Effects of Stimulated or Immunologically Activated Macrophages on the Induction of Immune Responses to Listeria monocytogenes

lmmunobiol., vol. 180, pp. 124-137 (1990)

Department of Immunology, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan

Effects of Stimulated or Immunologically Activated Macrophages on the Induction of Immune Responses to Listeria monocytogenes YASUNOBU YOSHIKAI, SHOICHI OHGA, YASUYUKI TAKEDA,

and KIKUO NOMOTO Received May 10, 1989 . Accepted in Revised Form September 15, 1989

Abstract The influences of peritoneal macrophages induced by proteose peptone, Corynebacterium parvum (c. parvum) or Bacillus Calmette Guerin (BCG) on the initiation and development of immune responses and protection against Listeria monocytogenes infection were studied in mice. Mice treated intraperitoneally (i.p.) with proteose peptone 4 days previously showed much the same level of protection against an intraperitoneal infection with Listeria as untreated mice. Mice treated i.p. with C. parvum 4 days previously, of which peritoneal macrophages had increased abilities for intracellular killing of Listeria and O 2 - generation as compared with peptone-elicited macrophages, exhibited an enhanced resistance against the listerial infection. The degree of immune respon'ses, as assessed by delayed footpad reaction (DFR), was rather depressed in these mice because C. parvum-activated macrophages acting as scavenger cells reduced the amount of effective antigenic stimulation. BCG-activated peritoneal macrophages from mice treated i.p. with BCG 14 days previously showed a strong ability for antigen presentation in correlation with increases in the number of la-bearing macrophages and in the level of interleukin 1 (IL 1) production. These mice showed an early appearance of DFR response and a markedly enhanced resistance against the listerial infection. These results suggested that the differences in macrophage activities as scavenger cells, cytokine-secreting cells and antigen presenting cells may account for the differences in the responsiveness against listerial infection in peptone-, C. parvum- and BCG-treated mice.

Introduction

It is a well known fact that the macrophages play important roles in the initiation and development of immune responses against Listeria monocytogenes infection (1, 2). la-negative macrophages, acting as scavenger phagocytes, regulate the subsequent immune responses by altering the amount of listerial antigens (3, 4), while la-bearing macrophages, acting as antigen presenting cells (APC), are essential for cellular immunity induced by listerial infection (5). Macrophages also play crucial roles in the inducAbbreviations: C. parvum = Corynebacterium parvum; BCG = Bacillus Calmette Guerin; i.p. = intraperitoneally; DFR = delayed footpad reaction; APC = antigen presenting cells; PEC = peritoneal exudate cells.

Induced Macrophages and Listerial Infection . 125

tion of immune responses by secreting a considerable number of soluble products such as interleukin 1 (IL 1) and tumor necrosis factor (TNF) (6, 7), all having different effects on lymphocyte functions. Elicitation of peritoneal macrophages with sterile inflammatory agents or infections with various intracellular pathogens yield macrophages which are very distinct in their biochemical and functional properties from resident peritoneal macrophages (8-10). Both proteose peptone- or Corynebacterium parvum-elicited macrophages show an increase in size, content of intracellular granules, spreading, phagocytosis, oxidation of glucose through the hexose-monophosphate shunt (11). On the other hand, activation of macrophages by BCG is considered to be augmented by macrophage activating factors (MAF)linterferon-y liberated from T lymphocytes (12). In addition to possessing the characteristics of stimulated macrophages, the immunologically activated macrophages comprise a large number of la-bearing macrophages (13, 14). In this paper, we have investigated various properties of peritoneal exudate macrophages elicited with proteose peptone, C. parvum or BCG and compared the initiation and development of immune responses and protection against intraperitoneallisterial infection among mice pretreated intraperitoneally (i.p.) with peptone, C. parvum or BCG. Our data suggest that differences in macrophage activities as scavenger cells, APC and cytokine-secreting cells may be mainly responsible for the differences in responsiveness against listerial infection in peptone-, C. parvum- or BCGtreated mice.

