Vol. 11, No. 1 Printed in U.S.A.
INFECTION AND IMMUNITY, Jan. 1975, p. 80-85 Copyright © 1975 American Society for Microbiology
Effect of Corynebacterium acnes on Interferon Production in Mouse Peritoneal Exudate Cells J. FISCHBACH AND L. A. GLASGOW Departments of Pediatrics and Microbiology, University of Utah College of Medicine, Salt Lake City, Utah 84132
Received for publication 19 July 1974
Corynebacterium acnes, an organism closely related to C. parvum, has been recognized to have a striking effect on the reticuloendothelial system, as well as on both humoral and cellular immunity. In mice previously exposed to C. acnes, serum interferon levels induced by injection of Newcastle disease virus (NDV), Chikungunya virus (CV), and polyinosinic-polycytidylic acid are suppressed. When peritoneal macrophages and lymphocytes from animals exposed to C. acnes were cultivated in vitro, their capacity to produce interferon in r6sponse to NDV and CV was reduced. Furthermore, the interferon-producing capacity of these cells in tissue culture was inhibited after exposure to C. acnes in vitro. Exposure of separated populations of peritoneal macrophages and lymphocytes to C. acnes in vitro demonstrated that the interferon response to NDV by both cell types is inhibited. Peritoneal macrophages appear to be the major contributor to the interferon response in this system. Finally, this inhibitory effect was shown to occur after exposure to a purified cell wall preparation of C. acnes organisms, as well as a lipid extract of this preparation. showed that mice exposed to C. acnes formed significantly reduced levels of interferon in response to Newcastle disease virus (NDV), Chikungunya virus (CV) and the synthetic polynucleotide polyinosinic-polycytidylic acid (poly I:C), whereas serum interferon levels induced by endotoxin were enhanced. The purpose of this study was to determine: (i) whether this effect on interferon production was reflected in peritoneal exudate cells (PEC) obtained from mice exposed to C. acnes; (ii) which cell population, macrophages or lymphocytes, was affected by C. acnes; (iii) whether this effect could be produced in PEC taken from normal mice and exposed to C. acnes in vitro; and (iv) what components of the organism were responsible for this effect.
Anaerobic coryneforms, such as Corynebacterium parvum, have been shown to exert a stimulatory effect on the reticuloendothelial system in experimental animals, which is characterized by hypertrophy of lymphoid tissue, increased phagocytic activity as measured by carbon clearance, and antitumor activity (1, 6-8, 12-14, 16). Furthermore, C. parvum, which is currently being utilized as an immunotherapeutic agent in certain human malignancies, has been recognized to enhance the humoral immune response to certain antigens and yet to suppress cellular functions such as the mixed lymphocyte and graft-versus-host reactions as well as the phytohemagglutinin response (9, 15). C. acnes, a closely related bacterium, is the most common member of the normal microbial flora of our skin. Although it has been suggested that the microorganism be reclassified as Propionibacterium acnes (2, 4, 10), Cummins and Johnson (3) have been unable to distinguish P. acnes (C. acnes) from C. paruum on the basis of cell wall analysis, serological tests, and fermentation tests. Their data suggest that these strains are identical, or very closely related. Farber and Smith (Bacteriol. Proc., p. 83, 1969) demonstrated that C. acnes and C. parvum are capable of producing similar effects on the reticuloendothelial system in experimental animals. Subsequently, Farber and Glasgow (5)
MATERIALS AND METHODS
C. acnes, from the American Type Culture Collection (11828), was grown in dextrose-thioglycolate broth containing 2% peptone (Difco), 0.5% yeast extract (Difco), 0.5% dextrose, and 0.1% sodium thioglycolate. After 5 to 7 days of growth at 37 C, the organisms were killed by heating at 60 C for 1 h; the cells were collected by centrifugation and washed twice in phosphate-buffered saline (PBS). An inoculum of approximately 109 colony-forming units (CFU) was used throughout these experiments. Cell wall preparation. The wet weight of the organisms was determined and a 50% slurry was 80
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EFFECT OF C. ACNES ON INTERFERON PRODUCTION
made. The organisms were transferred to a Braun flask containing 0.1-mm glass beads three times the weight of the bacteria. The bacteria were shaken at 400 cycles/min for five 1-min intervals with CO2 cooling. The contents of the flask were then centrifuged at 350 x g for 10 min at 4 C to remove the glass beads and intact cells; the supernatant fluid containing the cell wall material was centrifuged at 10,000 x g for 10 min at 4 C. This pellet was washed 12 times with PBS and resuspended in PBS at the original volume of the C. acnes suspension. Medium. All cell cultures were maintained in Eagle minimal essential medium (MEM) with 10% fetal calf serum (FCS). Interferon inducers. NDV, Herts strain, grown in the allantoic cavity of embryonated hen's eggs, titered approximately 3 x 109 plaque-forming units (PFU)/ml in primary chick embryo fibroblasts. CV, originally obtained from the Walter Reed Medical Center Arbovirus Unit, was prepared from the brains of infected suckling mice and titered approximately 107 PFU/ml when assayed on primary chick 5.0 embryo fibroblasts. An inoculum of 0.1 ml of undiluted stock virus (NDV or CV) was used to induce interferon. Poly I:C was obtained from P-L Biochemicals, Milwaukee, Wis. A dose of 100 ,g was used in these experiments. Endotoxin, Escherichia coli 0111:B4 lipopolysaccharide prepared by the Westphal method (Difco Laboratories), was used at dosages of 100 gg. Mice. Female CD-1 mice obtained from Charles River Breeding Laboratories (Brookline, Mass.) were housed six/cage and received food and water ad libitum. PEC. PEC were collected aseptically from the peritoneal cavities of normal mice which had received intraperitoneal (ip) injections of approximately 109 CFU of heat-killed C. acnes 7 to 14 days prior to collection by washing with cold MEM without FCS. Three days prior to collection, all mice were injected ip with 2.0 ml of 1% peptone. The peritoneal washings were centrifuged at 350 x g and resuspended in MEM with 10% FCS at a concentration of 106 cells/ml. The cells were then distributed in 2.0-ml portions into 35-mm plastic tissue culture dishes (Falcon Plastics) and challenged with NDV, CV, poly I:C, endotoxin, or C. acnes. A preparation of normal control PEC processed simultaneously in an identical manner served as the control. After 18 h at 37 C, the medium was collected from each plate and stored at -20 C until assayed. In experiments in which the effect of C. acnes on PEC from normal mice was determined, a cellular response was stimulated with peptone, and cells were collected and prepared in the manner just described. Prior to challenge with an interferon inducer, plates containing normal PEC were inoculated with heatkilled whole C. acnes or the cell wall preparation and incubated at 37 C for 48 h. At this time, cell preparations exposed to C. acnes and untreated controls were challenged with NDV. Medium from each plate was collected after 18 h to be assayed for interferon. Cell viability was determined by the trypan blue dye exclusion method on all PEC cultures. x
