INFECTION AND IMMUNITY, Mar. 1992, p. 1193-1201
Vol. 60, No. 3
0019-9567/92/031193-10$02.00/0 Copyright C) 1992, American Society for Microbiology
Macrophage Activation during Plasmodium chabaudi AS Infection in Resistant C57BL/6 and Susceptible A/J Mice MARY M. STEVENSON,* DIANA Y. HUANG, JOHN E. PODOBA, AND MARGARET E. NOWOTARSKI
Centre for the Study of Host Resistance, McGill University and the Montreal General Hospital Research Institute, 1650 Cedar Avenue, Montreal, Quebec H3G IA4, Canada Received 11 September 1991/Accepted 18 December 1991
Macrophage activation was examined in resistant C57BL/6 and susceptible A/J mice during the course of blood-stage infection with Plasmodium chabaudi AS. Three parameters of macrophage activation (lipopolysaccharide [LPS]- and malaria antigen-induced tumor necrosis factor [TNF] production in vitro, phorbol myristate acetate [PMAJ-induced production of oxygen metabolites in vitro, and Ia antigen expression) were assessed during infection in populations of peritoneal and splenic macrophages recovered from infected mice of the two strains. The peak level of LPS-induced TNF production in vitro by splenic macrophages from both infected C57BL/6 and infected A/J mice occurred on day 7, which was 3 days before the peak of parasitemia. Although the kinetics of TNF production in vitro in response to either LPS, soluble malaria antigen, or intact parasitized erythrocytes varied in some of the other macrophage populations during infection, there was no significant difference in the peak level of production. Peritoneal and splenic macrophages from infected C57BL/6 mice exhibited significantly increased PMA-induced production of H202 in vitro on day 7. Peritoneal macrophages from infected AJJ mice also exhibited significant PMA-induced H202 production on day 7, while production by splenic macrophages from these hosts was not increased in comparison with production by cells from normal animals. Only peritoneal macrophages from infected C57BL/6 mice produced significantly increased levels of 2-, and this occurred on day 7 postinfection. Ia antigen expression by both peritoneal and splenic macrophages from resistant C57BL/6 and susceptible ANJ mice was significantly increased during P. chabaudi AS infection. However, the percentage of Ia' peritoneal macrophages on days 8 and 10 postinfection and Ia' splenic macrophages on day 3 postinfection was significantly higher in C57BV6 than in ANJ mice. Thus, these results demonstrate that macrophages from P. chabaudi AS-infected AJJ mice exhibit defects in oxygen metabolism and Ia antigen expression which may contribute to the susceptibility of these hosts to this intraerythrocytic parasite. The cause-and-effect relationship between these defects and the susceptibility of A/J mice to P. chabaudi AS is unknown. cules such as IFN--y or tumor necrosis factor (TNF), results in increased resistance to infection (4, 6, 7, 23, 25, 37). Furthermore, results from our laboratory (26) as well as from others (19) demonstrated that in vivo elimination of macrophages by treatment with the macrophage poison silica results in increased susceptibility to infection apparent as significantly higher parasitemia and death. The results of these studies suggest that macrophages play a role in resistance to blood-stage malaria. However, they do not identify the exact contribution of these cells either to innate resistance or to the development of acquired immunity to malaria as antigen-presenting affector or as activated effector cells. The contribution of other cells to the effector arm of the cellular immune response to blood-stage malaria is quite likely. Results of in vitro studies suggest that eosinophils and neutrophils which, like macrophages, are of myeloid origin can destroy blood-stage parasites or inhibit their growth (20). Studies by Cavacini and colleagues (2) demonstrated that P/J mice, which do not exhibit macrophage activation during infection with Plasmodium chabaudi adami as assessed by oxidative and tumoricidal activities, can resolve an acute blood-stage infection with this parasite as efficiently as can BALB/c mice, which develop activated macrophages during infection. These results, thus, suggest that effector cells other than activated macrophages may contribute to the development of protective immunity to blood-stage malaria. The availability of inbred strains of mice which are either resistant or susceptible to blood-stage malaria offers an ideal tool with which to evaluate the contribution of activated
It is now generally accepted that cell-mediated immune mechanisms contribute to acquired immunity to blood-stage malaria. Evidence from in vitro and in vivo studies in experimental animal models as well as in patients with malaria suggests that CD4+ T cells, activated during infection, produce macrophage-activating factors, of which the most potent molecule is gamma interferon (IFN-y) (reviewed in references 30, 38, and 39). As a result of exposure to such T-cell-derived factors in vitro, macrophages express enhanced microbicidal activity and are able to destroy the intraerythrocytic parasites by the production of cytotoxic molecules such as H202 (22). However, while the activation of effector macrophages by soluble products of parasitespecific CD4+ T cells resulting in the production of cytotoxic molecules appears to be an efficient mechanism of antiplasmodial activity in vitro, the identity of the major effector cell(s) mediating destruction of the parasite in vivo as well as the mechanism(s) are not yet known. The concept that macrophages play a role in cell-mediated immunity against blood-stage malaria is based on the following experimental evidence. It has been observed in several experimental models, including the one used in the studies presented here, that pretreatment of mice with a variety of immunomodulators, such as Mycobacterium bovis (BCG), Propionibactenum acnes, liposome-encapsulated muramyl dipeptide derived from mycobacteria, or recombinant mole*
Corresponding author. 1193
1194
STEVENSON ET AL.