Materials and Methods Animals

Male mice of AKR strain were obtained from the Breeding Unit of Kyushu University. Tenweek-old mice were used for the experiments. Microorganisms

Pasteur strain of BCG and L. monocytogenes (EGD) were prepared as described previously (15). A suspension of heat-killed and formalin-preserved Corynebacterium parvum (Propionibacterium avidum) was supplied by the Institute Merieux, Lyon, France. The 50 % lethal dose (LDso) was determined by using the method of REED and MUENCH (16). Mice were injected intraperitoneally (i.p.) with 1 ml of 10 % proteose peptone or 0.25 ml of C. parvum suspension (equivalent to 0.5 mg dry weight of organism) 4 days prior to the experiments or with 1 mg of wet weight of BCG (1 x 107 CFU/mg wet weight) 14 days previously. Preparation of peritoneal cells

Peritoneal exudate cells (PEC) were recovered by lavage of the peritoneal cavity with Hanks' balanced salt solution (HBSS) containing 10 units heparin/m!. The cells were collected by centrifugation at 110 x g for 10 min, washed once with HBSS, and counted in a hemocytometer. PEC suspended in HBSS were purified as adherent cells by removal of plasticnonadherent cells after 2 h of incubation at 37°C. Ia antigens on cells adherent to glass covers lips were examined as described previously (17). Briefly, 1 x 106 cells in medium were

126 . YASUNOBU YOSHIKAI et a!. incubated with 5 Itg/ml of anti-IAk antibody (Becton Dickinson Co., Sunnyvale, CA, U.S.A.) for 30 min at O°C. The cells were washed twice with medium and exposed to an optimal concentration of fluorescence-conjugated immunoglobulin F(ab)' anti-mouse IgG for an additional 30 min. Measurement of O2 - release from macrophages

Superoxide anion (0 2-) released from· peritoneal macrophages was measured by the ferricytochrome C reduction method, using phorbol myristate acetate (PMA) as a trigger. Cells were suspended in Krebs-Ringer phosphate solution with glucose (137 mM NaCl, 4.9 mM KCI, 1.2 mM MgS04, 0.5 mM CaClz, 5.7 mM Na2P04, 5.5 mM glucose, pH 7.35) at a concentration of 1 x 106 cells per m!. Cells were held in a photometric cuvette and preincubated at 3rc for 5 min after addition of 80 ItM ferricytochrome C (type VI; Sigma Chemical Co., St. Louis, MO, U.S.A.) and then added with 100 ng of PMA (Sigma) per ml dissolved in dimethyl sulfoxide (Aldrich Chemical Co., Milwaukee, WI, U.S.A.). The final concentration of dimethyl sulfoxide was 0.033 %. The cuvette was placed in a thermostated cuvette holder at 37°C, and cytochrome C reduction was measured with a two-wave-Iength spectrophotometer (Hitachi 200-20) at 540 to 550 nM. The amount of O 2- released after triggering with PMA was calculated from the linear reduction of ferricytochrome C (change in optical density per minute) by taking the molar extinction coefficient as 19.1 x 103/M. Results were expressed as nanomoles of O 2- per minute (18). In vitro intracellular killing of Listeria In vitro intracellular killing of Listeria by peritoneal cells was determined according to the methods of VAN FURTH and VAN ZWET (19). Briefly, a I-ml suspension of Listeria (1 x 106 ) in HBSS containing 0.1 % (w/v) gelatin and 10 % (v/v) FCS was injected i.p. into mice treated with peptone on day -4, mice treated with C. parvum on day -4 or mice treated with BeG on day -14. Mice were sacrificed exactly 3 min later by cervical dislocation, and 1 min later,S ml PBS with heparin were injected for collection of peritoneal leukocytes. Cell suspensions from five mice were pooled in a polypropylene tube (no. 2063, Falcon, Oxnard, CA, U.S.A.). To remove the extracellular bacteria, the suspensions were centrifuged at 110 x g for 5 min and washed three times with HBSS. Cells containing ingested bacteria were then resuspended in gelatin-HBSS at a concentration of 2 x 106 leukocytes/ml. After 0 and 60 min of reincubation, the cells were lysed by adding 1.0 ml distilled water and by freezing and thawing. The number of viable bacteria was then determined by culture on nutrient agar. The rate of intracellular killing was determined by the following formula: % intracellular killing = 100 x [(no. of bacteria after 0 min of incubation) - (no. of bacteria after 60 min of incubation)J/(no. of bacteria after 0 min of incubation). IL 1 assay