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Separation of macrophages and lymphocytes. Cells were separated into macrophage and lymphocyte populations by placing a suspension of normal PEC, at a concentration of 106 cells/ml, into tissue culture dishes and incubating them for 3 h at 37 C. After incubation, the medium from each plate was removed, pooled, and centrifuged at 350 x g to collect the nonadherent cell population. The nonadherent cells (predominantly lymphocytes) were resuspended in MEM with 10% FCS at a concentration equivalent to the adherent cell population. The plates containing the adherent cells (predominantly macrophages) were refed with fresh MEM with 10% FCS. Prior to challenge with an interferon inducer, plates from each group were inoculated with heat-killed whole C. acnes and incubated at 37 C for 48 h. At this time, cell preparations exposed to C. acnes and untreated controls were challenged with NDV. Medium from each plate was collected after 18 h to be assayed for interferon. Interferon assay. Virus-induced samples to be assayed for interferon were adjusted to pH 2.0 with concentrated HCl. After 2 days (CV) or 5 days (NDV) at 4 C, samples were returned to pH 7.0 with NaOH. Assays were performed on L-cell monolayers with approximately 30 to 50 PFU of vesicular stomatitis virus (VSV; Indiana strain) as the challenge virus (11). The concentration of interferon was defined as the reciprocal of the dilution which produced a 50% plaque inhibition of the control VSV inoculum. A laboratory interferon standard was included in each assay to serve as a guide to the sensitivity of our assay system; results were not adjusted by comparison with this standard interferon preparation. Periodically, the sensitivity of our assay was compared with the international mouse interferon reference preparation from the National Institutes of Health. In our system, the international standard had a titer of approximately 300 units/ml. Lipid extraction. A suspension of heat-inactivated C. acnes at a concentration of 109 CFU/ml was washed two times in PBS, resuspended in twice the original volume of chloroform-methanol (2:1, vol/vol), and mixed well. This mixture was allowed to stand at room temperature for 30 min and then was centrifuged at 350 x g for 10 min. The supernatant-crude extract was removed and mixed thoroughly with a 0.73% NaCl solution (5:1, vol/vol). The two phases were separated by centrifugation at 1,000 x g for 15 min. The upper, aqueous phase (predominantly nonlipid material) was removed, the interface was rinsed three times with small amounts of chloroformmethanol-water (3:48:47, vol/vol) so as not to disturb the lower phase, and methanol was added dropwise until one phase was obtained. The solvents were then removed by evaporation and the lipid material was resuspended in PBS. The original C. acnes suspension was extracted four times and the extracts were combined. RESULTS
To determine the effect of C. acnes on the capacity of PEC to produce interferon, we
FISCHBACH AND GLASGOW
82
collected PEC from mice 14 days after they had received a single ip injection of approximately 109 CFU of nonviable C. acnes. The cell preparation was then incubated in vitro and exposed to a representative interferon-inducing agent. The collection time was selected to coincide with the period of suppression of the interferon response by C. acnes in the whole animal. The culture medium from PEC obtained from C. acnes-exposed and control animals was collected 18 h after addition of the inducing agent and assayed for the presence of interferon. The results of four experiments are summarized in Fig. 1. Prior exposure to C. acnes resulted in suppression of the interferon response of PEC to both NDV and CV; levels of interferon produced in response to poly I:C were variable. No detectable interferon was produced in response to endotoxin by PEC from C. acnes-exposed or normal mice. These data indicate that the altered capacity of mice exposed to C. acnes to produce interferon in response to NDV and CV in vivo is reflected in the decreased capability of a mixed population of macrophages and lymphocytes from these animals to respond to these same viral inducing agents in vitro. Although our previous data showed that exposure to C.
INFECT. IMMUN.
acnes resulted in an enhanced interferon response to endotoxin in the intact animal (5), this observation could not be confirmed since detectable levels of interferon were not induced by endotoxin under these in vitro conditions. To determine whether the suppression of the interferon response would also occur in normal PEC exposed to C. acnes in cultures, we collected PEC harvested from normal mice and exposed them to preparations of heat-killed C. acnes. These cultures were then incubated for 48 h before being challenged with NDV. The results from three experiments are illustrated in Fig. 2. NDV-induced interferon produced by normal PEC exposed in vitro to C. acnes was inhibited to the same degree as levels of interferon produced by PEC harvested from mice inoculated with C. acnes. These data suggest that the direct exposure of a mixed population of macrophages and lymphocytes to C. acnes results in a decreased capacity to produce interferon in response to at least one representative viral inducer. W
120-
0 _
CONTROL
C. Acnes WHOLE ORGANISM C. Acnes CELL WALL
1000
z
O 80u
0
,,; 60w -i
z
0 cru
40-
.z
20-
v
INDUCER:
Newcastle Disease
Chikungunya Virus
Poly I:C
Endotoxin
virus
FIG. 1. Suppression of mean interferon levels (expressed as a percentage of the control) produced by peritoneal macrophages and lymphocytes from control and C. acnes-exposed mice after induction with Newcastle disease virus, Chikungunya virus, poly I:C, and endotoxin.
