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The Journal of Immunology, 1998, 161: 5507-5515.
Copyright © 1998 by The American Association of Immunologists

Experimental Murine Trypanosoma congolense Infections. I. Administration of Anti-IFN-{gamma} Antibodies Alters Trypanosome-Susceptible Mice to a Resistant-Like Phenotype1

Jude E. Uzonna, Radhey S. Kaushik, John R. Gordon and Henry Tabel2

Department of Veterinary Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The mechanisms regulating resistance or susceptibility to African trypanosomes have been enigmatic. In this study, we assessed the production of several cytokines (IL-4, IFN-{gamma}, and TNF-{alpha}) in vivo and in vitro using genetically susceptible (BALB/c) or resistant (C57BL/6) mice infected with cloned Trypanosoma congolense and the role of these cytokines in pathogenesis of this infection. Plasma of infected BALB/c mice contained higher levels of IL-4 and IFN-{gamma} than the plasma of infected C57BL/6 mice. Conversely, plasma TNF-{alpha} levels were elevated significantly in the resistant mice relative to the susceptible ones. Splenic IFN-{gamma} mRNA appeared earlier and were maintained at higher levels in infected BALB/c than in C57BL/6 mice. Both spontaneous and Con A-induced secretions of IL-4 and IFN-{gamma} by splenocytes from infected BALB/c mice were significantly higher than those from their C57BL/6 counterparts. Con A-induced proliferation of splenocytes from infected BALB/c mice was progressively suppressed. Nitric oxide was not involved in this suppression, but the suppression was positively correlated with IFN-{gamma} secretion. Addition of neutralizing Abs to IFN-{gamma} to cultures of Con A-stimulated spleen cells from infected BALB/c mice effectively reversed this suppression. Furthermore, administration of anti-IFN-{gamma} Abs to BALB/c mice early during infection dramatically shifted the phenotype of these susceptible mice to a more resistant-like phenotype, as expressed by a low and undulating parasitemia and a >300% increase in survival period. These results strongly suggest that the enhanced induction and secretion of IFN-{gamma} during T. congolense infections contribute to the relative susceptibility of BALB/c mice to the disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
African trypanosomes are extracellular hemoprotozoan parasites that cause disease and death in humans and animals in the subSaharan Africa. A remarkable feature of salivarian trypanosomes is their ability to continually change their surface protein coat, and thereby effectively evade the host’s immune response (reviewed in Refs. 1–3). Infections are associated with profound aberrations in the host’s immune system, including a generalized immunosuppression that affects both the humoral and cellular immune compartments (reviewed in Refs. 4–7). This suppression encompasses responses to parasite-related and unrelated Ags and mitogens and is responsible for the increased susceptibility of infected animals to opportunistic infections as well as for the poor responsiveness of parasite-bearing animals to vaccinations against unrelated pathogens (8).

The indigenous West African N’dama breed of cattle are more resistant than the Zebu breed to both natural and experimental infections with Trypanosoma congolense (reviewed in Refs. 9 and 10). Similarly, in a mouse model system, BALB/c mice are highly susceptible to experimental T. congolense infections, whereas C57BL/6 mice are relatively resistant, as measured by the levels of parasitemia and immunosuppression and survival periods (11, 12, 13). Genetic analyses have shown that differences in resistance to related Trypanosoma brucei infections in mice are not due to differences in the MHC genes (10, 14), and that the survival period of BALB/c mice infected with T. congolense is significantly correlated with the speed of control of the first wave of parasitemia (13). The mechanisms underlying the differences in resistance among different breeds of cattle or among inbred mouse strains are poorly understood.

