912
Endogenous Interferon-y, Macrophage Activation, and Murine Host Defense against Acute Infection with Trypanosoma cruzi Robert E. McCabe, Stephen G. Meagher, and Brian T. Mullins
Medical Service, Martinez VA Medical Center, Martinez, California; Department of Medicine, University of California Medical School, Davis
Activated macrophages are considered important for host defense against a number of human intracellular pathogens, including Trypanosoma cruzi, Listeria monocytogenes, Chlamydia, Leishmania, and Plasmodium species, and Toxoplasma gondii [1]. The importance of activated macrophages is indicated by their ability to kill or inhibit multiplication of organisms in vitro and the presence of activated macro phages in hosts with active infection ~ Lymphokines, particularly interferon-v (lFN-1'), can activate macrophages in vivo and in vitro [1]. The availability of antibody that selectively neutralizes IFN -1''s ability to activate macrophages in vivo makes it possible to evaluate more directly the role of endogenous IFN-1' in macrophage activation and control of infection. We examined the role of endogenous IFN-1' and macrophage activation in the pathogenesis of early acute rhurine infection with
T. cruzi.
Materials and Methods Mice. BALB/c (Bantin and Kingman, Fremont, CA), Swiss Webster (Bantin and Kingman; National Cancer Institute, Bethesda, MD), and C57Bl/6 and C3H/HEN (National Cancer Institute) mice weighing 18-22 g each were used in all experiments. T. cruzi. The Y strain was used in all experiments [2]. Bloodform trypomastigotes obtained from Swiss Webster mice were used
Received 17 April 1990; revised 16 November 1990. Presented in part: 29th Interscience Conference on Antimicrobial Agents and Chemotherapy, Houston, September 1989 (abstract 1205). Financial support: Research Service, Department of Veterans Affairs. Reprintsor correspondence: Dr. Robert McCabe, Medical Service (111F), Martinez VAMC, 150 Muir Rd., Martinez, CA 94553. The Journal of Infectious Diseases 1991;163:912-915 © 1991 by The University of Chicago. AU rights reserved. 0022-1899/91/6304-0045$01.00
to infect mice. Parasitemias were determined by inspection of blood smears with a light microscope [3]. Tissue culture organisms used to infect macrophages were obtained from cultures of L929 cells (ATCC, Rockville, MD) as previously described [4]. Anti-IFN-y Anti-IFN-')' was prepared from a rat hybridpma that secretes IgGI antibody to murine IFN-')' (R4-6A2, HB170; ATCC) [5]. The ammonium sulfate precipitate of hybridom a media was dialyzed against PBS and then filter sterilized. The protein concentration of the final preparation, determined with a kit (Bio-Rad, Richmond, CA), was 21.5 mg/ml. The preparation was titered for neutralizing activity against recombinant IFN-')' (gift of Genentech, South San Francisco, CA) (see IFN Assay). Neutralization was defined as reduction of antiviral activity of a specimen containing 10 units ofIFN-')' to undetectable levels (no activity in the 1:2 dilution of specimen). The preparation used in the experiments reported here had 25,000 units of anti-IFN-')' activity per milligram of protein. Anti-IFN-')' was injected intraperitoneally (ip) or intravenously (iv) into mice. Rat serum, PBS, boiled anti-If'Nvy, or an irrelevant monoclonal antibody from a hybridoma (anti-DR5; ATCC) prepared similarly to anti-IFN-')' were used as controls for injection. Preliminary experiments (data not shown) showed no differences between PBS, rat serum, boiled anti-If'Ns-y, or the irrelevant monoclonal antibody with respect to either levels of parasitemia or activation of macrophages. IFN assay. A microtiter photometric assay [6] in which threefold dilutions of the sample to be tested for IFN are added to confluent L929 cells in 96-well microculture plates (Costar, Cambridge, MA) and then challenged with encephalomyocarditis virus was used. After 90 % destruction of control monolayers by virus, monolayers were stained and air dried, and retained dye was quantitated by optical density readings (Titertek Multiskan; Flow Laboratories, McLean, VA) with a 600-nm filter. Anti-IFN-')' (ATCC) and anti-o/d antiserum (Lee Biologics, San Diego) were used to identify the type of IFN. Infection ofmacrophage mono/ayers with T. cruzi. Mouse peritoneal macrophage monolayers were cultured in duplicate replicates in eight-well tissue culture chambers (Labtek Products, Naperville, IL) with RPMI 1640 tissue culture medium (GmCO, Grand Island,
Downloaded from http://jid.oxfordjournals.org/ at University of Sussex on September 6, 2015
Parenteral interferon-v (IFN-')') activates murine macrophages to inhibit Trypanosoma cruz; multiplication and diminishes parasitemia and mortality in acute infection. To investigate the role of endogenous IFN-')' in acute infection, monoclonal antibody to IFN-')' was injected intraperitoneally into mice. The 6250 neutralizing units given 24 and 96 h after infection reproducibly increased mortality (P < .05). Histology sections showed markedly more nests of T. cruz; in treated mice. BALB/c, Swiss Webster, C57Bl/6, and C3H/HEN mice were susceptible to the effects of anti-IFN-')'. Peritoneal macrophages from mice 4 days after infection and a single dose of 6250 units of anti-IFN-')' had significantly reduced ability to inhibit T. cruz; multiplication. Multiple doses of anti-IFN-')' delayed but did not prevent macrophage activation. These results indicate the critical role of endogenous IFN-')' for macrophage activation and host defense against acute T. cruz; infection in mice.