INDUCER: NEWCASTLE DISEASE VIRUS FIG. 2. Suppression of mean interferon levels (expressed as a percentage of the control) produced by peritoneal macrophages and lymphocytes from normal mice exposed in vitro to a nonviable C. acnes preparation or a cell wall fraction prepared from C. acnes prior to induction with Newcastle disease virus.
Control peritoneal exudate cells received no C. acnes exposure.
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EFFECT OF C. ACNES ON INTERFERON PRODUCTION
To examine which specific cell types were being altered in their ability to produce interferon, we separated PEC from normal mice into either predominantly macrophage or lymphocyte populations on the basis of their adherence properties. These cells, in equivalent concentrations, were then exposed to heat-killed C. acnes in vitro and incubated for 48 h before being challenged with NDV. The culture medium was collected at 18 h and assayed for the presence of interferon. As shown in Fig. 3. the interferon response of both the macrophage and, to a lesser degree, the lymphocyte populations was suppressed by prior exposure to C. acnes. The greater levels of interferon produced by the macrophage population (3,000 units/ml compared with 100 units/ml) indicate that the macrophage is the major contributor to the interferon response in this system. The effect of C. acnes on mouse cells derived from a source other than the reticuloendothelial system was determined by exposing monolayers of mouse embryo fibroblasts (MEF) to C. acnes in the same manner as PEC and challenging them with NDV. The culture medium was collected at 18 h and assayed for the presence of interferon. MEF which had been previously exposed to C. acnes produced approximately 8,500 units of interferon/ml and control MEF produced approximately 8,000 units/ml. There appears to be no inhibitory effect on interferon production by this cell of nonreticuloendothelial origin. No toxicity was observed in MEF which had been exposed to C. acnes. The results of these experiments made it possible to explore the components of the C. acnes organism which were responsible for the observed effects on interferon synthesis. It was assumed that neither multiplication nor metabolic activity of C. acnes was responsible for the interferon inhibition since a nonviable, heatkilled preparation of C. acnes was as effective as viable organisms in these experiments. To determine whether a component of C. acnes was responsible for this effect, we first prepared a purified cell wall fraction. Normal PEC were incubated with this cell wall preparation in the same manner as in previous experiments in which the whole organism was used. After challenge with NDV as the interferon-inducing agent, culture medium was collected and assayed for interferon. As can be seen in Fig. 2, the cell wall preparation from C. acnes suppressed the interferon response to the same degree as the whole organism. Because the Corynebacterium and Propionibacterium groups of microorganisms are characterized by the lipids present in their cell wall, we next examined the ability
83
= CONTROL E C Acnes
cr-
z
MACROPHAGE
(Gloss- adherent)
LYMPHOCYTE (Not Gloss-odherent)
FIG. 3. Interferon levels produced by control and C. acnes-exposed peritoneal exudate cells separated into macrophage and lymphocyte populations, after induction by Newcastle disease virus.
of a lipid extract and the cell wall preparation from which it was removed to suppress the capacity of cells to produce interferon. The activity of these two preparations was determined in the same manner used with the whole organism and the cell wall. Again, cultures of PEC exposed to nonviable whole organisms and similar cultures which were not exposed to C. acnes served as controls. The ability to suppress the production of interferon was present in the lipid component removed from the cell wall of C. acnes, but the cell wall preparation from which the lipid was extracted also retained the ability to suppress interferon production to the same degree (Fig. 4). These data support the hypothesis that a component of the cell wall is responsible for the observed inhibition of interferon production. In spite of the fact that the lipid extract inhibited interferon synthesis, it is not clear whether that property resides solely in the lipid component of the cell wall. Although the cell wall preparation from which the lipid was extracted also suppressed interferon production, it is possible that some lipid remained bound to the cell wall preparation and that the observed effect was due to residual amounts of lipid material in the cell wall which were not removed by the extraction procedure.
84
FISCHBACH AND GLASGOW W CONTROL CELL WALL LIPID COM PON ENT CELL WALL WITH M LIPID COMPONENT
120-~~~~~~~::
120-
REMOVED
WHOLE ORGANISM 100 0
°) 800 -
En
, 60uJ z 0
uw
40-
20L
INDUCER: NEWCASTLE DISEASE VIRUS FIG. 4. Suppression of mean interferon levels (expressed as a percentage of the control) in vitro in response to Newcastle disease virus produced by normal peritoneal exudate cells exposed to a lipid component of C. acnes cell wall, the cell wall with a lipid component removed, and the whole organism, in comparison with control peritoneal exudate cells.
DISCUSSION Microorganisms of the genera Corynebacterium and Propionibacterium have been recognized to have a wide range of effects on the host's defense mechanisms, including phagocytosis by the reticuloendothelial system as well as both humoral and cellular immunity. C.
parvum was originally observed to have a striking effect on the lymphoreticular system (8). Hepatosplenomegaly in animals inoculated with suspensions of these organisms was associated with proliferation of both lymphocytes and macrophages and with an enhanced capacity to clear carbon particles from the blood. More specifically, it has been demonstrated that C. parvum possesses adjuvant activity, as evidenced by an increased humoral antibody production in response to certain antigens, and could enhance host resistance in animals challenged with bacteria, protozoa, or tumor cells. Recently, it has been recognized that these properties are shared by other members of the
INFECT. IMMUN.