The peritoneal exudates were recovered by lavage of the peritoneal cavity with 5 ml phosphate-buffered saline (PBS). The supernatants of the peritoneal exudates were collected by centrifugation at 110 x g for 10 min, and the IL 1 activities of the diluted samples of the supernatants were tested on thymocytes from C3H/HeJ mice (The Jackson Laboratory, Bar Harbor, ME, U.S.A.). Thymocytes (1 x 106 ) in RPMI 1640 medium (200 !!1/well) supplemented with 1 % L-glutamine, 1 x 10-5 M 2-mercaptoethanol (2-ME) and 5 % fetal calf serum (FCS) were incubated in flat-bottomed microtiter plates for 72 h in the presence of phytohemagglutinin (PHA) 0.5 ltg/well. Sixteen h before harvest, 0.5 ItCi of eH)-thymidine (5.0 Ci/mmol) was added per well (20). For assay of mRNA encoding IL la, total cellular RNA were extracted from the peritoneal exudate adherent cells by the guanidine thiocyanate and CsCI gradient centrifuge procedure (21). Total RNA (30 Itg) was electrophoresed on 1 % agarose in 10 mM sodium-phosphate buffer at pH 7.0 and transferred to Gene Screen Plus (NEN, Boston, MA) and hybridized to 32P-Iabeled probe of IL la cDNA in 1 % SDS, 1 M NaCI and 50 % dextran sulfate (22). After hybridization at 65°C for 24 h, the filter was washed in 3 x sse, 1 % SDS at 65°C and exposed

Induced Macrophages and Listerial Infection' 127 to X-ray films at -70 DC in the presence of intensifying screens. To confirm that equal amounts of RNA were blotted to the screen, murine ~-actin genomic DNA probe was hybridized to the same filter after washing 3 times with boiling water. Antigen presenting activity of peritoneal macrophages Listeria-immune T cells were prepared according to the method of FARR and UNANUE with minor modifications (23). Mice were inoculated i.p. with 1 x 103 viable Listeria, and 7 days after inoculation, the mice were injected i.p. with 10 % proteose peptone. Listeria-immune T cells were purified from PEC obtained from the mice by double passages over nylon wool column. A variety of numbers of macrophages in 0.2 ml RPMI medium supplemented with 10 % FCS, 100 U penicillin, 100 [,Ig streptomycin, and 5 x 10-5 2-ME were incubated in microtiter plates (3040 Micro test II, Falcon) for 2 h at 37 DC, then nonadherent cells were removed by vigorous washing with medium. Heat-killed Listeria (1 x 107 ) and Listeriaimmune T cells (2 x 105) were added to the culture of macrophage monolayers in a total volume of 0.2 ml. The cultures were incubated for 4 days at 37 DC in a humidified atmosphere of 5 % CO 2 and air. A 20 [,II amount of medium containing 10 [,ICi/ml eH)-thymidine (Nippon Isotope Co.) was added to each well 18 h before harvesting cells on filter papers with a Labo Mash Semi Automatic Multiple Harvester. The samples were counted in a liquid scintillation counter. Detection of bacterial growth

Mice treated with proteose peptone on day -4, mice treated with C. parvum on day -4, and mice treated with BCG on day -14 were inoculated i.p. with various doses of Listeria on day 0 and sacrificed by cutting the femoral artery at intervals after inoculation. To observe bacterial growth in the peritoneal cavity, contents were washed out with PBS. Peritoneal leukocytes were lysed by freezing and thawing, and the fluids were diluted 10-fold with PBS. One tenth ml of each dilution was spread on nutrient agar plates containing 0.4 % (w/v) glucose. Colonies were counted after incubation for 20 h at 37 D C. Bacterial growth in spleen homogenates was determined as described previously (24). Assay of footpad reaction

For elicitation of DFR, 1 x 10 7 killed bacteria in SO [,11 of PBS were injected into the left hind footpads. The right hind footpad was injected with SO [,II of PBS as control. Swelling of these footpads was measured 24 h later with a dial thickness gauge. The difference in thickness between the right and left hind footpad was expressed as the degree of reaction. Statistical analysis

The statistical significance of the data was determined by the Student's t-test. A P value of less than 0.05 was considered significant.