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NY) supplemented with 10% fetal calf serum and penicillin (100 units/ml; Sigma, St. Louis) and streptomycin (100 Itg/ml; Sigma) as previously described [7]. Monolayers were coincubated for 1 h with tissue culture organisms, washed to remove extracellular organisms, and then incubated an additional 48-72 h. After Giemsa staining, the percentage of macrophages infected and number of organisms per infected macrophage were calculated. Pathology. Sections of brain, heart, skeletal muscle, diaphragm, liver, spleen, lung, stomach, pancreas, kidney, ureter, and omental fat were stained with hematoxylin and eosin and examined for nests of T. cruzi by a blinded observer. Statistics. The Mann-Whitney U test was used to determine differences in mortality, and Student's t test was used to assess differences in parasitemia and infection of macrophages.
Effectofanti-IFN-"( on parasitemia and mortality. Administration of one or two doses of 25,000 units of anti-If'Ney after infection with 50,000 bloodform trypomastigotes produced a five- to eightfold increase in peak parasitemia in BALB/c mice. This increase was sustained until the mice died on days 11-13 of infection (figure lA). The pattern of parasitemia in anti-If'Nev-treated mice was similar to that in untreated controls, but peak parasitemias were much higher in the treated mice. All mice treated with anti-IFN-"( died 1-2 days before all control mice died (P < .001). Reduction of the inoculum of T. cruzi to 100 ttypomastigotes produced similar results in that treatment with anti-IFN-"( produced significantly higher (P < .001) parasitemia (data not shown); mortality was not evaluated since mice were sacrificed for histology. Doses of anti-IFN-"( as small as 100 units given ip 24 h after infection significantly increased parasitemia on days 7 and 9 of infection, although there was not a clear-cut doseresponse relationship when 160r>, 400, and 100 units were 2000
0
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compared (data not shown). In an experiment in which mice were treated with 25,000 units ip, 6250 units ip, 6250 units iv, 1600 units ip, and 400 units ip, all but one mouse treated with anti-If'Nvy died before any control mouse died (figure IB; P < .01). Administration of 6250 units 4 and 7 days after infection also significantly increased parasitemia. ip and it administration were about equally effective. Three experiments were done to determine whether administration of 6250 units of anti-IFN-"( 24 h before, at the time of, or 24 h after infection differentially affected parasitemia. The timing of the injection of anti-Il-Nvy did not notably affect parasitemia. Significantly higher parasitemias were present in treated mice compared with controls (data not shown). A single experiment was done to compare different strains of mice with respect to sensitivity to anti-IFN-,,(. The three inbred strains tested, C3H/HEN, BALB/c, and C57Bl/6, all had significantly higher parasitemias on day 7 of infection when treated with 6250 units of anti-IFN -"(than controls (data not shown), as did outbred Swiss Webster mice. All groups of mice treated with anti-IFN-,,( tended to die before controls, C3H/HEN most dramatically. The small numbers of mice (three per group) limited statistical evaluation. However, when results from all mice treated with anti-IFN-,,( were combined and compared with controls, the difference in mortality was significant (P = .01; data not shown). Effect ofanti-IFN-"( on histology. Groups of two BALB/c mice were infected by ip injection with 50,000 bloodform trypomastigotes, treated 24 h later with 6250 units of antiIFN-"(, and sacrificed on day 8 of infection. Liver, spleen, and omental fat from anti-If'Nev-treated mice had 13, 182, and 747 nests of T. cruzi/25 fields (x400), respectively, compared with 3, 18, and 253 nests, respectively, in controls. There were no remarkable differences for heart, skeletal muscle,
controls 25,000 U 2 h post in! 25,000 U 2 hand 96 h post in!
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Figure 1. A, Effect of anti-interferon-v (lFN-')') on parasitemias of BALB/c mice (five per group) treated with 25,000 or 6250 units of . anti-IFN-')' 24 or 24 and 96 h after infection. Compared with controls, P < .001 for all differences in parasitemia of treated mice for days 7, 8, 9, 10, and 11. B, Mortality of BALB/c mice treated intraperitoneally with 25,000, 6250, 1600, and 400 units of anti-If'Nsv (four groups, three mice each) or 6250 units intravenously (three mice) 24 h after infection with 50,000 trypomastigotes; P < .01.
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diaphragm, stomach, brain, lung, pancreas, bladder and ureter, and kidney histology sections with respect to number of nests of T. cruzi, although mice treated with anti-IFN-'Y tended to have more nests of T. cruzi.
