutaneously to patients in increasing doses from 50/zL to 500/zL (maximum), immunological examination revealed decreased IgG and IgA levels, significantly lower numbers of T lymphocytes which became normal within two months after the beginning of therapy; also, the total complement activity and phagocytic activity decreased (Kyn~lov~ 1984). STAVA induces a dose-dependent stimulation of chemiluminescence and may act as mitogen T (Pillich 1986).
M A T E R I A L S A N D METHODS 77~e vaccines. (1) Stava (SS) - a bacteriophage lyzate (Institute of Biophysics, Czech. Acad. Sci., Brno) is an immunomodulator which was prepared from two S. attreus strains grown individually up to the concentration of 2 - 3 • 108 bacteria per mL of tryptone broth. The bacteria were lyzed with polyvalent bacteriophage at a concentration of 8 x 109/mL without physical or chemical treatment which might denature important immunogenic and antigenic components. (2) Staphylococcus aureus autovaccine (SA) - (Department of Medical Microbiology and hnmunology, Comenius University, Bratislava) was prepared by cultivating the bacterial strain isolated from the sputum of a patient suffering from chronic bronchitis on blood agar with a cellophane foil. It contains exoproducts of bacteria and a suspension of bacterial cells at a concentration of 3 x 108/mL. (3) Staphyllococcus aureus -
1,54 J. TAKA~OV,~, et al.
Vol. 37
autovaccine (ST) - (Department o f Medical Microbiology and Immunology, Comenius University, Bratislava) contains bacterial cells only (concentration 3 x 108/mL) without exoproducts. (4)Proteus mirabilis - autovaecine (PA) - (Department o f Medical Microbiology a n d Immunology, Comenius University, Bratislava) was prepared from a strain isolated from a patient's sputum similarly as the Staphylococcus aureus autovaccine. Guhlea-pigperitoneal macrophages. Groups of guinea pigs (eight animals in each group) were injected subcutaneously with S. attreus vaccines (SS, SA, ST) and P. mirabilis autovaccine for the period of 6 weeks in 12 doses equivalent to 5 ~L, up to 60 g L of the original undiluted vaccine. The first control group received an equivalent amount of physiological solution (PS), the second control group was not immunized (C). After obtaining the blood sample by cardiac puncture the animals were sacrificed by air embolism and mononuclear phagocytes were obtained by washing the peritoneal cavity with phosphate-buffered saline (PBS) supplemented with heparin (5 U / m L ) . The cells were then centrifuged and washed and the suspension was adjusted to the final concentration of 107/mL. Metabolic activity. The INT-reductase activity was determined according to Lokaj and Obfirkov~i (1975) employing microtiter plates (Feren~fk et al. 1988). Reduction of 3-(4-iodophenyl)-2(4-nitrophenyl)-5-phenyltetrazoliumchloride (INT) by resting macropahges and by macrophages phagocytosing zymosan or opsonized zymosan was determined. The results were expressed in fmol formazan reduced by a single phagocyte (fmol/Ma). Functional activities. The phagocytic activity (PA) was examined with the aid of heat-inactivated yeast Candida albicans (Kotulov~i and ~tefanovi6. 1983). Phagocytes were incubated in the presence of autologous serum and five aliquots of yeast suspension under permanent shaking. After a 30-min incubation smears were prepared, dried and stained according to Wright. 200 phagocytes were examined microscopically and the phagocytic activity (i.e. proportion of phagocytosing macrophages) and phagocytic index (i.e. mean number of yeasts engulfed by a single macrophage) were determined. Candidacidal activity was determined by incubating phagocytes with yeasts (ratio 1:1) in the presence of autologous serum. Proportion of intracellularly killed candidas was determined after a 1-h incubation and after lyzing the phagocytic cells by means of vital staining with methylene blue (Kotulov~i 1986).
RESULTS AND DISCUSSION Results concerning the total INT reduction by peritoneal macropahges of quinea pigs injected with various vaccines are given in Table I. The vaccines produced decreased INT reductase activity but, following further stimulation with zymosan and zymosan preopsonized with homologous serum, an increase of activity could be observed. Thus, one may presume that peritoneal macrophages can Table
I. INT reduction (• SD) in guinea pig peritoneal macrophages
Guinea pigsa group
pMa
pMa + Z p
Functional and Metabolic Changes of Guinea Pig Peritoneal Macrophages Induced by Bacterial Vaccines J. TAKe,OVAa, D. KOTULOVAb, A. KA~I(ZKOVAand M. FEREN~[Kc aHospital with Policlinic, Department of Clinical Biochemistry, 054 11 Levoda, Czechoslovakia bDepartment of Medical Microbiology and bmmmology, Comenius Unh,ersity, 811 08 Bratislava, Czechoslovakia Clnstitute of lmmttnology, Comenius University 811 08 Bratislava, Czechoslovakia Received May 29, 1991
ABSTRACT. Phagocytic activity of peritoneal macrophages of guinea pigs injected subcutaneously with bacterial vaccines was found to be increased. The phagocytic index remained unchanged or was decreased. In addition, a decreased candidacidal activity was observed. Metabolic activation of macrophages, measured by the INT test, was inhibited in unstimulated cells while cells stimulated with zymosan or opsonized zymosan exhibited higher values of the INT test as compared with control animals. After injection of vaccines the number of peritoneal macrophages was increased, but the spleen mass decreased.