macrophages to host resistance to Plasmodium spp. In this study, therefore, we compared three parameters of macrophage activation, namely, lipopolysaccharide (LPS)- and malaria antigen-induced TNF production in vitro, phorbol myristate acetate (PMA)-induced production of oxygen metabolites in vitro, and Ia antigen expression, during infection with P. chabaudi AS in resistant C57BL/6 and susceptible A/J mice (27-29, 31).
MATERLALS AND METHODS Mice. Mice, 8 to 12 weeks old, were age and sex matched in all experiments. C57BL/6 mice were purchased from Charles River, St. Constant, Quebec, Canada, and ANJ mice were purchased from Jackson Laboratory, Bar Harbor, Maine. Parasite. P. chabaudi AS was maintained by weekly passage in female C57BL/6 mice. After 12 passages, a fresh inoculum was prepared and a new passage was initiated from frozen stock cultures which were stored at -70°C. For passage or infection of experimental animals, blood was collected via the retro-orbital plexus from two infected C57BL/6 mice and pooled. Total erythrocyte (RBC) counts were determined. The percent parasitemia was determined by counting the percentage of parasitized RBC (PRBC) per 100 RBC on duplicate, Dif-Quik (American Scientific Products, McGaw Park, Ill.)-stained thin blood smears. RBC, diluted in sterile phosphate-buffered saline (PBS), were adjusted to the desired concentration of PRBC and injected intraperitoneally into passage or experimental mice. For passage, mice were injected with a dose of 107 PRBC. Experimental infections were initiated with a dose of 106 P. chabaudi AS PRBC. Determination of parasitemia. To determine the course of infection, blood samples were collected from experimental mice by bleeding via the tail vein at the times indicated. Duplicate thin blood smears were prepared and stained with Dif-Quik. Parasitemia was determined by counting the percentage of infected cells per 100 RBC per slide. The parasitemia is expressed as mean percent PRBC + standard error of the mean (SEM) for each group of mice. Malaria antigens. Mice with high parasitemia (>50% PRBC) were bled by cardiac puncture. RBC were washed twice in Hanks' balanced salt solution (HBSS) without phenol red (GIBCO Laboratories, Grand Island, N.Y.) to remove serum proteins. Packed RBC were lysed in 0.2% NaCl and then restored to isotonicity by addition of 1.6% NaCl. The preparation was sonicated in a Fisher Sonic Dismembrator (Fisher Scientific, Canada, Montreal, Quebec, Canada) until membranes were disrupted. Protein concentration was determined with Bio-Rad reagents (Bio-Rad Laboratories, Richmond, Calif.), using hemoglobin as a standard. RBC collected from normal, uninfected animals (ghost antigen) were prepared by the same procedure. Cell preparation. Macrophages from the peritoneum and the spleen of normal and infected C57BL/6 and A/J mice were used in all experiments. For the TNF experiments, peritoneal macrophages were obtained by peritoneal lavage of mice injected intraperitoneally with 1.5 ml of 10% proteose peptone 3 days previously. Otherwise, resident peritoneal macrophages were harvested. Washed cells were suspended in Dulbecco's modified Eagle medium (GIBCO) containing 10% fetal calf serum (HyClone Laboratories, Inc., Logan, Utah) and 0.12% gentamicin (Schering Canada Inc., Montreal, Quebec, Canada). Splenic macrophages were isolated by perfusion of spleens with RPMI 1640 (Flow
INFECT. IMMUN.