Table 1. Effect of administration of anti-interferon-v (IFN-y) on
Effectofadministration ofanti-IFN-'Y on serum IFN activity. Mice infected with T. cruzi consistently had antiviral
Day of infection, macrophage source (mice)
activity in serum by day 3 of infection that persisted until at least day 7. The antiviral titers were very low (9-30 units/50Jll serum sample tested) and did not correlate with treatment with anti-If'Nev, Neutralization experiments indicated that the antiviral activity in control mice was at least partly IFN al {3.
cavity [8]. Peritoneal macrophages harvested 4 and 6 days after infection (50,000 trypomastigotes ip) from Swiss Webster mice treated with 6250 units of anti -IFN-'Y 24 h after infection had significantly diminished ability to inhibit multiplication of T. cruzi in vitro compared with activated macrophages from sham-treated, infected mice (experiment 1, table 1). The anti-To cruzi activity was not completely abrogated by this dose (6250 units) of anti-IFN-'Y in this experiment, since macrophages from anti-If'Nvy-treated mice inhibited multiplication significantly better than resident peritoneal macrophages from sham-treated and sham-infected mice. However, in two additional experiments, the effect of anti-If'N'-y on peritoneal macrophage killing of T. cruzi was complete in that no difference existed between the numbers oforganisms in macrophages from treated infected (for 4 days) mice and resident macrophages from uninfected mice. For example, resident peritoneal macrophages, macrophages from anti-If'Nsv-treated (6250 units) infected mice, and macrophages from untreated infected mice had means (SE) of 9.3 (1.2), 10.2 (0.7), and 1.2 (0.2) amastigotes per infected macrophage, respectively, 48 h after infection in vitro. Since the number of macrophages infected and number of organisms per infected macrophage did not differ significantly immediately after washing extracellular organisms from the monolayers, the results of the foregoing experiments cannot be explained on the basis of ingestion of differing numbers of T. cruzi. The effect of treatment with 6250 units of anti -IFN-'Y 24 h after infection of mice with 50,000 trypomastigotes was not permanent, and macrophages obtained 8 days after infection were activated as assessed by inhibition of multiplication of T. cruzi in vitro. To determine if repeated administration of anti-If'Nvv could prevent activation of macrophages, two separate doses of anti-IFN-'Y were given to mice 24 and 96 h after infection with the lowest inoculum of T. cruzi (1000 bloodform trypomastigotes) sufficient to activate macrophages to inhibit T. cruzi replication in vitro by day 4 of infection. The results (experiment 2, table 1) indicate that the second dose of anti-IFN-'Y significantly inhibited anti-To cruzi activity of macrophages harvested on day 8 of infection. However,
Experiment 1, anti-IFN-y 24 h after infection Day 4 Uninfected Control infected Anti-IFN-y treated Day 6 Uninfected Control infected Anti-IFN-y treated Experiment 2, anti-IFN-y 24 and 96 h after infection Day 8 Uninfected Control infected Anti-IFN-y treated Experiment 3, anti-IFN-y 24, 96, and 192 h after infection Day 13 Uninfected Control infected Anti-IFN-y treated
% macrophages infected
No. of organisms/ infected macrophage
53 ± 7 16 ± 8 38 ± 2
7.3 ± 0.3 2.0 ± 0.3 4.8 ± 0.7
46 ± 0.8 3 ± 0.8 34 ± 11
4.3 ± 0.2 1.1 ± 0.1 1.8 ± 0.3
27 ± 2 3 ± 2 28 ± 5
8.2 ± 0.5 1.0 ± 0.02 2.8 ± 0.4
30 ± 2 2 ± 0.1
6.7.± 1.1 1.2 ± 0.1 1.0 ± 0.1
4
± 1
NOTE. Data are representative results of two experiments each (mean ± SE). In each experiment, 6250 units of anti-IFN-y per injection were given intraperitoneally.
a third dose on day 8 did not significantly diminish 'the inhibitory activity of activated macrophages on the multiplication of T. cruzi in macrophages harvested 13 days after infection. Experiments by ourselves (data not shown) and others [9] indicate that IFN-'Y-neutralizing activity persists in serum for at least 4-5 days after ip injection of 6250 units of anti-If'Nvy.
Discussion Administration of recombinant IFN-'Y to mice infected with
T. cruzi has been shown to diminish parasitemia, prolong survival, and prevent death in acute T. cruzi infection [8, 10]. Peritoneal macrophages from mice injected with recombinant IFN-'Y become activated to inhibit replication of T. cruzi in vitro, and incubation of murine macrophages with IFN-'Y enhances intracellular killing of T. cruzi [8, 10]. Anti-IFN-'Y has been used to define the relationship between endogenous IFN-'Y and protection against pathogens. For example, treatment with anti-IFN-'Y caused 100% mortality from challenge with a relatively avirulent strain of Tox. gondii [11]. Peritoneal macrophages from infected mice treated with anti-IFN-'Y allowed replication of Tox. gondii in vitro, indicating diminished macrophage activation. This study indicates that endogenous IFN-'Y is critical to
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Effect ofadministration ofanti-IFN-'Y on capacity ofmacrophages to inhibit T. cruzi replication. After 4 days of infection with the Y strain of T. cruzi, macrophages that can kill T..cruzi in vitro appear in abundance in the peritoneal
capacity of peritoneal macrophages from infected mice to inhibit Trypanosoma cruzi replication in vitro.
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Acknowledgment We thank Jon Green for establishing and interpreting the interferon assays and for reviewing the manuscript.