The patterns of cytokine responses during some parasite infections determine or are at least strongly correlated to the relative susceptibility of the host (reviewed in 15 , although the role(s) of individual cytokines in resistance to trypanosomal infections remains equivocal. IFN-{gamma} is expressed just preceding the peak of parasitemia in mice infected with T. brucei (16). It has been associated with immunosuppression mediated, in part, by stimulation of nitric oxide (NO)3 production by macrophages (17, 18), although Darji et al. (19) did not observe any NO-mediated immunosuppression caused by IFN-{gamma}. IFN-{gamma} has also been associated with enhanced host susceptibility to T. brucei infections, and this effect is related to its direct stimulatory effect on the growth of T. brucei (20). On the other hand, IFN-{gamma} also has been reported to mediate parasite control and host protection (21). TNF-{alpha} appears to play a protective role during the initial phase of experimental infection with T. brucei brucei in mice (22). It has been shown to have a direct lytic effect on T. brucei (23, 24). More recently, the expression of IL-4 mRNA was shown to be higher in the relatively resistant C57BL/6 than in susceptible C3H mice infected with T. brucei brucei, and it was suggested that the differential expression of this cytokine might be responsible for the observed differences in resistance between these inbred mouse strains (25). To further document these interstrain differences, we examined the expression of IL-4, IFN-{gamma}, and TNF-{alpha} in mice infected with T. congolense and the roles of the former two cytokines in susceptibility to this parasite. We show in this study that following infection with T. congolense, BALB/c and C57BL/6 mice differentially express each of these cytokines, such that the plasma of the highly susceptible BALB/c mice contained significantly more IL-4 and IFN-{gamma} and less TNF-{alpha} than that of the relatively resistant C57BL/6 mice. The splenocytes from infected BALB/c mice contained more IFN-{gamma} mRNA and produced much more IL-4 and IFN-{gamma} protein than those of the resistant C57BL/6 mice. The proliferative responses to Con A of splenocytes from infected BALB/c mice were progressively suppressed during infection (26) and showed an inverse relationship with IFN-{gamma} production. The addition of neutralizing anti-IFN-{gamma} Abs into such cultures restored the proliferative responses. Furthermore, in vivo, anti-IFN-{gamma} Ab treatments, but not anti-IL-4 treatments, of infected BALB/c mice significantly reduced the parasitemia of these highly susceptible mice and increased their life span relative to untreated or control Ab-treated mice by more than 300%.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Female BALB/cAnNCrlBR (BALB/c) and outbred CD1 mice were obtained from the Animal Resource Center of the University of Saskatchewan (Saskatoon, Canada). Female C57BL/6NCrlBR (C57BL/6) mice were purchased from Charles River Laboratories (St. Constant, Quebec, Canada). The BALB/c and C57BL/6 mice were 8–10 wk of age and the CD1 mice were 5–6 wk old. All mice were maintained according to the recommendations of the Guide for the Care and Use of Experimental Animals of the Canadian Council of Animal Care.

Parasites

The origin of the T. congolense, variant antigenic type TC13 used in the present study, has been previously described (27). Cloned trypanosome populations were stored as frozen stabilates in liquid nitrogen. Parasites were passaged in CD1 mice, as previously described (27). The parasites for infection of BALB/c and C57BL/6 mice were isolated from the blood of CD1 mice 3 days after passage by DEAE anion-exchange chromatography (28).

Experimental design

Groups of four to six BALB/c or C57BL/6 mice were infected i.p. with 103 organisms of T. congolense variant antigenic type TC13 and killed with CO2 on days 1 to 10 postinfection. In some experiments, infected mice were treated with Berenil (14 mg/kg of diminazene aceturate; Sigma, St. Louis, MO) on days 6 and 7 postinfection to cure their infections. On each day indicated, blood was withdrawn from the caudal vena cava into syringes containing heparin (final concentration ~20 IU/ml), and the plasma was collected after centrifuging the blood at 1,000 x g for 30 min. The plasma was centrifuged at 13,000 x g for 15 min, and the supernatant plasma was stored at -35°C until used. Each experiment (and cytokine determination) was performed at least twice.

Estimation of parasitemia and survival period

To estimate the circulating parasite numbers, a drop of blood from the tail vein of each infected mouse was examined at x400 magnification by phase-contrast microscopy. Parasitemia was most often estimated by counting the number of parasites present in at least 10 fields, but unusually heavy parasite loads were quantified according to Herbert and Lumsden (29). The survival period was defined as the number of days postinfection that the infected mice remained alive. Moribund mice were euthanized with CO2.

Splenocyte cultures for measurement of cytokine synthesis

Single cell suspensions of spleen cells from normal and infected mice were adjusted to 5 x 106 cells/ml of complete medium (RPMI 1640 supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 50 mM 2-ME, and 100 IU/ml penicillin/streptomycin) and cultured in 96-well plates (Falcon VWR, Edmonton, Alberta, Canada) at 0.2 ml/well in a humidified 37°C, 5% CO2 atmosphere. Some wells were stimulated with 5 µg/ml Con A (Calbiochem, Behring Diagnostics, La Jolla, CA). All culture fluids were collected after 48 h and centrifuged at 1500 x g for 10 min, and the supernatant fluids were stored at -35°C until used.