Laboratories, Inc., Mississauga, Ontario, Canada) supplemented with 10% fetal calf serum and 0.12% gentamicin. RBC were lysed with cold 0.17 M NH4Cl, and the cells were washed three times in fresh medium. Total and differential counts were determined on peritoneal and splenic macrophages. In addition, the viability of splenic macrophages was determined by trypan blue exclusion and was always greater than 90%. TNF. Peritoneal and splenic macrophages were adjusted to 106 macrophages per ml, and aliquots of 1.0 ml of each cell suspension were added to 24-well plates (Nunc, Inc., Roskilde, Denmark) and allowed to adhere for 2 h at 37°C in an atmosphere of 5% CO2 in air. Nonadherent cells were removed by washing with warm HBSS. Macrophage monolayers were stimulated with either 10 ,ug of Escherichia coli 0127:B8 LPS (Difco, Detroit, Mich.) per ml, 100 ,ug of malaria or ghost antigen per ml, or 107 washed normal or PRBC. Macrophages incubated with medium alone were always included as controls. Supernatants were collected 20 h later and stored at -20°C until assayed for TNF. A double-sandwich enzyme-linked immunosorbent assay (ELISA), as described by Sheehan et al. (24), was used to determine the quantity of TNF in the supematants of macrophages treated as described above. Hamster monoclonal antibody to murine TNF was purchased from Genzyme, Boston, Mass., and rabbit polyclonal antimurine TNF was prepared and purified by standard procedures (12). The level of TNF was calculated from a standard curve established in every experiment using recombinant TNF, and the results are expressed as mean picograms per milliliter + SEM, calculated from the formula 1.0 IU = 25 pg/ml. H202 and °2- Production of H202 and °2 by peritoneal and spleen macrophages was determined by the micro methods described by Pick and Mizel (18). Briefly, 100 ,ul per well containing 106 peritoneal macrophages per ml and 107 splenic macrophages per ml was dispensed into 96-well flat-bottom tissue culture plates (Falcon Microtest II Plate; Fisher Scientific). After 2 h of incubation at 37°C, 5% CO2 in air, nonadherent cells were removed by washing with warm HBSS without phenol red. For determination of H202 production, macrophage monolayers in each well were incubated for 1 h with 100 ,ul of HBSS containing 1% phenol red (Aldrich Chemical Co., Inc., Milwaukee, Wis.), 10% glucose (Fisher Scientific), and 5 mg of horseradish peroxidase (Sigma Chemical Co., St. Louis, Mo.) per ml with or without 500 ng of PMA (Sigma) per ml. The reaction was interrupted by adding 10 ,ul of 1 N NaOH per well. A620 was read with an ELISA reader. A standard curve with a known concentration of H202 was established for each experiment. The amount of H202 produced was calculated from the following formula: nanomoles of H202 per well = A620 nm x 16.7. Results are expressed as mean nanomoles of H202 per milligram of macrophage protein per hour + SEM. For determination of 02 production, macrophage monolayers in each well were incubated with 100 p,l of a 160 ,uM solution of ferricytochrome c (type III; Sigma) in HBSS without phenol red (GIBCO) with or without 500 ng of PMA per ml. After incubation for 1 h, A550 was read in an ELISA reader. The amount of 02- produced was calculated from the following formula: nanomoles of 02 per well = A550 x 15.87. Results are expressed as mean nanomoles Of 02 per milligram of macrophage protein per hour + SEM. Protein concentration of macrophage monolayers was determined by using Bio-Rad reagents. Quantification of Ia antigens. Peritoneal and splenic macrophages were adjusted to a concentration of 106 macro-
VOL. 60, 1992
MACROPHAGE ACTIVATION DURING P. CHABAUDI AS INFECTION IN MICE
phages per ml, and aliquots of 200 pl per well were cultured on Titertech 8 chamber slides (Nunc) for 2 h at 37°C, 5% CO2 in air. After removal of nonadherent cells by washing, adherent cells were fixed in 1% paraformaldehyde for 15 min at room temperature, and the slides were washed with PBS. For detection of Ia antigens, 200 pl of a monoclonal antimouse antibody (I-A' immunoglobulin G2a/k [IgG2a/k] antibody [clone 9030; Cedar Lane Laboratories, Homby, Ontario, Canada] for macrophages from C57BL/6 mice or I-Ak IgG2a/k antibody [clone 8709; Cedar Lane] for macrophages from A/J mice) was added to each well at a 1:100 dilution in PBS containing 3% normal goat serum (Cedar Lane), and the wells were incubated for 45 min at 4°C. The slides were washed with PBS, 200 pl of fluorescein-conjugated F(ab')2 goat anti-mouse IgG (Cedar Lane) was added to each well at a dilution of 1:20 in PBS, and the wells were incubated for 45 min at 4°C. After staining, the slides were washed and stored in PBS until the time of microscopic examination. Immediately before visualization under a fluorescence microscope, the slides were washed in double-distilled H20 to prevent salt crystal formation. Ia antigen expression is expressed as mean percentage of Ia+ macrophages ± SEM per each group of mice. Statistical analysis. Differences in mean values of production of TNF, H202, and 2- and of Ia antigen expression between normal and infected animals as well as between infected A/J and C57BL/6 mice were analyzed by Student's t test. A probability of less than 0.05 was considered significant.