References
1. Murray HW. Interferon-gamma, the activated macrophage, and host defense against microbial challenge. Ann Intern Med 1988;108:595-608. 2. Melo RC, Brener Z. TIssuetropism of different Trypanosoma cruz; strains. 1 Parasitol 1978;64:475-82. 3. Brener Z. Therapeutic activity and criterion of cure in mice experimentally infected with Trypanosoma cruti. Rev Inst Med Trop Sao Paulo 1962;4:389-96. 4. McCabe RE, Remington IS, Araujo FG. Ketoconazole inhibition of intracellular multiplication of Trypanosoma cruzi and protection of mice against lethal infection with the organism. 1 Infect Dis 1984;150: 594-601. 5. Spitalny GL. Monoclonal antibody to murine gamma interferon inhibits lymphokine-induced antiviral and macrophage tumoricidal activities. 1 Exp Med 1984;159:1560-5. 6. Yeh TJ, McBride PT, Overall lC, Green lA. Automated, quantitative cytopathic effect reduction assay for interferon. 1 Clin Microbiol 1982;16:413-5. 7. McLeod R, Remington IS. Studies on the specificity of killing of intracellular pathogens by macrophages. Cell ImmunoI1977;34:156-74. 8. McCabe RE, Remington IS, Araujo FG. Enhancement of resistance to Trypanosoma cruzi infection by recombinant interferon gamma. In: Byrne G, Turco 1, eds. Interferon and nonviral pathegens, New York: Marcel Dekker, 1988:203-15. 9. Li H, lerrells TR, Spitalny GL, Walker DH. Gamma interferon as a crucial host defense against Rickettsiaconorii in vivo. Infect Immun 1987;55:1252-5. 10. Reed SG. In vivo administration of recombinant IFN-gamma induces macrophage activation and prevents acute disease, immune suppression, and death in experimental Trypanosoma cruzi infections. J Immunol 1988;140:4342-7. 11. Suzuki Y, Orellana MA, Schreiber RD, Remington JS. Interferon-gamma: the major mediator of resistance against Toxoplasma gondii. Science 1988;240:516-8. 12. McCabeRE, MeagherS, MullinsB. Trypanosoma cruzi: explant organ cultures from mice with chronic Chagas' disease. Exp Parasitol 1989;68:462-9. 13. Schofield L, Villaquiran J, Ferreira A, Schellekens H, Nussenzweig R, Nussenzweig V. Gamma interferon, CDS+ T cells and antibodies required for immunity to malaria sporozoites. Nature 1987;330:664-6. 14. Kierszenbaum F, Sonnenfeld G. Characterization of the antiviral activity produced during Trypanosoma cruii infection and protective effects of exogenous interferon against experimental Chagas' disease. J Parasitol 1982;68:194-8. 15. Quan PC, Rager-Zisman B, Wittner M, Tanowitz HB. Interferon and natural killer cells in murine Chagas' disease. J Parasitol 1983;69: 1164-6.
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murine host defense against acute T. cruzi infection. Treatment with anti-IFN--y exacerbated parasitemia and significantly accelerated time to death. The route or timing of administration of anti-IFN-"( near the time of infection did not markedly affect results, and four strains of mice, including outbred mice, which have different susceptibilities to T. cruzi [12], responded similarly. Of interest is that the doses of anti-IFN-"( necessary to exacerbate parasitemia and to accelerate time to death were comparable to doses of IFN-"( needed to protect against T. cruzi in vivo [8, 10]. Tissue parasitization with T. cruzi was greater for liver, spleen, and omental fat in anti-If'Ney-treated mice. Differences in parasitization of other organs was not as apparent. IFN-"( may be more critical for control of infection in reticuloendothelial organs such as liver and spleen than in other organs. Rickettsial infection of liver and spleen previously was found to be greater in anti-If'Nvy-treated mice [9], and the effectof anti-IFN-"(was on organisms in the liver in the malaria model [13]. Alternatively, T. cruzi infection might be more intense in liver and spleen, as suggested by the presenct? of more organisms in spleen and liver sections than in other organs (data not shown). Anti-IFN-"( may disproportionately promote replication in more heavily parasitized tissues. Anti-IFN-"( treatment delayed activation of peritoneal macrophages to inhibit T. cruzi replication in vitro. In some experiments, macrophage ability to limit T. cruzi replication appeared to be completely abolished by 6250 units of antiIFN-"( when macrophages were tested early in the course of infection. However, with the doses of anti-If'Nvy used, ultimate activation was not prevented even by repeated administration of anti-If'Ney. Thus initial activation of macrophages by T. cruzi infection appears to be mediated primarily by IFN-"(. Later in the course of infection ojher macrophage-activating factors may play roles, for example, tumor necrosis factor. Antiviral activity was detected in serum of both anti-IFN"(-treated and untreated infected mice but at very low titers. Low serum titers of IFN activity, suspected to be primarily IFN a/{3, have been detected by other investigators [14, 15] in mice acutely infected with T. cruzi. Our results indicate that acute infection results in detectable serum levels of IFN a/{3 but do not eliminate the possibility that IFN-"( is present in serum in amounts below the sensitivity of our assay system.