Results

Characterization of peritoneal exudate macrophages The number of PEe was increased to 7.3 x 106 , 6.1 X 106 , or 5.0 x 106 cells after treatment with proteose peptone 4 days previously, with C. parvum 4 days previously, or with BCG 14 days previously, respectively. Approximately 75 % of the PEe from peptone-treated mice were macrophages as judged by morphological characteristics and latex ingestion, while 60 % and 45 % were macrophages in the PEe from C. parvumtreated mice and BCG-treated mice, respectively. The percentage of Ia-

75.6 ± 2.3 59.6 ± 2.8 44.0 ± 4.1

7.3 ± 1.8 6.1 ± 1.6 5.0 ± 0.9

peptone C. parvum BCG

9.0 ± 5.1 13.0 ± 3.7 56.0 ± 7.9"

la-bearing cells per M (%) 0.97 ± 0.15 6.01 ± 0.47" 3.38 ± 0.34'"

Amount of O 2 (nmol/min per 106 M
± 9.5 ± 18.5*" ± 9.3"~c';

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4.5 x 103 7.7 x 105 1.8 x 10 7

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Listeria. Mean of five mice ± S.E. "Significantly different from value for peptone-treated mice: P < 0.001. "". P < 0.005, *"". P < 0.01.

"The peritoneal cells were collected from mice treated i.p. with 1 ml of 10 % proteose peptone on day -4, mice treated i.p. with 0.5 mg of C. parvum on day -4 and mice treated i.p. with 1 mg of BeG on day -14. b % intracellular killing = 100 x [(no. of bacteria after 0 min of incubation) - (no. of bacteria after 60 min of incubation)]/(no. of bacteria after 0 min of incubation). C LDso was determined by the mortality rate in peptone-treated, C. parvum-treated and BeG-treated mice after i.p. infection with various doses of viable

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Induced Macrophages and Listerial Infection . 129

positive macrophages increased to 55.6 % in BeG-treated mice (P < 0.001), but there were no differences in the proportion of la-positive macrophages in the PEe between peptone-treated and C. parvum-treated mice (Table 1). Scavenger activity of peritoneal exudate macrophages in vitro

To assess the scavenger activities of the peritoneal macrophages, we first examined the amount of O 2 - release by the peritoneal macrophages elicited by proteose peptone, C. parvum or BeG. Superoxide anion generation by C. parvum- or BeG-activated macrophages was significantly higher than that by peptone-induced macrophages (P < 0.001, Table 1). Next, the ability of the peritoneal exudate macrophages for intracellular killing of Listeria was assessed in vitro. The PEe were prepared after i.p. injection of 1 x 106 Listeria, washed and then incubated at 3rC in vitro for oand 60 min. When incubated at 4°C, the numbers of viable bacteria were constant during the incubation period of 60 min (data not shown). The degree of in vitro intracellular killing activity was significantly higher in PEe of C. parvum- or BeG-treated mice than that of peptone-treated mice (P
First, we examined the capabilities of the supernatants of the peritoneal exudates from peptone-, C. parvum- or BeG-treated mice to induce a proliferative response in mouse thymocytes in the presence of PHA. As shown in Figure 1, the supernatants from BeG-treated mice or from C. parvum-treated mice were clearly effective in enhancing thymocyte proliferation. On the contrary, the supernatants from peptone-treated mice showed a low level of lL 1 activities. Since the thymocytes proliferation we used here for IL 1 assay may not be an IL 1 specific read-out system, we also examined the expression of IL la mRNA in the peritoneal exudate macrophages, using Northern blot analysis. The highest level of IL 1 mRNA expression was detected in the peritoneal exudate macrophages from BeG-treated mice. C. parvumactivated macrophages manifested an appreciable level of IL la mRNA expression, whereas peptone-induced macrophages expressed a low amount of IL la mRNA. Nearly equal amounts of ~-actin messages were expressed in peptone-elicited, C. parvum- and BeG-activated macrophages (Figure 2). These results indicated that BeG-activated macrophages displayed the strongest activities to secrete IL 1, followed by C. parvum-activated macrophages. The ability to secrete IL 1 was not increased in peptone-elicited macrophages. Antigen presenting activity of peritoneal exudate macrophages

Antigen presenting ability of the peritoneal exudate macrophages was assessed by Listeria-immune T cell proliferation in vitro. The peritoneal

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Figure 1. The proliferative responses of C3H/HeJ thymocytes (1 x 106 /200 [!l) to different dilutions of supernatants of PEC induced by peptone (0), C. parvum (6) or BeG (0) in the presence of PHA (0.5 [!g/well). Results are expressed as cpm ± S.E. The bottom shaded area represents S.E. above and below the mean of control cultures (thymocytes ± PHA). " Significantly different from value for peptone-elicited macrophages; P < 0.005. "." P < 0.01.

exudate T cells from Listeria-immune mice and heat-killed Listeria were added to cultures of various numbers of peptone-elicited macrophages, C. parvum-activated macrophages, or BCG-activated macrophages. BCGactivated macrophages showed an enhanced ability to stimulate T cell proliferative responses as compared with peptone-elicited macrophages (P < 0.001), while C. parvum-activated macrophages exhibited slightly higher level of APC activity than peptone-elicited macrophages did (P < 0.01, Fig. 3).