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RESULTS TNF production during P. chabaudi AS infection. At various times after infection with 106 P. chabaudi AS PRBC, proteose peptone-elicited peritoneal and splenic macrophages were recovered from resistant C57BL/6 and susceptible ANJ mice and examined for the ability to produce TNF in vitro in response to LPS or parasite antigens. The kinetics of TNF production in vitro in response to LPS by these macrophage populations are presented in Fig. 1. Proteose peptone-elicited peritoneal and splenic macrophages were also prepared from normal, uninfected mice of both strains. There was no significant difference in LPS-induced TNF production by proteose peptone-stimulated peritoneal macrophages from normal A/J mice (1,350 + 150 pg/ml) in comparison with the response of cells from normal C57BL/6 mice (1,975 +- 312; P < 0.08). Similarly, there was no significant difference in TNF production between splenic macrophages from normal ANJ mice (1,041 + 158) and normal C57BL/6 mice (1,664 + 107; P < 0.08). In the case of P. chabaudi AS-resistant C57BL/6 mice, in vitro production of TNF was determined throughout the course of infection (Fig. la); macrophages were recovered from infected animals on days 3, 5, 7, 10, 14, 21, and 28 postinfection. There was marked and significant production of TNF by peritoneal macrophages early in infection on day 3, when the parasitemia level was approximately 2% (P < 0.006). However, there was no significant difference in TNF production between peritoneal macrophages from infected and normal C57BL/6 mice at any other time. On the other hand, splenic macrophages recovered from infected C57BL/6 animals on day 5 through day 28 produced significantly higher levels of TNF, which were 1.3 to 4.3 times as much as that produced by cells recovered from normal mice. On day 7 postinfection, which was 3 days before the peak parasitemia of 32.8 ± 1.9% PRBC, the peak of TNF produc-
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FIG. 1. Kinetics of TNF production in vitro by macrophages from C57BL/6 (a) and A/J (b) mice during P. chabaudi AS infection. In each experiment, macrophage monolayers were prepared from peritoneal cells (J) recovered and pooled from groups of three normal (day 0) or P. chabaudi AS-infected mice injected intraperitoneally with 1.5 ml of proteose peptone 3 days previously or from individual spleens (U) harvested from the same animals. Adherent cells were stimulated for 20 h with 10 ,ug of LPS per ml. Supernatants were collected, and the level of TNF was determined by using a double-sandwich ELISA. The course of parasitemia (0) was determined by quantitating the percent PRBC in the peripheral blood on the days indicated. Pooled data from three separate experiments are presented as mean + SEM.
tion by splenic macrophages occurred and 7,091 + 518 pg of TNF per ml was produced in comparison with cells from normal C57BL/6 mice (P < 0.0001). After this time, TNF production by splenic macrophages from P. chabaudi ASinfected C57BL/6 mice decreased but was still significant. Macrophages from infected C57BL/6 mice were, therefore, still activated on day 28 postinfection, as demonstrated by their ability to produce approximately 3,000 pg of TNF per ml (P < 0.03 in comparison with normal). In vitro production of TNF by proteose peptone-elicited peritoneal and splenic macrophages from P. chabaudi ASsusceptible A/J mice in response to LPS was also determined but only on days 3, 5, 7, and 10 postinfection, since mice of
1196
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STEVENSON ET AL.