915
Endogenous Interferon-y, Macrophage Activation, and Murine Host Defense against Acute Infection with Trypanosoma cruzi Robert E. McCabe, Stephen G. Meagher, and Brian T. Mullins
Medical Service, Martinez VA Medical Center, Martinez, California; Department of Medicine, University of California Medical School, Davis
Activated macrophages are considered important for host defense against a number of human intracellular pathogens, including Trypanosoma cruzi, Listeria monocytogenes, Chlamydia, Leishmania, and Plasmodium species, and Toxoplasma gondii [1]. The importance of activated macrophages is indicated by their ability to kill or inhibit multiplication of organisms in vitro and the presence of activated macro phages in hosts with active infection ~ Lymphokines, particularly interferon-v (lFN-1'), can activate macrophages in vivo and in vitro [1]. The availability of antibody that selectively neutralizes IFN -1''s ability to activate macrophages in vivo makes it possible to evaluate more directly the role of endogenous IFN-1' in macrophage activation and control of infection. We examined the role of endogenous IFN-1' and macrophage activation in the pathogenesis of early acute rhurine infection with
T. cruzi.
Materials and Methods Mice. BALB/c (Bantin and Kingman, Fremont, CA), Swiss Webster (Bantin and Kingman; National Cancer Institute, Bethesda, MD), and C57Bl/6 and C3H/HEN (National Cancer Institute) mice weighing 18-22 g each were used in all experiments. T. cruzi. The Y strain was used in all experiments [2]. Bloodform trypomastigotes obtained from Swiss Webster mice were used
Received 17 April 1990; revised 16 November 1990. Presented in part: 29th Interscience Conference on Antimicrobial Agents and Chemotherapy, Houston, September 1989 (abstract 1205). Financial support: Research Service, Department of Veterans Affairs. Reprintsor correspondence: Dr. Robert McCabe, Medical Service (111F), Martinez VAMC, 150 Muir Rd., Martinez, CA 94553. The Journal of Infectious Diseases 1991;163:912-915 © 1991 by The University of Chicago. AU rights reserved. 0022-1899/91/6304-0045$01.00
to infect mice. Parasitemias were determined by inspection of blood smears with a light microscope [3]. Tissue culture organisms used to infect macrophages were obtained from cultures of L929 cells (ATCC, Rockville, MD) as previously described [4]. Anti-IFN-y Anti-IFN-')' was prepared from a rat hybridpma that secretes IgGI antibody to murine IFN-')' (R4-6A2, HB170; ATCC) [5]. The ammonium sulfate precipitate of hybridom a media was dialyzed against PBS and then filter sterilized. The protein concentration of the final preparation, determined with a kit (Bio-Rad, Richmond, CA), was 21.5 mg/ml. The preparation was titered for neutralizing activity against recombinant IFN-')' (gift of Genentech, South San Francisco, CA) (see IFN Assay). Neutralization was defined as reduction of antiviral activity of a specimen containing 10 units ofIFN-')' to undetectable levels (no activity in the 1:2 dilution of specimen). The preparation used in the experiments reported here had 25,000 units of anti-IFN-')' activity per milligram of protein. Anti-IFN-')' was injected intraperitoneally (ip) or intravenously (iv) into mice. Rat serum, PBS, boiled anti-If'Nvy, or an irrelevant monoclonal antibody from a hybridoma (anti-DR5; ATCC) prepared similarly to anti-IFN-')' were used as controls for injection. Preliminary experiments (data not shown) showed no differences between PBS, rat serum, boiled anti-If'Ns-y, or the irrelevant monoclonal antibody with respect to either levels of parasitemia or activation of macrophages. IFN assay. A microtiter photometric assay [6] in which threefold dilutions of the sample to be tested for IFN are added to confluent L929 cells in 96-well microculture plates (Costar, Cambridge, MA) and then challenged with encephalomyocarditis virus was used. After 90 % destruction of control monolayers by virus, monolayers were stained and air dried, and retained dye was quantitated by optical density readings (Titertek Multiskan; Flow Laboratories, McLean, VA) with a 600-nm filter. Anti-IFN-')' (ATCC) and anti-o/d antiserum (Lee Biologics, San Diego) were used to identify the type of IFN. Infection ofmacrophage mono/ayers with T. cruzi. Mouse peritoneal macrophage monolayers were cultured in duplicate replicates in eight-well tissue culture chambers (Labtek Products, Naperville, IL) with RPMI 1640 tissue culture medium (GmCO, Grand Island,
Downloaded from http://jid.oxfordjournals.org/ at University of Sussex on September 6, 2015
Parenteral interferon-v (IFN-')') activates murine macrophages to inhibit Trypanosoma cruz; multiplication and diminishes parasitemia and mortality in acute infection. To investigate the role of endogenous IFN-')' in acute infection, monoclonal antibody to IFN-')' was injected intraperitoneally into mice. The 6250 neutralizing units given 24 and 96 h after infection reproducibly increased mortality (P < .05). Histology sections showed markedly more nests of T. cruz; in treated mice. BALB/c, Swiss Webster, C57Bl/6, and C3H/HEN mice were susceptible to the effects of anti-IFN-')'. Peritoneal macrophages from mice 4 days after infection and a single dose of 6250 units of anti-IFN-')' had significantly reduced ability to inhibit T. cruz; multiplication. Multiple doses of anti-IFN-')' delayed but did not prevent macrophage activation. These results indicate the critical role of endogenous IFN-')' for macrophage activation and host defense against acute T. cruz; infection in mice.