genus Corynebacterium. One such organism, C. acnes, not only has a stimulatory effect on the lymphoreticular system but also causes a significant alteration in interferon production in response to a representative group of inducing agents in mice. Interferon production in response to NDV, CV, and poly I:C was significantly suppressed, whereas animals produced enhanced quantities of interferon after inoculation with endotoxin (5). Because of the use of C. parvum as an immunotherapeutic agent in humans with cancer and the similarity of the effects of C. acnes and C. parvum, these studies were initiated to develop an in vitro system which would permit investigation of the mechanism of action of these organisms under more controlled conditions. The data presented clearly demonstrate that macrophages and lymphocytes harvested from animals exposed to C. acnes reflect in vitro the decreased capacity of the donor animals to produce interferon in vivo. This effect was observed when macrophages and lymphocytes were both present in culture or when the cell populations were separated and studied independently. These results further support the concept that cells of the lymphoreticular system are major sites of interferon production in response to NDV and CV in vivo. The data also demonstrate that macrophages contribute proportionately greater levels of interferon in vitro in response to the inducing agent NDV than the lymphocyte population in this system. The failure of C. acnes to inhibit interferon production by MEF provides additional evidence that this function is not affected in other cell types. Although it is not possible to extrapolate to the in vivo situation, the data do suggest that the observed alteration in the animal's capacity to produce interferon is more likely to reflect an effect on phagocytic cells of the lymphoreticular system. It is interesting to note that under in vitro conditions these cells reflected the capacity of the donor animal to produce interferon in vivo in response to the viral inducers NDV and CV, but failed to do so when a synthetic double-stranded nucleic acid (poly I:C) was utilized. It is possible that this inconsistency reflects the fact that our preparation of PEC is not representative of that cell population which is the principal source of interferon in vivo in response to poly I:C. The observation that peritoneal cells in vitro could be utilized as an experimental system made possible further studies to better define the mechanism of interferon inhibition. Initially, we documented that macrophages and lymphocytes collected from normal mice and
VOL. 11, 1975
EFFECT OF C. ACNES ON INTERFERON PRODUCTION
then exposed to a preparation of nonviable C. acnes organisms after culture in vitro also manifested a suppressed capacity to produce interferon in response to NDV. The next series of experiments were designed to try to identify the components of the Corynebacterium organisms responsible for the observed effects. Because this group of organisms is characterized by the lipid content in their cell wall, a purified cell wall preparation was developed and its effect on interferon production by PEC was determined. The results clearly indicated that a cell wall preparation contained the factors responsible for the suppression of interferon production. Further, the lipid extracted from the cell walls could interfere with interferon synthesis, as could the cell wall preparation after lipid extraction. If the fraction (or fractions) which is responsible for many of the biological effects of this group of organisms can be separated, identified, and characterized, more specific studies concerning the mechanism of action of Corynebacterium should become possible. Furthermore, it could possibly permit the utilization of the active component of C. acnes or C. parvum in stimulating or inhibiting the host response to microbial infections as well as to malignant cells. ACKNOWLEDGMENT This work was supported by Public Health Service grant AI 10217 from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Adlam, C., and M. T. Scott. 1973. Lympho-reticular stimulatory properties of Corynebacterium parvum and related bacteria. Med. Microbiol. 6:261-274. 2. Barksdale, L. 1970. Corynebacterium diphtheriae and its relatives. Bacteriol. Rev. 34:378-422. 3. Cummins, C. S., and J. L. Johnson. 1974. Corynebacterium parvum: a synonym for Propionibacterium acnes? J. Gen. Microbiol. 80:433-442. 4. Douglas, H. C., and S. E. Gunter. 1946. The taxonomic position of Corynebacterium acnes. J. Bacteriol. 52:15.
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5. Farber, P. A., and L. A. Glasgow. 1972. Effect of Corynebacterium acnes on interferon production in mice. Infect. Immunity 6:272-276. 6. Fisher, J. C., W. R. Grace, and J. A. Mannick. 1970. The effect of nonspecific immune stimulation with Corynebacterium parvum on patterns of tumour growth. Cancer 26:1379-1382. 7. Halpern, B. N., G. Biozzi, C. Stiffel, and D. Mouton. 1966. Inhibition of tumour growth by administration of killed Corynebacterium parvum. Nature (London) 212:853-854. 8. Halpern, B. N., A. R. Prevot, G. Biozzi, C. Stiffel, D. Mouton, J. C. Monard, Y. Bouthillier, and C. De Creusefond. 1964. Stimulation de lactivite phagocytaire du systeme reticuloendothelial provoquee par Corynebacterium parvum. J. Reticuloendothel. Soc. 1:77-96. 9. Howard, J. G., G. Biozzi, C. Stiffel, D. Mouton, and P. Liacopaulos. 1967. An analysis of the inhibitory effect of Corynebacterium parvum on graft-versus-host disease. Transplantation 5:1510-1524. 10. Johnson, J. L., and C. S. Cummins. 1972. Cell wall composition and deoxyribonucleic acid similarities among the anaerobic coryneforms, classical proprionibacteria and strains of Arachnia propionica. J. Bacteriol. 109:1047-1066. 11. Murphy, B. R., and L. A. Glasgow. 1968. Factors modifying host resistance to viral infections. III. Effect of whole-body x-irradiation on experimental encephalomyocarditis virus infection in mice. J. Exp. Med. 127:1035-1052. 12. Neveu, T., A. Branellec, and G. Biozzi. 1964. Proprietes adjuvantes de Corynebacterium parvum et sur l'induction de l'hypersensibilite retardee envers les proteines conjugees. Ann. Inst. Pasteur (Paris) 106:771-777. 13. O'Neill, G. J., D. C. Henderson, and R. G. White. 1973. The role of anaerobic coryneforms on specific and non-specific immunological reactions. I. Effect on particle clearance and humoral and cell-mediated immunological responses. Immunology 24:977-995. 14. Prevot, A. R., and J. Tran Van Phi. 1964. ltude comparative de la stimulation du systeme reticuloendothelial par differentes souches de corynebacteries anaerobies et d'espbces voisines. C.R. Acad. Sci. 258:4619. 15. Scott, M. T. 1972. Biological effects of the adjuvant Corynebacterium parvum. I. Inhibition of PHA, mixed lymphocyte and GVH reactivity. Cell Immunol. 5:459-468. 16. Smith, L. H., and M. F. A. Woodruff. 1968. Comparative effect of two strains of Corynebacterium parvum on phagocytic activity and tumour growth. Nature (London) 219:197-198.