Splenocyte proliferation assay

Splenocyte proliferation was quantified by the MTT assay, as described by Mosmann (30). Briefly, cells from infected mice were cultured for 72 h in 200 µl of complete medium at a density of 105 cells/well in 96-well flat-bottom plates (Falcon; Becton Dickinson, Lincoln Park, NJ), using quadruplicate cultures of pooled cells from groups of four mice. Cells were cultured either without or with Con A (5 µg/ml). Some wells were treated with either 5 µg/ml of purified rat anti-mouse IFN-{gamma} mAbs (XMG 1.2; American Type Tissue Collection (ATCC), Manassas, VA), isotype-matched rat IgG1 Ab (Cappel, Organon Teknika, Scarborough, ON, Canada), or 500 µM (final concentration) of NO synthase (NOS) inhibitor, NG-monomethyl-L-arginine (NGMMA; Sigma). After 72 h, we added MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide; Sigma) to a final concentration of 0.48 µg/ml for an additional 3 h. Then the cells were lysed with acidified isopropanol, and the dissolved mitochondrial formazan precipitates were quantified with an ELISA plate reader by measuring the absorbance at 595 nm wavelength. Data are presented as percentage of proliferation relative to splenocytes from uninfected mice stimulated with Con A only.

Recombinant cytokines and cytokine assays

Recombinant murine IL-4 and ELISA kits for the determination of plasma IL-4 were purchased from Endogen (Boston, MA). ELISA kits used for determination of plasma levels of IFN-{gamma} and TNF-{alpha} were purchased from Genzyme Diagnostics (Cambridge, MA). Recombinant murine IL-4, IL-10, IFN-{gamma}, and TNF-{alpha}, and the paired Abs used in our sandwich ELISAs for the determination of IL-4, IL-10, IFN-{gamma}, and TNF-{alpha} in spleen culture supernatants were produced by PharMingen (San Diego, CA). ELISA assays were done according to the manufacturer’s suggested protocols. The functional activity of IFN-{gamma} in the spleen culture supernatants was quantified by the viral cytopathic effect reduction assay, using L929 cells and endomyocarditis virus, as described by Familletti et al. (31). A functional assay was used to detect the plasma TNF-{alpha} bioactivities, as previously described (32).

Quantification of nitrite

NO rapidly reacts with oxygen to form nitrate and nitrite, which are relatively stable in vitro and are therefore useful for NO quantification (33). The concentration of nitrite in supernatant fluids from splenocyte cultures was determined by a standard Griess reaction (34). The detection limit of the test was 1 µM nitrite.

Northern blot analysis

Total cellular RNA was extracted from the spleens of normal and T. congolense-infected BALB/c and C57BL/6 mice from days 1 to 9 postinfection using the guanidinium isothiocyanate/cesium chloride method (35). For each sample, 20 µg of RNA was electrophoresed in a 1.2% denaturing agarose gel, transferred to a nylon membrane (Zetabind; CUNO Lab., Meridien, CT) by capillary action, and cross-linked by UV irradiation (Stratalinker; Stratagene, La Jolla, CA). The membranes were blocked with 0.1x SSC/0.5% SDS for 1 h at 65°C and prehybridized overnight at 42°C in a solution containing 50% deionized formamide, 0.1 M HEPES, 0.5 M NaCl, 5 mM EDTA (pH 8), 5x Denhardt’s solution, 1% SDS, 0.5% sodium pyrophosphate, and 12.5 µg/ml salmon sperm DNA. 32P-labeled cDNA probes were prepared by the random hexamer-labeling technique (Oligolabeling kit; Pharmacia, Uppsala, Sweden), according to the manufacturer’s suggested protocol. The mouse IFN-{gamma} and TNF-{alpha} cDNAs consisted of 550-bp PstI fragment and 990-bp EcoRI fragments, respectively (kindly provided by Dr. John Elliot, University of Alberta, and Dr. Rik Derynck, Genentech, South San Francisco, CA, respectively), while the human {gamma}-actin cDNA comprised a 910-bp EcoRI/HindIII fragment (provided by Drs. B. Murphy and V. Misra, University of Saskatchewan). The membranes were hybridized overnight at 42°C in prehybridization buffer containing 1.5 x 106 cpm/ml of labeled probes, and then washed twice with 2x SSC/0.1% SDS for 5 min at room temperature and another two times for 30 min each with 0.2x SSC/0.1% SDS at 42°C. Autoradiography was performed at -80°C for 1 to 10 days. Densitometric analysis of the autoradiograph signals was conducted using the Image 1 program from Universal Imaging (Westchester, PA).