TABLE 1. Production of TNF by macrophages from P. chabaudi AS-infected C57BL/6 and A/J mice in response to malaria antigens A/J
TNF level (mean pg/ml + SEM)a C57BL/6
Days postinfection
0 3 5 7 9 10 14 21 28
Parasitemia (%)
Malaria antigens
1.5 + 0.39 3.8 ± 0.54 17.2 0.98
Vol. 60, No. 3
0019-9567/92/031193-10$02.00/0 Copyright C) 1992, American Society for Microbiology
Macrophage Activation during Plasmodium chabaudi AS Infection in Resistant C57BL/6 and Susceptible A/J Mice MARY M. STEVENSON,* DIANA Y. HUANG, JOHN E. PODOBA, AND MARGARET E. NOWOTARSKI
Centre for the Study of Host Resistance, McGill University and the Montreal General Hospital Research Institute, 1650 Cedar Avenue, Montreal, Quebec H3G IA4, Canada Received 11 September 1991/Accepted 18 December 1991
Macrophage activation was examined in resistant C57BL/6 and susceptible A/J mice during the course of blood-stage infection with Plasmodium chabaudi AS. Three parameters of macrophage activation (lipopolysaccharide [LPS]- and malaria antigen-induced tumor necrosis factor [TNF] production in vitro, phorbol myristate acetate [PMAJ-induced production of oxygen metabolites in vitro, and Ia antigen expression) were assessed during infection in populations of peritoneal and splenic macrophages recovered from infected mice of the two strains. The peak level of LPS-induced TNF production in vitro by splenic macrophages from both infected C57BL/6 and infected A/J mice occurred on day 7, which was 3 days before the peak of parasitemia. Although the kinetics of TNF production in vitro in response to either LPS, soluble malaria antigen, or intact parasitized erythrocytes varied in some of the other macrophage populations during infection, there was no significant difference in the peak level of production. Peritoneal and splenic macrophages from infected C57BL/6 mice exhibited significantly increased PMA-induced production of H202 in vitro on day 7. Peritoneal macrophages from infected AJJ mice also exhibited significant PMA-induced H202 production on day 7, while production by splenic macrophages from these hosts was not increased in comparison with production by cells from normal animals. Only peritoneal macrophages from infected C57BL/6 mice produced significantly increased levels of 2-, and this occurred on day 7 postinfection. Ia antigen expression by both peritoneal and splenic macrophages from resistant C57BL/6 and susceptible ANJ mice was significantly increased during P. chabaudi AS infection. However, the percentage of Ia' peritoneal macrophages on days 8 and 10 postinfection and Ia' splenic macrophages on day 3 postinfection was significantly higher in C57BV6 than in ANJ mice. Thus, these results demonstrate that macrophages from P. chabaudi AS-infected AJJ mice exhibit defects in oxygen metabolism and Ia antigen expression which may contribute to the susceptibility of these hosts to this intraerythrocytic parasite. The cause-and-effect relationship between these defects and the susceptibility of A/J mice to P. chabaudi AS is unknown. cules such as IFN--y or tumor necrosis factor (TNF), results in increased resistance to infection (4, 6, 7, 23, 25, 37). Furthermore, results from our laboratory (26) as well as from others (19) demonstrated that in vivo elimination of macrophages by treatment with the macrophage poison silica results in increased susceptibility to infection apparent as significantly higher parasitemia and death. The results of these studies suggest that macrophages play a role in resistance to blood-stage malaria. However, they do not identify the exact contribution of these cells either to innate resistance or to the development of acquired immunity to malaria as antigen-presenting affector or as activated effector cells. The contribution of other cells to the effector arm of the cellular immune response to blood-stage malaria is quite likely. Results of in vitro studies suggest that eosinophils and neutrophils which, like macrophages, are of myeloid origin can destroy blood-stage parasites or inhibit their growth (20). Studies by Cavacini and colleagues (2) demonstrated that P/J mice, which do not exhibit macrophage activation during infection with Plasmodium chabaudi adami as assessed by oxidative and tumoricidal activities, can resolve an acute blood-stage infection with this parasite as efficiently as can BALB/c mice, which develop activated macrophages during infection. These results, thus, suggest that effector cells other than activated macrophages may contribute to the development of protective immunity to blood-stage malaria. The availability of inbred strains of mice which are either resistant or susceptible to blood-stage malaria offers an ideal tool with which to evaluate the contribution of activated
It is now generally accepted that cell-mediated immune mechanisms contribute to acquired immunity to blood-stage malaria. Evidence from in vitro and in vivo studies in experimental animal models as well as in patients with malaria suggests that CD4+ T cells, activated during infection, produce macrophage-activating factors, of which the most potent molecule is gamma interferon (IFN-y) (reviewed in references 30, 38, and 39). As a result of exposure to such T-cell-derived factors in vitro, macrophages express enhanced microbicidal activity and are able to destroy the intraerythrocytic parasites by the production of cytotoxic molecules such as H202 (22). However, while the activation of effector macrophages by soluble products of parasitespecific CD4+ T cells resulting in the production of cytotoxic molecules appears to be an efficient mechanism of antiplasmodial activity in vitro, the identity of the major effector cell(s) mediating destruction of the parasite in vivo as well as the mechanism(s) are not yet known. The concept that macrophages play a role in cell-mediated immunity against blood-stage malaria is based on the following experimental evidence. It has been observed in several experimental models, including the one used in the studies presented here, that pretreatment of mice with a variety of immunomodulators, such as Mycobacterium bovis (BCG), Propionibactenum acnes, liposome-encapsulated muramyl dipeptide derived from mycobacteria, or recombinant mole*
Corresponding author. 1193
1194
STEVENSON ET AL.