INFECTION AND IMMUNITY, Jan. 1975, p. 80-85 Copyright © 1975 American Society for Microbiology
Effect of Corynebacterium acnes on Interferon Production in Mouse Peritoneal Exudate Cells J. FISCHBACH AND L. A. GLASGOW Departments of Pediatrics and Microbiology, University of Utah College of Medicine, Salt Lake City, Utah 84132
Received for publication 19 July 1974
Corynebacterium acnes, an organism closely related to C. parvum, has been recognized to have a striking effect on the reticuloendothelial system, as well as on both humoral and cellular immunity. In mice previously exposed to C. acnes, serum interferon levels induced by injection of Newcastle disease virus (NDV), Chikungunya virus (CV), and polyinosinic-polycytidylic acid are suppressed. When peritoneal macrophages and lymphocytes from animals exposed to C. acnes were cultivated in vitro, their capacity to produce interferon in r6sponse to NDV and CV was reduced. Furthermore, the interferon-producing capacity of these cells in tissue culture was inhibited after exposure to C. acnes in vitro. Exposure of separated populations of peritoneal macrophages and lymphocytes to C. acnes in vitro demonstrated that the interferon response to NDV by both cell types is inhibited. Peritoneal macrophages appear to be the major contributor to the interferon response in this system. Finally, this inhibitory effect was shown to occur after exposure to a purified cell wall preparation of C. acnes organisms, as well as a lipid extract of this preparation. showed that mice exposed to C. acnes formed significantly reduced levels of interferon in response to Newcastle disease virus (NDV), Chikungunya virus (CV) and the synthetic polynucleotide polyinosinic-polycytidylic acid (poly I:C), whereas serum interferon levels induced by endotoxin were enhanced. The purpose of this study was to determine: (i) whether this effect on interferon production was reflected in peritoneal exudate cells (PEC) obtained from mice exposed to C. acnes; (ii) which cell population, macrophages or lymphocytes, was affected by C. acnes; (iii) whether this effect could be produced in PEC taken from normal mice and exposed to C. acnes in vitro; and (iv) what components of the organism were responsible for this effect.
Anaerobic coryneforms, such as Corynebacterium parvum, have been shown to exert a stimulatory effect on the reticuloendothelial system in experimental animals, which is characterized by hypertrophy of lymphoid tissue, increased phagocytic activity as measured by carbon clearance, and antitumor activity (1, 6-8, 12-14, 16). Furthermore, C. parvum, which is currently being utilized as an immunotherapeutic agent in certain human malignancies, has been recognized to enhance the humoral immune response to certain antigens and yet to suppress cellular functions such as the mixed lymphocyte and graft-versus-host reactions as well as the phytohemagglutinin response (9, 15). C. acnes, a closely related bacterium, is the most common member of the normal microbial flora of our skin. Although it has been suggested that the microorganism be reclassified as Propionibacterium acnes (2, 4, 10), Cummins and Johnson (3) have been unable to distinguish P. acnes (C. acnes) from C. paruum on the basis of cell wall analysis, serological tests, and fermentation tests. Their data suggest that these strains are identical, or very closely related. Farber and Smith (Bacteriol. Proc., p. 83, 1969) demonstrated that C. acnes and C. parvum are capable of producing similar effects on the reticuloendothelial system in experimental animals. Subsequently, Farber and Glasgow (5)
MATERIALS AND METHODS
C. acnes, from the American Type Culture Collection (11828), was grown in dextrose-thioglycolate broth containing 2% peptone (Difco), 0.5% yeast extract (Difco), 0.5% dextrose, and 0.1% sodium thioglycolate. After 5 to 7 days of growth at 37 C, the organisms were killed by heating at 60 C for 1 h; the cells were collected by centrifugation and washed twice in phosphate-buffered saline (PBS). An inoculum of approximately 109 colony-forming units (CFU) was used throughout these experiments. Cell wall preparation. The wet weight of the organisms was determined and a 50% slurry was 80
VOL. 11, 1975
EFFECT OF C. ACNES ON INTERFERON PRODUCTION
made. The organisms were transferred to a Braun flask containing 0.1-mm glass beads three times the weight of the bacteria. The bacteria were shaken at 400 cycles/min for five 1-min intervals with CO2 cooling. The contents of the flask were then centrifuged at 350 x g for 10 min at 4 C to remove the glass beads and intact cells; the supernatant fluid containing the cell wall material was centrifuged at 10,000 x g for 10 min at 4 C. This pellet was washed 12 times with PBS and resuspended in PBS at the original volume of the C. acnes suspension. Medium. All cell cultures were maintained in Eagle minimal essential medium (MEM) with 10% fetal calf serum (FCS). Interferon inducers. NDV, Herts strain, grown in the allantoic cavity of embryonated hen's eggs, titered approximately 3 x 109 plaque-forming units (PFU)/ml in primary chick embryo fibroblasts. CV, originally obtained from the Walter Reed Medical Center Arbovirus Unit, was prepared from the brains of infected suckling mice and titered approximately 107 PFU/ml when assayed on primary chick 5.0 embryo fibroblasts. An inoculum of 0.1 ml of undiluted stock virus (NDV or CV) was used to induce interferon. Poly I:C was obtained from P-L Biochemicals, Milwaukee, Wis. A dose of 100 ,g was used in these experiments. Endotoxin, Escherichia coli 0111:B4 lipopolysaccharide prepared by the Westphal method (Difco Laboratories), was used at dosages of 100 gg. Mice. Female CD-1 mice obtained from Charles River Breeding Laboratories (Brookline, Mass.) were housed six/cage and received food and water ad libitum. PEC. PEC were collected aseptically from the peritoneal cavities of normal mice which had received intraperitoneal (ip) injections of approximately 109 CFU of heat-killed C. acnes 7 to 14 days prior to collection by washing with cold MEM without FCS. Three days prior to collection, all mice were injected ip with 2.0 ml of 1% peptone. The peritoneal washings were centrifuged at 350 x g and resuspended in MEM with 10% FCS at a concentration of 106 cells/ml. The cells were then distributed in 2.0-ml portions into 35-mm plastic tissue culture dishes (Falcon Plastics) and challenged with NDV, CV, poly I:C, endotoxin, or C. acnes. A preparation of normal control PEC processed simultaneously in an identical manner served as the control. After 18 h at 37 C, the medium was collected from each plate and stored at -20 C until assayed. In experiments in which the effect of C. acnes on PEC from normal mice was determined, a cellular response was stimulated with peptone, and cells were collected and prepared in the manner just described. Prior to challenge with an interferon inducer, plates containing normal PEC were inoculated with heatkilled whole C. acnes or the cell wall preparation and incubated at 37 C for 48 h. At this time, cell preparations exposed to C. acnes and untreated controls were challenged with NDV. Medium from each plate was collected after 18 h to be assayed for interferon. Cell viability was determined by the trypan blue dye exclusion method on all PEC cultures. x