Vaccinotherapy belongs to the oldest methods used in the therapy of bacterial allergic diseases and chronic microbial infections that are resistant to therapy by means of antibiotics and chemotherapeutics. Thus, bacterial vaccines are supposed to possess immunizing and hyposensitizing effects. The immunizing effect results in enhanced specific immune resistance and the hyposensitizing effect is manifested by decrease intensity of the hypersensitivity reaction to the given allergen. It seems that the decisive role is played by immunostimulatory effects that are manifested by activation of macrophages and NK cells, enhancement of specific cell-mediated immunity reactions, B-lymphocyte stimulation and increased biosynthesis and secretion of immunoglobulins, particularly secretory IgA (Girard and Fleury 1979; Milatovic 1982; Kr~l et al. 1984; Maestroni and Losa 1984; Rosenthal 1986). Stock vaccines are indicated primarily for hyposensitization reasons, but obviously they possess also an immunopreventive capacity in terms of immunostimulation (Beneg 1986). Also, such vaccines stimulate TH lymphocytes ((~ip and Buransk~ 1986). The Staphylococctts attretts vaccine induces, after a short-term administration to guinea pigs, an increased number of peritoneal macrophages as well as enhancement of their phagocytic index. The candidacidal and phagocytic activity of these macrophages was significantly decreased. As regards b~ vitro experiments, after a short-term contact of human granulocytes with the Staphylococcus vaccine, stimulation of INT reduction, superoxide production and liberation of extracellular enzymes such as myeloperoxidase, lysozyme and fl-D-glucuronidase was observed (Kotulov~i et al. 1988). The Propionibacterium ache vaccines possess a stimulatory effect for phagocytes and potentiate the secretory function of murine peritoneal macrophages releasing elastase, glucuronidase and hydrogen peroxide (Drews 1985). The staphylococcal lyzate STAVA contains important antigenic components of staphylococci and living phage particles. When injected subcutaneously to patients in increasing doses from 50/zL to 500/zL (maximum), immunological examination revealed decreased IgG and IgA levels, significantly lower numbers of T lymphocytes which became normal within two months after the beginning of therapy; also, the total complement activity and phagocytic activity decreased (Kyn~lov~ 1984). STAVA induces a dose-dependent stimulation of chemiluminescence and may act as mitogen T (Pillich 1986).
M A T E R I A L S A N D METHODS 77~e vaccines. (1) Stava (SS) - a bacteriophage lyzate (Institute of Biophysics, Czech. Acad. Sci., Brno) is an immunomodulator which was prepared from two S. attreus strains grown individually up to the concentration of 2 - 3 • 108 bacteria per mL of tryptone broth. The bacteria were lyzed with polyvalent bacteriophage at a concentration of 8 x 109/mL without physical or chemical treatment which might denature important immunogenic and antigenic components. (2) Staphylococcus aureus autovaccine (SA) - (Department of Medical Microbiology and hnmunology, Comenius University, Bratislava) was prepared by cultivating the bacterial strain isolated from the sputum of a patient suffering from chronic bronchitis on blood agar with a cellophane foil. It contains exoproducts of bacteria and a suspension of bacterial cells at a concentration of 3 x 108/mL. (3) Staphyllococcus aureus -
1,54 J. TAKA~OV,~, et al.
Vol. 37
autovaccine (ST) - (Department o f Medical Microbiology and Immunology, Comenius University, Bratislava) contains bacterial cells only (concentration 3 x 108/mL) without exoproducts. (4)Proteus mirabilis - autovaecine (PA) - (Department o f Medical Microbiology a n d Immunology, Comenius University, Bratislava) was prepared from a strain isolated from a patient's sputum similarly as the Staphylococcus aureus autovaccine. Guhlea-pigperitoneal macrophages. Groups of guinea pigs (eight animals in each group) were injected subcutaneously with S. attreus vaccines (SS, SA, ST) and P. mirabilis autovaccine for the period of 6 weeks in 12 doses equivalent to 5 ~L, up to 60 g L of the original undiluted vaccine. The first control group received an equivalent amount of physiological solution (PS), the second control group was not immunized (C). After obtaining the blood sample by cardiac puncture the animals were sacrificed by air embolism and mononuclear phagocytes were obtained by washing the peritoneal cavity with phosphate-buffered saline (PBS) supplemented with heparin (5 U / m L ) . The cells were then centrifuged and washed and the suspension was adjusted to the final concentration of 107/mL. Metabolic activity. The INT-reductase activity was determined according to Lokaj and Obfirkov~i (1975) employing microtiter plates (Feren~fk et al. 1988). Reduction of 3-(4-iodophenyl)-2(4-nitrophenyl)-5-phenyltetrazoliumchloride (INT) by resting macropahges and by macrophages phagocytosing zymosan or opsonized zymosan was determined. The results were expressed in fmol formazan reduced by a single phagocyte (fmol/Ma). Functional activities. The phagocytic activity (PA) was examined with the aid of heat-inactivated yeast Candida albicans (Kotulov~i and ~tefanovi6. 1983). Phagocytes were incubated in the presence of autologous serum and five aliquots of yeast suspension under permanent shaking. After a 30-min incubation smears were prepared, dried and stained according to Wright. 200 phagocytes were examined microscopically and the phagocytic activity (i.e. proportion of phagocytosing macrophages) and phagocytic index (i.e. mean number of yeasts engulfed by a single macrophage) were determined. Candidacidal activity was determined by incubating phagocytes with yeasts (ratio 1:1) in the presence of autologous serum. Proportion of intracellularly killed candidas was determined after a 1-h incubation and after lyzing the phagocytic cells by means of vital staining with methylene blue (Kotulov~i 1986).
RESULTS AND DISCUSSION Results concerning the total INT reduction by peritoneal macropahges of quinea pigs injected with various vaccines are given in Table I. The vaccines produced decreased INT reductase activity but, following further stimulation with zymosan and zymosan preopsonized with homologous serum, an increase of activity could be observed. Thus, one may presume that peritoneal macrophages can Table
I. INT reduction (• SD) in guinea pig peritoneal macrophages
Guinea pigsa group
pMa
pMa + Z p