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NY) supplemented with 10% fetal calf serum and penicillin (100 units/ml; Sigma, St. Louis) and streptomycin (100 Itg/ml; Sigma) as previously described [7]. Monolayers were coincubated for 1 h with tissue culture organisms, washed to remove extracellular organisms, and then incubated an additional 48-72 h. After Giemsa staining, the percentage of macrophages infected and number of organisms per infected macrophage were calculated. Pathology. Sections of brain, heart, skeletal muscle, diaphragm, liver, spleen, lung, stomach, pancreas, kidney, ureter, and omental fat were stained with hematoxylin and eosin and examined for nests of T. cruzi by a blinded observer. Statistics. The Mann-Whitney U test was used to determine differences in mortality, and Student's t test was used to assess differences in parasitemia and infection of macrophages.
Effectofanti-IFN-"( on parasitemia and mortality. Administration of one or two doses of 25,000 units of anti-If'Ney after infection with 50,000 bloodform trypomastigotes produced a five- to eightfold increase in peak parasitemia in BALB/c mice. This increase was sustained until the mice died on days 11-13 of infection (figure lA). The pattern of parasitemia in anti-If'Nev-treated mice was similar to that in untreated controls, but peak parasitemias were much higher in the treated mice. All mice treated with anti-IFN-"( died 1-2 days before all control mice died (P < .001). Reduction of the inoculum of T. cruzi to 100 ttypomastigotes produced similar results in that treatment with anti-IFN-"( produced significantly higher (P < .001) parasitemia (data not shown); mortality was not evaluated since mice were sacrificed for histology. Doses of anti-IFN-"( as small as 100 units given ip 24 h after infection significantly increased parasitemia on days 7 and 9 of infection, although there was not a clear-cut doseresponse relationship when 160r>, 400, and 100 units were 2000
0
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compared (data not shown). In an experiment in which mice were treated with 25,000 units ip, 6250 units ip, 6250 units iv, 1600 units ip, and 400 units ip, all but one mouse treated with anti-If'Nvy died before any control mouse died (figure IB; P < .01). Administration of 6250 units 4 and 7 days after infection also significantly increased parasitemia. ip and it administration were about equally effective. Three experiments were done to determine whether administration of 6250 units of anti-IFN-"( 24 h before, at the time of, or 24 h after infection differentially affected parasitemia. The timing of the injection of anti-Il-Nvy did not notably affect parasitemia. Significantly higher parasitemias were present in treated mice compared with controls (data not shown). A single experiment was done to compare different strains of mice with respect to sensitivity to anti-IFN-,,(. The three inbred strains tested, C3H/HEN, BALB/c, and C57Bl/6, all had significantly higher parasitemias on day 7 of infection when treated with 6250 units of anti-IFN -"(than controls (data not shown), as did outbred Swiss Webster mice. All groups of mice treated with anti-IFN-,,( tended to die before controls, C3H/HEN most dramatically. The small numbers of mice (three per group) limited statistical evaluation. However, when results from all mice treated with anti-IFN-,,( were combined and compared with controls, the difference in mortality was significant (P = .01; data not shown). Effect ofanti-IFN-"( on histology. Groups of two BALB/c mice were infected by ip injection with 50,000 bloodform trypomastigotes, treated 24 h later with 6250 units of antiIFN-"(, and sacrificed on day 8 of infection. Liver, spleen, and omental fat from anti-If'Nev-treated mice had 13, 182, and 747 nests of T. cruzi/25 fields (x400), respectively, compared with 3, 18, and 253 nests, respectively, in controls. There were no remarkable differences for heart, skeletal muscle,
controls 25,000 U 2 h post in! 25,000 U 2 hand 96 h post in!
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Figure 1. A, Effect of anti-interferon-v (lFN-')') on parasitemias of BALB/c mice (five per group) treated with 25,000 or 6250 units of . anti-IFN-')' 24 or 24 and 96 h after infection. Compared with controls, P < .001 for all differences in parasitemia of treated mice for days 7, 8, 9, 10, and 11. B, Mortality of BALB/c mice treated intraperitoneally with 25,000, 6250, 1600, and 400 units of anti-If'Nsv (four groups, three mice each) or 6250 units intravenously (three mice) 24 h after infection with 50,000 trypomastigotes; P < .01.
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diaphragm, stomach, brain, lung, pancreas, bladder and ureter, and kidney histology sections with respect to number of nests of T. cruzi, although mice treated with anti-IFN-'Y tended to have more nests of T. cruzi.