81
Separation of macrophages and lymphocytes. Cells were separated into macrophage and lymphocyte populations by placing a suspension of normal PEC, at a concentration of 106 cells/ml, into tissue culture dishes and incubating them for 3 h at 37 C. After incubation, the medium from each plate was removed, pooled, and centrifuged at 350 x g to collect the nonadherent cell population. The nonadherent cells (predominantly lymphocytes) were resuspended in MEM with 10% FCS at a concentration equivalent to the adherent cell population. The plates containing the adherent cells (predominantly macrophages) were refed with fresh MEM with 10% FCS. Prior to challenge with an interferon inducer, plates from each group were inoculated with heat-killed whole C. acnes and incubated at 37 C for 48 h. At this time, cell preparations exposed to C. acnes and untreated controls were challenged with NDV. Medium from each plate was collected after 18 h to be assayed for interferon. Interferon assay. Virus-induced samples to be assayed for interferon were adjusted to pH 2.0 with concentrated HCl. After 2 days (CV) or 5 days (NDV) at 4 C, samples were returned to pH 7.0 with NaOH. Assays were performed on L-cell monolayers with approximately 30 to 50 PFU of vesicular stomatitis virus (VSV; Indiana strain) as the challenge virus (11). The concentration of interferon was defined as the reciprocal of the dilution which produced a 50% plaque inhibition of the control VSV inoculum. A laboratory interferon standard was included in each assay to serve as a guide to the sensitivity of our assay system; results were not adjusted by comparison with this standard interferon preparation. Periodically, the sensitivity of our assay was compared with the international mouse interferon reference preparation from the National Institutes of Health. In our system, the international standard had a titer of approximately 300 units/ml. Lipid extraction. A suspension of heat-inactivated C. acnes at a concentration of 109 CFU/ml was washed two times in PBS, resuspended in twice the original volume of chloroform-methanol (2:1, vol/vol), and mixed well. This mixture was allowed to stand at room temperature for 30 min and then was centrifuged at 350 x g for 10 min. The supernatant-crude extract was removed and mixed thoroughly with a 0.73% NaCl solution (5:1, vol/vol). The two phases were separated by centrifugation at 1,000 x g for 15 min. The upper, aqueous phase (predominantly nonlipid material) was removed, the interface was rinsed three times with small amounts of chloroformmethanol-water (3:48:47, vol/vol) so as not to disturb the lower phase, and methanol was added dropwise until one phase was obtained. The solvents were then removed by evaporation and the lipid material was resuspended in PBS. The original C. acnes suspension was extracted four times and the extracts were combined. RESULTS
To determine the effect of C. acnes on the capacity of PEC to produce interferon, we
FISCHBACH AND GLASGOW
82
collected PEC from mice 14 days after they had received a single ip injection of approximately 109 CFU of nonviable C. acnes. The cell preparation was then incubated in vitro and exposed to a representative interferon-inducing agent. The collection time was selected to coincide with the period of suppression of the interferon response by C. acnes in the whole animal. The culture medium from PEC obtained from C. acnes-exposed and control animals was collected 18 h after addition of the inducing agent and assayed for the presence of interferon. The results of four experiments are summarized in Fig. 1. Prior exposure to C. acnes resulted in suppression of the interferon response of PEC to both NDV and CV; levels of interferon produced in response to poly I:C were variable. No detectable interferon was produced in response to endotoxin by PEC from C. acnes-exposed or normal mice. These data indicate that the altered capacity of mice exposed to C. acnes to produce interferon in response to NDV and CV in vivo is reflected in the decreased capability of a mixed population of macrophages and lymphocytes from these animals to respond to these same viral inducing agents in vitro. Although our previous data showed that exposure to C.
INFECT. IMMUN.
acnes resulted in an enhanced interferon response to endotoxin in the intact animal (5), this observation could not be confirmed since detectable levels of interferon were not induced by endotoxin under these in vitro conditions. To determine whether the suppression of the interferon response would also occur in normal PEC exposed to C. acnes in cultures, we collected PEC harvested from normal mice and exposed them to preparations of heat-killed C. acnes. These cultures were then incubated for 48 h before being challenged with NDV. The results from three experiments are illustrated in Fig. 2. NDV-induced interferon produced by normal PEC exposed in vitro to C. acnes was inhibited to the same degree as levels of interferon produced by PEC harvested from mice inoculated with C. acnes. These data suggest that the direct exposure of a mixed population of macrophages and lymphocytes to C. acnes results in a decreased capacity to produce interferon in response to at least one representative viral inducer. W
120-
0 _
CONTROL
C. Acnes WHOLE ORGANISM C. Acnes CELL WALL
1000
z
O 80u
0
,,; 60w -i
z
0 cru
40-
.z
20-
v
INDUCER:
Newcastle Disease
Chikungunya Virus
Poly I:C
Endotoxin
virus
FIG. 1. Suppression of mean interferon levels (expressed as a percentage of the control) produced by peritoneal macrophages and lymphocytes from control and C. acnes-exposed mice after induction with Newcastle disease virus, Chikungunya virus, poly I:C, and endotoxin.