Resistance against listeria} infection in peptone-, C. parvum- or BCGtreated mice To assess resistance against primary infection with Listeria, mice treated i.p. with peptone on day -4, mice treated i.p. with C. parvum on day -4 or mice treated i.p. with BCG on day -14 were challenged i.p. with high and moderate doses of viable bacteria. After 10 days of observation, the cumulative survival rate was calculated for each group of mice and LDso was determined by using the method of REED and MUENCH (16). LDso of Listeria inoculated i.p. increased to 7.7 x lOs in C. parvum-treated mice and

Induced Macrophages and Listeria! Infection . 131

1.8 X 107 in BeG-treated mIce from 4.5 x 103 in peptone-treated mice (Table 1).

The kinetics of bacterial growth in spleen also was investigated in peptone-, C. parvum- or BeG-treated mice (Fig. 4). The number of bacteria increased to reach the maximal level by day 5 and decreased from day 7 to day 10 after infection in peptone-treated mice when inoculated with a sublethal dose of Listeria (5 x 102). In C. parvum-treated mice and BeG-treated mice, no bacteria were detectable on day 1 after inoculation with 5 x 102 Listeria (data not shown). When challenge dose increased to 5 x 10\ the growth curve in C. parvum-treated mice showed much the same pattern as that of peptone-treated mice inoculated with 5 x 102 Listeria. On the other hand, the number of bacteria in BeG-treated mice decreased more rapidly from day 3 to day 7 than those in peptone-treated or C. parvum-treated mice (Fig. 4). Kinetics in DFR response in peptone-treated mice, C. parvum-treated mice or BeG-treated mice

The DFR was elicited on various days after inoculation with various doses of viable Listeria. When mice were challenged with a 5 x 102 viable

Figure 2. Northern blot analysis of IL 1a mRNA in peptone-, C. parvum- and BGG-induced macrophages. Thirty !-Ig of total RNA was electrophoresed, transferred to Gene Screen Plus® and probed with 32P-labeled IL 1a cDNA. The same filter was rehybridized to 32P-labeled pactin genomic DNA after washed with boiling water. Approximate sizes of the hybridizing RNA were estimated mouse ribosomal RNA (1.9 kb and 4.8 kb) as molecular size markers.

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Listeria, a significant degree of DFR was observed on day 7, reached its peak on day 10 in peptone-treated mice. On the other hand, no significant level of DFR response was detected in C. parvum-treated mice or in BCGtreated mice when 5 x 102 Listeria was challenged. When the challenge dose was increased to 5 x 104, the maximal level of DFR was observed in C. parvum-treated mice on day 10. The pattern in appearance of DFR response in C. parvum-treated mice was much the same in that seen in peptone-treated mice immunized with 5 x 102 Listeria. In BCG-treated mice, the maximal response was induced on day 7 after immunization with a high dose of Listeria (5 x 105) and the DFR response rapidly decreased to an undetectable level by day 10. Thus, BCG-treated mice showed an early appearing immune response to Listeria.

Discussion

The results described in this report demonstrated that functions of macrophages acting as scavenger cells, as cytokine-secreting cells and as

Induced Macrophages and Listerial Infection' 133

APC may play important roles in the induction of immune responses and protection against listerial infection. Macrophages activated by killed C. parvum have shown an increased bactericidal activity in addition to possessing the characteristics of peptoneelicited macrophages which show an increase in content of intracellular granules, oxidation of glucose-hexose monophosphate shunt as compared with peritoneal resident macrophages (25-28). In the present study, C. parvum-treated mice showed an enhanced resistance to the lethal effect of listerial infection, although the immune response against Listeria was not augmented in these mice. The level of the immune response was rather depressed in C. parvum-treated mice when these mice were immunized with a low dose of Listeria. We have previously reported that macrophages acting as scavenger cells regulate subsequent immune responses by altering the amounts of antigen (4, 15, 29). The activity of C. parvum-activated macrophages acting as scavenger cells was significantly increased as assessed by in vitro intracellular killing of Listeria and O 2 - generation, while the APC activity was much the same as that in peptone-elicited macrophages. Therefore, the increased activity of C. parvum-activated macrophages mainly as scavenger cells may contribute to the enhanced resistance against the bacterial infection in C. parvum-treated mice and may reduce the amount of effective listerial antigenic stimulation and depress the subsequent immune response. 6J::