Anti-IFN-{gamma} and anti-IL-4 Abs for in vivo experiments

Anti-IFN-{gamma} (XMG 1.2, IgG1 isotype; ATCC) and rat anti-mouse IL-4 (11B11, IgG1 isotype; ATCC) hybridomas were grown either in serum- and protein-free medium (Life Technologies, Mississauga, ON, Canada) or complete medium. The hybridoma supernatants, as well as control culture medium (i.e., complete medium), were precipitated with 45% v/v saturated ammonium sulfate solution and dialyzed extensively against PBS (pH 7). Abs were further concentrated with Amicon Centriprep 100 concentrators (Amicon, W. R. Grace and Co., Beverly, MA) and filtered through 0.2-µm syringe filters (Amicon), and protein levels were quantified using the Bradford method (Bio-Rad Protein Assay; Bio-Rad Laboratories, Richmond, CA). The levels of IgG1 Abs in the concentrates were determined by single radial immunodiffusion using goat anti-rat IgG Abs (Cappel) and purified rat IgG1 (Cappel) standards.

Treatment of mice with mAbs

For estimation of parasitemia and survival periods, groups of six BALB/c mice were injected i.p. on days 0, 2, 4, and 6 postinfection with 200 µg of purified anti-IL-4 or anti-IFN-{gamma} Abs in 200–400 µl of PBS. Control mice received PBS alone. Treated and control mice were monitored daily for parasitemia and mortality. To determine the effect of in vivo injections of anti-IFN-{gamma} Abs on cytokine and NO production, groups of four BALB/c mice were injected i.p. with either PBS alone or 200 µg of purified anti-IFN-{gamma} Abs in PBS on days 0, 2, and 4 postinfection. Treated and control mice were killed on day 7 postinfection. Collection of plasma and spleens for splenocyte cultures was conducted as previously described. The levels of IL-10 in the plasma and IL-10 and IFN-{gamma} in culture supernatants were determined by sandwich ELISA. The levels of nitrite in the culture supernatant fluids were determined by a standard Griess reaction (34).

Statistical analysis

Data are represented as means ± SE of the mean. Significance of differences was mostly determined by Student’s t test. Significance of differences in the survival periods was determined by the Kruskal-Wallis one-way nonparametric ANOVA using the StatView.SE 1988 Software (Abacus Concepts, Berkeley, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Parasitemia and survival period

BALB/c mice infected with 103 organisms of T. congolense were unable to control their parasitemia and succumbed with a mean survival time of 8.5 ± 0.5 days (Fig. 1GoA). In contrast, infected C57BL/6 mice controlled their first parasitemia by day 9 postinfection (Fig. 1GoA). The survival time of infected C57BL/6 mice was not determined in this study, but was previously found to be 163 ± 12 days (13).



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  		FIGURE 1. Parasitemia (A) and plasma levels of IL-4 (B), IFN-{gamma} (C), and TNF-{alpha} (D) in BALB/c (closed squares) and C57BL/6 (open squares) mice experimentally infected with 103 organisms of T. congolense. Daily parasitemia was estimated by examining a drop of blood from the tail vein of infected mice on a microscopic slide at x400 magnification by phase-contrast microscopy and counting the number of parasites present in at least 10 fields. Dotted line (A) indicates limit of parasite detection by this method. Plasma samples were collected from four mice on each day postinfection. IL-4, IFN-{gamma}, and TNF-{alpha} levels were determined by sandwich ELISA. Data are presented as mean ± SE (some error bars in B and C are too small to be visible). The results presented are from one of two similar experiments.