INDUCER: NEWCASTLE DISEASE VIRUS FIG. 2. Suppression of mean interferon levels (expressed as a percentage of the control) produced by peritoneal macrophages and lymphocytes from normal mice exposed in vitro to a nonviable C. acnes preparation or a cell wall fraction prepared from C. acnes prior to induction with Newcastle disease virus.
Control peritoneal exudate cells received no C. acnes exposure.
VOL. 11, 1975
EFFECT OF C. ACNES ON INTERFERON PRODUCTION
To examine which specific cell types were being altered in their ability to produce interferon, we separated PEC from normal mice into either predominantly macrophage or lymphocyte populations on the basis of their adherence properties. These cells, in equivalent concentrations, were then exposed to heat-killed C. acnes in vitro and incubated for 48 h before being challenged with NDV. The culture medium was collected at 18 h and assayed for the presence of interferon. As shown in Fig. 3. the interferon response of both the macrophage and, to a lesser degree, the lymphocyte populations was suppressed by prior exposure to C. acnes. The greater levels of interferon produced by the macrophage population (3,000 units/ml compared with 100 units/ml) indicate that the macrophage is the major contributor to the interferon response in this system. The effect of C. acnes on mouse cells derived from a source other than the reticuloendothelial system was determined by exposing monolayers of mouse embryo fibroblasts (MEF) to C. acnes in the same manner as PEC and challenging them with NDV. The culture medium was collected at 18 h and assayed for the presence of interferon. MEF which had been previously exposed to C. acnes produced approximately 8,500 units of interferon/ml and control MEF produced approximately 8,000 units/ml. There appears to be no inhibitory effect on interferon production by this cell of nonreticuloendothelial origin. No toxicity was observed in MEF which had been exposed to C. acnes. The results of these experiments made it possible to explore the components of the C. acnes organism which were responsible for the observed effects on interferon synthesis. It was assumed that neither multiplication nor metabolic activity of C. acnes was responsible for the interferon inhibition since a nonviable, heatkilled preparation of C. acnes was as effective as viable organisms in these experiments. To determine whether a component of C. acnes was responsible for this effect, we first prepared a purified cell wall fraction. Normal PEC were incubated with this cell wall preparation in the same manner as in previous experiments in which the whole organism was used. After challenge with NDV as the interferon-inducing agent, culture medium was collected and assayed for interferon. As can be seen in Fig. 2, the cell wall preparation from C. acnes suppressed the interferon response to the same degree as the whole organism. Because the Corynebacterium and Propionibacterium groups of microorganisms are characterized by the lipids present in their cell wall, we next examined the ability
83
= CONTROL E C Acnes
cr-
z
MACROPHAGE
(Gloss- adherent)
LYMPHOCYTE (Not Gloss-odherent)
FIG. 3. Interferon levels produced by control and C. acnes-exposed peritoneal exudate cells separated into macrophage and lymphocyte populations, after induction by Newcastle disease virus.
of a lipid extract and the cell wall preparation from which it was removed to suppress the capacity of cells to produce interferon. The activity of these two preparations was determined in the same manner used with the whole organism and the cell wall. Again, cultures of PEC exposed to nonviable whole organisms and similar cultures which were not exposed to C. acnes served as controls. The ability to suppress the production of interferon was present in the lipid component removed from the cell wall of C. acnes, but the cell wall preparation from which the lipid was extracted also retained the ability to suppress interferon production to the same degree (Fig. 4). These data support the hypothesis that a component of the cell wall is responsible for the observed inhibition of interferon production. In spite of the fact that the lipid extract inhibited interferon synthesis, it is not clear whether that property resides solely in the lipid component of the cell wall. Although the cell wall preparation from which the lipid was extracted also suppressed interferon production, it is possible that some lipid remained bound to the cell wall preparation and that the observed effect was due to residual amounts of lipid material in the cell wall which were not removed by the extraction procedure.
84
FISCHBACH AND GLASGOW W CONTROL CELL WALL LIPID COM PON ENT CELL WALL WITH M LIPID COMPONENT
120-~~~~~~~::
120-
REMOVED
WHOLE ORGANISM 100 0
°) 800 -
En
, 60uJ z 0
uw
40-
20L
INDUCER: NEWCASTLE DISEASE VIRUS FIG. 4. Suppression of mean interferon levels (expressed as a percentage of the control) in vitro in response to Newcastle disease virus produced by normal peritoneal exudate cells exposed to a lipid component of C. acnes cell wall, the cell wall with a lipid component removed, and the whole organism, in comparison with control peritoneal exudate cells.
DISCUSSION Microorganisms of the genera Corynebacterium and Propionibacterium have been recognized to have a wide range of effects on the host's defense mechanisms, including phagocytosis by the reticuloendothelial system as well as both humoral and cellular immunity. C.
parvum was originally observed to have a striking effect on the lymphoreticular system (8). Hepatosplenomegaly in animals inoculated with suspensions of these organisms was associated with proliferation of both lymphocytes and macrophages and with an enhanced capacity to clear carbon particles from the blood. More specifically, it has been demonstrated that C. parvum possesses adjuvant activity, as evidenced by an increased humoral antibody production in response to certain antigens, and could enhance host resistance in animals challenged with bacteria, protozoa, or tumor cells. Recently, it has been recognized that these properties are shared by other members of the
INFECT. IMMUN.