Table 1. Effect of administration of anti-interferon-v (IFN-y) on
Effectofadministration ofanti-IFN-'Y on serum IFN activity. Mice infected with T. cruzi consistently had antiviral
Day of infection, macrophage source (mice)
activity in serum by day 3 of infection that persisted until at least day 7. The antiviral titers were very low (9-30 units/50Jll serum sample tested) and did not correlate with treatment with anti-If'Nev, Neutralization experiments indicated that the antiviral activity in control mice was at least partly IFN al {3.
cavity [8]. Peritoneal macrophages harvested 4 and 6 days after infection (50,000 trypomastigotes ip) from Swiss Webster mice treated with 6250 units of anti -IFN-'Y 24 h after infection had significantly diminished ability to inhibit multiplication of T. cruzi in vitro compared with activated macrophages from sham-treated, infected mice (experiment 1, table 1). The anti-To cruzi activity was not completely abrogated by this dose (6250 units) of anti-IFN-'Y in this experiment, since macrophages from anti-If'Nvy-treated mice inhibited multiplication significantly better than resident peritoneal macrophages from sham-treated and sham-infected mice. However, in two additional experiments, the effect of anti-If'N'-y on peritoneal macrophage killing of T. cruzi was complete in that no difference existed between the numbers oforganisms in macrophages from treated infected (for 4 days) mice and resident macrophages from uninfected mice. For example, resident peritoneal macrophages, macrophages from anti-If'Nsv-treated (6250 units) infected mice, and macrophages from untreated infected mice had means (SE) of 9.3 (1.2), 10.2 (0.7), and 1.2 (0.2) amastigotes per infected macrophage, respectively, 48 h after infection in vitro. Since the number of macrophages infected and number of organisms per infected macrophage did not differ significantly immediately after washing extracellular organisms from the monolayers, the results of the foregoing experiments cannot be explained on the basis of ingestion of differing numbers of T. cruzi. The effect of treatment with 6250 units of anti -IFN-'Y 24 h after infection of mice with 50,000 trypomastigotes was not permanent, and macrophages obtained 8 days after infection were activated as assessed by inhibition of multiplication of T. cruzi in vitro. To determine if repeated administration of anti-If'Nvv could prevent activation of macrophages, two separate doses of anti-IFN-'Y were given to mice 24 and 96 h after infection with the lowest inoculum of T. cruzi (1000 bloodform trypomastigotes) sufficient to activate macrophages to inhibit T. cruzi replication in vitro by day 4 of infection. The results (experiment 2, table 1) indicate that the second dose of anti-IFN-'Y significantly inhibited anti-To cruzi activity of macrophages harvested on day 8 of infection. However,
Experiment 1, anti-IFN-y 24 h after infection Day 4 Uninfected Control infected Anti-IFN-y treated Day 6 Uninfected Control infected Anti-IFN-y treated Experiment 2, anti-IFN-y 24 and 96 h after infection Day 8 Uninfected Control infected Anti-IFN-y treated Experiment 3, anti-IFN-y 24, 96, and 192 h after infection Day 13 Uninfected Control infected Anti-IFN-y treated
% macrophages infected
No. of organisms/ infected macrophage
53 ± 7 16 ± 8 38 ± 2
7.3 ± 0.3 2.0 ± 0.3 4.8 ± 0.7
46 ± 0.8 3 ± 0.8 34 ± 11
4.3 ± 0.2 1.1 ± 0.1 1.8 ± 0.3
27 ± 2 3 ± 2 28 ± 5
8.2 ± 0.5 1.0 ± 0.02 2.8 ± 0.4
30 ± 2 2 ± 0.1
6.7.± 1.1 1.2 ± 0.1 1.0 ± 0.1
4
± 1
NOTE. Data are representative results of two experiments each (mean ± SE). In each experiment, 6250 units of anti-IFN-y per injection were given intraperitoneally.
a third dose on day 8 did not significantly diminish 'the inhibitory activity of activated macrophages on the multiplication of T. cruzi in macrophages harvested 13 days after infection. Experiments by ourselves (data not shown) and others [9] indicate that IFN-'Y-neutralizing activity persists in serum for at least 4-5 days after ip injection of 6250 units of anti-If'Nvy.
Discussion Administration of recombinant IFN-'Y to mice infected with
T. cruzi has been shown to diminish parasitemia, prolong survival, and prevent death in acute T. cruzi infection [8, 10]. Peritoneal macrophages from mice injected with recombinant IFN-'Y become activated to inhibit replication of T. cruzi in vitro, and incubation of murine macrophages with IFN-'Y enhances intracellular killing of T. cruzi [8, 10]. Anti-IFN-'Y has been used to define the relationship between endogenous IFN-'Y and protection against pathogens. For example, treatment with anti-IFN-'Y caused 100% mortality from challenge with a relatively avirulent strain of Tox. gondii [11]. Peritoneal macrophages from infected mice treated with anti-IFN-'Y allowed replication of Tox. gondii in vitro, indicating diminished macrophage activation. This study indicates that endogenous IFN-'Y is critical to
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Effect ofadministration ofanti-IFN-'Y on capacity ofmacrophages to inhibit T. cruzi replication. After 4 days of infection with the Y strain of T. cruzi, macrophages that can kill T..cruzi in vitro appear in abundance in the peritoneal
capacity of peritoneal macrophages from infected mice to inhibit Trypanosoma cruzi replication in vitro.
JID 1991;163 (April)
Concise Communications
Acknowledgment We thank Jon Green for establishing and interpreting the interferon assays and for reviewing the manuscript.