macrophages to host resistance to Plasmodium spp. In this study, therefore, we compared three parameters of macrophage activation, namely, lipopolysaccharide (LPS)- and malaria antigen-induced TNF production in vitro, phorbol myristate acetate (PMA)-induced production of oxygen metabolites in vitro, and Ia antigen expression, during infection with P. chabaudi AS in resistant C57BL/6 and susceptible A/J mice (27-29, 31).
MATERLALS AND METHODS Mice. Mice, 8 to 12 weeks old, were age and sex matched in all experiments. C57BL/6 mice were purchased from Charles River, St. Constant, Quebec, Canada, and ANJ mice were purchased from Jackson Laboratory, Bar Harbor, Maine. Parasite. P. chabaudi AS was maintained by weekly passage in female C57BL/6 mice. After 12 passages, a fresh inoculum was prepared and a new passage was initiated from frozen stock cultures which were stored at -70°C. For passage or infection of experimental animals, blood was collected via the retro-orbital plexus from two infected C57BL/6 mice and pooled. Total erythrocyte (RBC) counts were determined. The percent parasitemia was determined by counting the percentage of parasitized RBC (PRBC) per 100 RBC on duplicate, Dif-Quik (American Scientific Products, McGaw Park, Ill.)-stained thin blood smears. RBC, diluted in sterile phosphate-buffered saline (PBS), were adjusted to the desired concentration of PRBC and injected intraperitoneally into passage or experimental mice. For passage, mice were injected with a dose of 107 PRBC. Experimental infections were initiated with a dose of 106 P. chabaudi AS PRBC. Determination of parasitemia. To determine the course of infection, blood samples were collected from experimental mice by bleeding via the tail vein at the times indicated. Duplicate thin blood smears were prepared and stained with Dif-Quik. Parasitemia was determined by counting the percentage of infected cells per 100 RBC per slide. The parasitemia is expressed as mean percent PRBC + standard error of the mean (SEM) for each group of mice. Malaria antigens. Mice with high parasitemia (>50% PRBC) were bled by cardiac puncture. RBC were washed twice in Hanks' balanced salt solution (HBSS) without phenol red (GIBCO Laboratories, Grand Island, N.Y.) to remove serum proteins. Packed RBC were lysed in 0.2% NaCl and then restored to isotonicity by addition of 1.6% NaCl. The preparation was sonicated in a Fisher Sonic Dismembrator (Fisher Scientific, Canada, Montreal, Quebec, Canada) until membranes were disrupted. Protein concentration was determined with Bio-Rad reagents (Bio-Rad Laboratories, Richmond, Calif.), using hemoglobin as a standard. RBC collected from normal, uninfected animals (ghost antigen) were prepared by the same procedure. Cell preparation. Macrophages from the peritoneum and the spleen of normal and infected C57BL/6 and A/J mice were used in all experiments. For the TNF experiments, peritoneal macrophages were obtained by peritoneal lavage of mice injected intraperitoneally with 1.5 ml of 10% proteose peptone 3 days previously. Otherwise, resident peritoneal macrophages were harvested. Washed cells were suspended in Dulbecco's modified Eagle medium (GIBCO) containing 10% fetal calf serum (HyClone Laboratories, Inc., Logan, Utah) and 0.12% gentamicin (Schering Canada Inc., Montreal, Quebec, Canada). Splenic macrophages were isolated by perfusion of spleens with RPMI 1640 (Flow
INFECT. IMMUN.