genus Corynebacterium. One such organism, C. acnes, not only has a stimulatory effect on the lymphoreticular system but also causes a significant alteration in interferon production in response to a representative group of inducing agents in mice. Interferon production in response to NDV, CV, and poly I:C was significantly suppressed, whereas animals produced enhanced quantities of interferon after inoculation with endotoxin (5). Because of the use of C. parvum as an immunotherapeutic agent in humans with cancer and the similarity of the effects of C. acnes and C. parvum, these studies were initiated to develop an in vitro system which would permit investigation of the mechanism of action of these organisms under more controlled conditions. The data presented clearly demonstrate that macrophages and lymphocytes harvested from animals exposed to C. acnes reflect in vitro the decreased capacity of the donor animals to produce interferon in vivo. This effect was observed when macrophages and lymphocytes were both present in culture or when the cell populations were separated and studied independently. These results further support the concept that cells of the lymphoreticular system are major sites of interferon production in response to NDV and CV in vivo. The data also demonstrate that macrophages contribute proportionately greater levels of interferon in vitro in response to the inducing agent NDV than the lymphocyte population in this system. The failure of C. acnes to inhibit interferon production by MEF provides additional evidence that this function is not affected in other cell types. Although it is not possible to extrapolate to the in vivo situation, the data do suggest that the observed alteration in the animal's capacity to produce interferon is more likely to reflect an effect on phagocytic cells of the lymphoreticular system. It is interesting to note that under in vitro conditions these cells reflected the capacity of the donor animal to produce interferon in vivo in response to the viral inducers NDV and CV, but failed to do so when a synthetic double-stranded nucleic acid (poly I:C) was utilized. It is possible that this inconsistency reflects the fact that our preparation of PEC is not representative of that cell population which is the principal source of interferon in vivo in response to poly I:C. The observation that peritoneal cells in vitro could be utilized as an experimental system made possible further studies to better define the mechanism of interferon inhibition. Initially, we documented that macrophages and lymphocytes collected from normal mice and
VOL. 11, 1975
EFFECT OF C. ACNES ON INTERFERON PRODUCTION
then exposed to a preparation of nonviable C. acnes organisms after culture in vitro also manifested a suppressed capacity to produce interferon in response to NDV. The next series of experiments were designed to try to identify the components of the Corynebacterium organisms responsible for the observed effects. Because this group of organisms is characterized by the lipid content in their cell wall, a purified cell wall preparation was developed and its effect on interferon production by PEC was determined. The results clearly indicated that a cell wall preparation contained the factors responsible for the suppression of interferon production. Further, the lipid extracted from the cell walls could interfere with interferon synthesis, as could the cell wall preparation after lipid extraction. If the fraction (or fractions) which is responsible for many of the biological effects of this group of organisms can be separated, identified, and characterized, more specific studies concerning the mechanism of action of Corynebacterium should become possible. Furthermore, it could possibly permit the utilization of the active component of C. acnes or C. parvum in stimulating or inhibiting the host response to microbial infections as well as to malignant cells. ACKNOWLEDGMENT This work was supported by Public Health Service grant AI 10217 from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Adlam, C., and M. T. Scott. 1973. Lympho-reticular stimulatory properties of Corynebacterium parvum and related bacteria. Med. Microbiol. 6:261-274. 2. Barksdale, L. 1970. Corynebacterium diphtheriae and its relatives. Bacteriol. Rev. 34:378-422. 3. Cummins, C. S., and J. L. Johnson. 1974. Corynebacterium parvum: a synonym for Propionibacterium acnes? J. Gen. Microbiol. 80:433-442. 4. Douglas, H. C., and S. E. Gunter. 1946. The taxonomic position of Corynebacterium acnes. J. Bacteriol. 52:15.
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5. Farber, P. A., and L. A. Glasgow. 1972. Effect of Corynebacterium acnes on interferon production in mice. Infect. Immunity 6:272-276. 6. Fisher, J. C., W. R. Grace, and J. A. Mannick. 1970. The effect of nonspecific immune stimulation with Corynebacterium parvum on patterns of tumour growth. Cancer 26:1379-1382. 7. Halpern, B. N., G. Biozzi, C. Stiffel, and D. Mouton. 1966. Inhibition of tumour growth by administration of killed Corynebacterium parvum. Nature (London) 212:853-854. 8. Halpern, B. N., A. R. Prevot, G. Biozzi, C. Stiffel, D. Mouton, J. C. Monard, Y. Bouthillier, and C. De Creusefond. 1964. Stimulation de lactivite phagocytaire du systeme reticuloendothelial provoquee par Corynebacterium parvum. J. Reticuloendothel. Soc. 1:77-96. 9. Howard, J. G., G. Biozzi, C. Stiffel, D. Mouton, and P. Liacopaulos. 1967. An analysis of the inhibitory effect of Corynebacterium parvum on graft-versus-host disease. Transplantation 5:1510-1524. 10. Johnson, J. L., and C. S. Cummins. 1972. Cell wall composition and deoxyribonucleic acid similarities among the anaerobic coryneforms, classical proprionibacteria and strains of Arachnia propionica. J. Bacteriol. 109:1047-1066. 11. Murphy, B. R., and L. A. Glasgow. 1968. Factors modifying host resistance to viral infections. III. Effect of whole-body x-irradiation on experimental encephalomyocarditis virus infection in mice. J. Exp. Med. 127:1035-1052. 12. Neveu, T., A. Branellec, and G. Biozzi. 1964. Proprietes adjuvantes de Corynebacterium parvum et sur l'induction de l'hypersensibilite retardee envers les proteines conjugees. Ann. Inst. Pasteur (Paris) 106:771-777. 13. O'Neill, G. J., D. C. Henderson, and R. G. White. 1973. The role of anaerobic coryneforms on specific and non-specific immunological reactions. I. Effect on particle clearance and humoral and cell-mediated immunological responses. Immunology 24:977-995. 14. Prevot, A. R., and J. Tran Van Phi. 1964. ltude comparative de la stimulation du systeme reticuloendothelial par differentes souches de corynebacteries anaerobies et d'espbces voisines. C.R. Acad. Sci. 258:4619. 15. Scott, M. T. 1972. Biological effects of the adjuvant Corynebacterium parvum. I. Inhibition of PHA, mixed lymphocyte and GVH reactivity. Cell Immunol. 5:459-468. 16. Smith, L. H., and M. F. A. Woodruff. 1968. Comparative effect of two strains of Corynebacterium parvum on phagocytic activity and tumour growth. Nature (London) 219:197-198.