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Figure 5. Kinetics of DFR responses to Listeria in peptone-treated mice, C. parvum-treated mice and BCG-treated mice. Mice treated with peptone on day -4 (0) were immunized i.p. with 5 x 102 Listeria on day O. Mice treated with C. parvum on day -4 (6) and mice treated with BCG on day -14 (D) were immunized i.p. with 5 x 104 and 5 x 105 Listeria on day 0, respectively. Each point and vertical bar indicate the mean number of five mice ± S.E.

BCG has been shown to intensify immune responses to some antigens by acting as a T cell mitogen, a polyclonal B cell mitogen and a direct stimulation of macrophages (30). In our results, an early appearing immune response to Listeria and a rapid decrease in number of bacteria in spleen were evident in BCG-treated mice. An increased APC activity was also demonstrated in BCG-activated macrophages. It is most likely that an increased APC activity in BCG-activated macrophages is partly responsible for the marked enhanced resistance against the listerial infection in BCGtreated mice. Three requirements are essential for a cell to function optimally in antigen presentation: the capacity to 1) express Ia antigens encoded by major histocompatibility complex (MHC) class II genes on its surface, 2) process antigen and 3) synthesize and release IL 1. After phagocytosis, antigens are processed by macrophages and antigen moieties appear 011 the plasma membrane. Antigenic moieties on the plasma membrane are exposed to lymphocytes in context with a product of the macrophages encoded by the MHC genes. T lymphocytes which recognize these structures become activated by additional stimulations such as IL 1 that is produced by macrophages. As shown in our results, the ability of BCG-activated macrophages for antigen processing, assessed by in vitro killing activity,

Induced Macrophages and Listerial Infection' 135

seemed to be augmented, and the number of la-bearing macrophages was significantly increased. Fluctuations in expression of Ia by the macrophages are shown to correlate with change in antigen presentation (31-33). In addition to macrophages, B cells (34), dendritic cells (35) and Langerhans cells (36) express Ia antigens on their surface and have antigen presenting activity. It is not clear whether the APC activity of these cells was affected by BeG treatment. However, most likely B cells and dendritic cells are found to have a limited capacity to present particulate antigens such as those on microbes (34). Therefore, la-positive macrophages appear to playa crucial role in the antigen presentation in the listerial infection. KURZJONES et al. reported that macrophage Ia and membrane IL 1 expression are critical for the induction of T cell responses to foreign antigens (37). A remarkable increase in IL 1 production was also detected in BeG-activated macrophages. It is most likely that increases both in the number of Iabearing macrophages and in the level of IL 1 production contribute to the increased ability for antigen presentation of BeG-activated macrophages, resulting in an increased level of immune responses and enhanced resistance against listerial infection in BeG-treated mice. Two types of helper T cells, THl and TH2, have been identified (38). TH1 were reported to secrete IL2 and y- IFN and play an important role in protection against Listeria infection. T H1 have also been shown to require cytokines other than IL 1 for their proliferation (39, 40). At the present time, we cannot rule out the participation of other cytokines produced by macrophages and T lymphocytes that might augment the cell-mediated immunity against Listeria in BeG-treated mice. In conclusion, macrophages play a crucial role in the initiation and development of immune responses. As various phases of antigen handling and regulation of lymphocyte activity by macrophages are susceptible to modulators such as C. parvum and BeG, it is likely that the modification of macrophage activities is a possible basis for immunomodulation. Acknowledgements We thank Dr. U. GUBLER in Hoffmann-La Roche, Inc. for providing IL-la cDNA (pIL 11301) and Dr. T. W. MAK for providing murine p-actin genomic DNA. This work was supported by grants alloted to Y. Yoshikai from the Ministry of Education, Science and Culture (62480167) and from Special Coordination Funds of the Science and Technology Agency of the Japanese Government.

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