References
1. Murray HW. Interferon-gamma, the activated macrophage, and host defense against microbial challenge. Ann Intern Med 1988;108:595-608. 2. Melo RC, Brener Z. TIssuetropism of different Trypanosoma cruz; strains. 1 Parasitol 1978;64:475-82. 3. Brener Z. Therapeutic activity and criterion of cure in mice experimentally infected with Trypanosoma cruti. Rev Inst Med Trop Sao Paulo 1962;4:389-96. 4. McCabe RE, Remington IS, Araujo FG. Ketoconazole inhibition of intracellular multiplication of Trypanosoma cruzi and protection of mice against lethal infection with the organism. 1 Infect Dis 1984;150: 594-601. 5. Spitalny GL. Monoclonal antibody to murine gamma interferon inhibits lymphokine-induced antiviral and macrophage tumoricidal activities. 1 Exp Med 1984;159:1560-5. 6. Yeh TJ, McBride PT, Overall lC, Green lA. Automated, quantitative cytopathic effect reduction assay for interferon. 1 Clin Microbiol 1982;16:413-5. 7. McLeod R, Remington IS. Studies on the specificity of killing of intracellular pathogens by macrophages. Cell ImmunoI1977;34:156-74. 8. McCabe RE, Remington IS, Araujo FG. Enhancement of resistance to Trypanosoma cruzi infection by recombinant interferon gamma. In: Byrne G, Turco 1, eds. Interferon and nonviral pathegens, New York: Marcel Dekker, 1988:203-15. 9. Li H, lerrells TR, Spitalny GL, Walker DH. Gamma interferon as a crucial host defense against Rickettsiaconorii in vivo. Infect Immun 1987;55:1252-5. 10. Reed SG. In vivo administration of recombinant IFN-gamma induces macrophage activation and prevents acute disease, immune suppression, and death in experimental Trypanosoma cruzi infections. J Immunol 1988;140:4342-7. 11. Suzuki Y, Orellana MA, Schreiber RD, Remington JS. Interferon-gamma: the major mediator of resistance against Toxoplasma gondii. Science 1988;240:516-8. 12. McCabeRE, MeagherS, MullinsB. Trypanosoma cruzi: explant organ cultures from mice with chronic Chagas' disease. Exp Parasitol 1989;68:462-9. 13. Schofield L, Villaquiran J, Ferreira A, Schellekens H, Nussenzweig R, Nussenzweig V. Gamma interferon, CDS+ T cells and antibodies required for immunity to malaria sporozoites. Nature 1987;330:664-6. 14. Kierszenbaum F, Sonnenfeld G. Characterization of the antiviral activity produced during Trypanosoma cruii infection and protective effects of exogenous interferon against experimental Chagas' disease. J Parasitol 1982;68:194-8. 15. Quan PC, Rager-Zisman B, Wittner M, Tanowitz HB. Interferon and natural killer cells in murine Chagas' disease. J Parasitol 1983;69: 1164-6.
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murine host defense against acute T. cruzi infection. Treatment with anti-IFN--y exacerbated parasitemia and significantly accelerated time to death. The route or timing of administration of anti-IFN-"( near the time of infection did not markedly affect results, and four strains of mice, including outbred mice, which have different susceptibilities to T. cruzi [12], responded similarly. Of interest is that the doses of anti-IFN-"( necessary to exacerbate parasitemia and to accelerate time to death were comparable to doses of IFN-"( needed to protect against T. cruzi in vivo [8, 10]. Tissue parasitization with T. cruzi was greater for liver, spleen, and omental fat in anti-If'Ney-treated mice. Differences in parasitization of other organs was not as apparent. IFN-"( may be more critical for control of infection in reticuloendothelial organs such as liver and spleen than in other organs. Rickettsial infection of liver and spleen previously was found to be greater in anti-If'Nvy-treated mice [9], and the effectof anti-IFN-"(was on organisms in the liver in the malaria model [13]. Alternatively, T. cruzi infection might be more intense in liver and spleen, as suggested by the presenct? of more organisms in spleen and liver sections than in other organs (data not shown). Anti-IFN-"( may disproportionately promote replication in more heavily parasitized tissues. Anti-IFN-"( treatment delayed activation of peritoneal macrophages to inhibit T. cruzi replication in vitro. In some experiments, macrophage ability to limit T. cruzi replication appeared to be completely abolished by 6250 units of antiIFN-"( when macrophages were tested early in the course of infection. However, with the doses of anti-If'Nvy used, ultimate activation was not prevented even by repeated administration of anti-If'Ney. Thus initial activation of macrophages by T. cruzi infection appears to be mediated primarily by IFN-"(. Later in the course of infection ojher macrophage-activating factors may play roles, for example, tumor necrosis factor. Antiviral activity was detected in serum of both anti-IFN"(-treated and untreated infected mice but at very low titers. Low serum titers of IFN activity, suspected to be primarily IFN a/{3, have been detected by other investigators [14, 15] in mice acutely infected with T. cruzi. Our results indicate that acute infection results in detectable serum levels of IFN a/{3 but do not eliminate the possibility that IFN-"( is present in serum in amounts below the sensitivity of our assay system.
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