Laboratories, Inc., Mississauga, Ontario, Canada) supplemented with 10% fetal calf serum and 0.12% gentamicin. RBC were lysed with cold 0.17 M NH4Cl, and the cells were washed three times in fresh medium. Total and differential counts were determined on peritoneal and splenic macrophages. In addition, the viability of splenic macrophages was determined by trypan blue exclusion and was always greater than 90%. TNF. Peritoneal and splenic macrophages were adjusted to 106 macrophages per ml, and aliquots of 1.0 ml of each cell suspension were added to 24-well plates (Nunc, Inc., Roskilde, Denmark) and allowed to adhere for 2 h at 37°C in an atmosphere of 5% CO2 in air. Nonadherent cells were removed by washing with warm HBSS. Macrophage monolayers were stimulated with either 10 ,ug of Escherichia coli 0127:B8 LPS (Difco, Detroit, Mich.) per ml, 100 ,ug of malaria or ghost antigen per ml, or 107 washed normal or PRBC. Macrophages incubated with medium alone were always included as controls. Supernatants were collected 20 h later and stored at -20°C until assayed for TNF. A double-sandwich enzyme-linked immunosorbent assay (ELISA), as described by Sheehan et al. (24), was used to determine the quantity of TNF in the supematants of macrophages treated as described above. Hamster monoclonal antibody to murine TNF was purchased from Genzyme, Boston, Mass., and rabbit polyclonal antimurine TNF was prepared and purified by standard procedures (12). The level of TNF was calculated from a standard curve established in every experiment using recombinant TNF, and the results are expressed as mean picograms per milliliter + SEM, calculated from the formula 1.0 IU = 25 pg/ml. H202 and °2- Production of H202 and °2 by peritoneal and spleen macrophages was determined by the micro methods described by Pick and Mizel (18). Briefly, 100 ,ul per well containing 106 peritoneal macrophages per ml and 107 splenic macrophages per ml was dispensed into 96-well flat-bottom tissue culture plates (Falcon Microtest II Plate; Fisher Scientific). After 2 h of incubation at 37°C, 5% CO2 in air, nonadherent cells were removed by washing with warm HBSS without phenol red. For determination of H202 production, macrophage monolayers in each well were incubated for 1 h with 100 ,ul of HBSS containing 1% phenol red (Aldrich Chemical Co., Inc., Milwaukee, Wis.), 10% glucose (Fisher Scientific), and 5 mg of horseradish peroxidase (Sigma Chemical Co., St. Louis, Mo.) per ml with or without 500 ng of PMA (Sigma) per ml. The reaction was interrupted by adding 10 ,ul of 1 N NaOH per well. A620 was read with an ELISA reader. A standard curve with a known concentration of H202 was established for each experiment. The amount of H202 produced was calculated from the following formula: nanomoles of H202 per well = A620 nm x 16.7. Results are expressed as mean nanomoles of H202 per milligram of macrophage protein per hour + SEM. For determination of 02 production, macrophage monolayers in each well were incubated with 100 p,l of a 160 ,uM solution of ferricytochrome c (type III; Sigma) in HBSS without phenol red (GIBCO) with or without 500 ng of PMA per ml. After incubation for 1 h, A550 was read in an ELISA reader. The amount of 02- produced was calculated from the following formula: nanomoles of 02 per well = A550 x 15.87. Results are expressed as mean nanomoles Of 02 per milligram of macrophage protein per hour + SEM. Protein concentration of macrophage monolayers was determined by using Bio-Rad reagents. Quantification of Ia antigens. Peritoneal and splenic macrophages were adjusted to a concentration of 106 macro-
VOL. 60, 1992
MACROPHAGE ACTIVATION DURING P. CHABAUDI AS INFECTION IN MICE
phages per ml, and aliquots of 200 pl per well were cultured on Titertech 8 chamber slides (Nunc) for 2 h at 37°C, 5% CO2 in air. After removal of nonadherent cells by washing, adherent cells were fixed in 1% paraformaldehyde for 15 min at room temperature, and the slides were washed with PBS. For detection of Ia antigens, 200 pl of a monoclonal antimouse antibody (I-A' immunoglobulin G2a/k [IgG2a/k] antibody [clone 9030; Cedar Lane Laboratories, Homby, Ontario, Canada] for macrophages from C57BL/6 mice or I-Ak IgG2a/k antibody [clone 8709; Cedar Lane] for macrophages from A/J mice) was added to each well at a 1:100 dilution in PBS containing 3% normal goat serum (Cedar Lane), and the wells were incubated for 45 min at 4°C. The slides were washed with PBS, 200 pl of fluorescein-conjugated F(ab')2 goat anti-mouse IgG (Cedar Lane) was added to each well at a dilution of 1:20 in PBS, and the wells were incubated for 45 min at 4°C. After staining, the slides were washed and stored in PBS until the time of microscopic examination. Immediately before visualization under a fluorescence microscope, the slides were washed in double-distilled H20 to prevent salt crystal formation. Ia antigen expression is expressed as mean percentage of Ia+ macrophages ± SEM per each group of mice. Statistical analysis. Differences in mean values of production of TNF, H202, and 2- and of Ia antigen expression between normal and infected animals as well as between infected A/J and C57BL/6 mice were analyzed by Student's t test. A probability of less than 0.05 was considered significant.
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RESULTS TNF production during P. chabaudi AS infection. At various times after infection with 106 P. chabaudi AS PRBC, proteose peptone-elicited peritoneal and splenic macrophages were recovered from resistant C57BL/6 and susceptible ANJ mice and examined for the ability to produce TNF in vitro in response to LPS or parasite antigens. The kinetics of TNF production in vitro in response to LPS by these macrophage populations are presented in Fig. 1. Proteose peptone-elicited peritoneal and splenic macrophages were also prepared from normal, uninfected mice of both strains. There was no significant difference in LPS-induced TNF production by proteose peptone-stimulated peritoneal macrophages from normal A/J mice (1,350 + 150 pg/ml) in comparison with the response of cells from normal C57BL/6 mice (1,975 +- 312; P < 0.08). Similarly, there was no significant difference in TNF production between splenic macrophages from normal ANJ mice (1,041 + 158) and normal C57BL/6 mice (1,664 + 107; P < 0.08). In the case of P. chabaudi AS-resistant C57BL/6 mice, in vitro production of TNF was determined throughout the course of infection (Fig. la); macrophages were recovered from infected animals on days 3, 5, 7, 10, 14, 21, and 28 postinfection. There was marked and significant production of TNF by peritoneal macrophages early in infection on day 3, when the parasitemia level was approximately 2% (P < 0.006). However, there was no significant difference in TNF production between peritoneal macrophages from infected and normal C57BL/6 mice at any other time. On the other hand, splenic macrophages recovered from infected C57BL/6 animals on day 5 through day 28 produced significantly higher levels of TNF, which were 1.3 to 4.3 times as much as that produced by cells recovered from normal mice. On day 7 postinfection, which was 3 days before the peak parasitemia of 32.8 ± 1.9% PRBC, the peak of TNF produc-
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FIG. 1. Kinetics of TNF production in vitro by macrophages from C57BL/6 (a) and A/J (b) mice during P. chabaudi AS infection. In each experiment, macrophage monolayers were prepared from peritoneal cells (J) recovered and pooled from groups of three normal (day 0) or P. chabaudi AS-infected mice injected intraperitoneally with 1.5 ml of proteose peptone 3 days previously or from individual spleens (U) harvested from the same animals. Adherent cells were stimulated for 20 h with 10 ,ug of LPS per ml. Supernatants were collected, and the level of TNF was determined by using a double-sandwich ELISA. The course of parasitemia (0) was determined by quantitating the percent PRBC in the peripheral blood on the days indicated. Pooled data from three separate experiments are presented as mean + SEM.
tion by splenic macrophages occurred and 7,091 + 518 pg of TNF per ml was produced in comparison with cells from normal C57BL/6 mice (P < 0.0001). After this time, TNF production by splenic macrophages from P. chabaudi ASinfected C57BL/6 mice decreased but was still significant. Macrophages from infected C57BL/6 mice were, therefore, still activated on day 28 postinfection, as demonstrated by their ability to produce approximately 3,000 pg of TNF per ml (P < 0.03 in comparison with normal). In vitro production of TNF by proteose peptone-elicited peritoneal and splenic macrophages from P. chabaudi ASsusceptible A/J mice in response to LPS was also determined but only on days 3, 5, 7, and 10 postinfection, since mice of
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TABLE 1. Production of TNF by macrophages from P. chabaudi AS-infected C57BL/6 and A/J mice in response to malaria antigens A/J
TNF level (mean pg/ml + SEM)a C57BL/6
Days postinfection
0 3 5 7 9 10 14 21 28
Parasitemia (%)
Malaria antigens
1.5 + 0.39 3.8 ± 0.54 17.2 0.98