 
The levels of IL-4, IFN-{gamma}, and TNF-{alpha} in the plasma of T. congolense-infected BALB/c and C57BL/6 mice differ significantly

The levels of IL-4, IFN-{gamma}, and TNF-{alpha} in the plasma of BALB/c and C57BL/6 mice infected with T. congolense were measured by sandwich ELISA. IL-4 was detected in the plasma of BALB/c mice starting at day 5 postinfection, and the levels rapidly increased to a peak at day 6 (138 pg/ml), and then gradually declined to insignificant levels by day 9 postinfection (Fig. 1GoB). In contrast, the IL-4 levels in the plasma of C57BL/6 mice remained very low or were undetectable throughout the infection period. IFN-{gamma} was first detected on day 5, also peaked on day 6, but then remained high until death in BALB/c mice (Fig. 1GoC). The levels of IFN-{gamma} in the BALB/c mice were consistently significantly higher (p < 0.05) than in those of the C57BL/6 mice. Treatment of infected BALB/c mice with Berenil on days 6 and 7 postinfection was associated with a dramatic decline in the plasma IFN-{gamma} levels on days 8 and 9 postinfection (504 ± 31 pg/ml for untreated mice versus 72 ± 9 pg/ml for treated animals on day 8, p < 0.01; and similarly, 741 ± 10 pg/ml versus 17 ± 9 pg/ml, p < 0.001, on day 9, respectively). TNF-{alpha} levels in plasma samples of normal BALB/c mice (as determined by a commercial ELISA kit) were surprisingly high (80 pg/ml, Fig. 1GoD), although TNF-{alpha} was not detectable in these samples using a functional assay sensitive to 10 pg/ml of TNF-{alpha} (data not shown). Neither the ELISA nor functional assays could detect TNF-{alpha} in the plasma of normal C57BL/6 mice (Fig. 1GoD). The levels of TNF-{alpha} in the infected animals of both strains of mice increased with the progression of the disease, although they were significantly higher (p < 0.05) in C57BL/6 mice than in BALB/c mice on days 6 to 9 postinfection (Fig. 1GoD). By day 9 postinfection, C57BL/6 mice had about 2.5 times more TNF-{alpha} in their plasma than the BALB/c mice. This period corresponds to the time of control of first the wave of parasitemia in C57BL/6 and the time of death in BALB/c mice. The TNF-{alpha} proteins detected in the plasma of infected C57BL/6 mice by ELISA were tested by functional assay and found to be biologically active (data not shown).

Splenic IFN-{gamma} and TNF-{alpha} and hepatic TNF-{alpha} mRNA expressions are differentially regulated in susceptible BALB/c and resistant C57BL/6 mice infected with T. congolense

The steady state levels of IFN-{gamma} and TNF-{alpha} mRNA in the spleens and TNF-{alpha} mRNA in the livers of infected BALB/c and C57BL/6 mice were assessed by Northern analysis. A significant difference was observed in the kinetics of expression of IFN-{gamma} mRNA by the splenocytes from infected BALB/c and C57BL/6 mice. IFN-{gamma} mRNA accumulated to high levels in the spleens of infected BALB/c mice by day 3 postinfection and remained high thereafter. In the C57BL/6 mice, IFN-{gamma} mRNA was not detected at very high levels until day 6, but then was quickly down-regulated thereafter, so that by day 8 the steady state levels were about 25% of those at day 6 (Fig. 2GoA). Treatment of infected BALB/c mice with Berenil on days 6 and 7 postinfection markedly reduced the subsequent expression of IFN-{gamma} mRNA in the spleen, such that on days 8 and 9 IFN-{gamma} mRNA was undetectable by Northern analysis (data not shown). The kinetics and patterns of expression of TNF-{alpha} mRNA in the spleens of infected mice were similar in the two strains (Fig. 2GoB). However, TNF-{alpha} mRNA expression in the livers of BALB/c mice gradually rose to a peak on day 7 and markedly declined on day 8 postinfection. This drop in TNF-{alpha} mRNA levels corresponded with the peak of parasitemia and the onset of morbidity in these animals. In contrast, in infected C57BL/6 mice and akin to the plasma levels (Fig. 1GoD), TNF-{alpha} mRNA levels steadily increased with the progression of the infection and were highest on day 9 postinfection, by which time the first wave of parasitemia was almost controlled (Fig. 2GoC).



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  		FIGURE 2. Expression of IFN-{gamma} mRNA by spleen tissues (A), and TNF-{alpha} mRNA by spleen (B) and liver (C) tissues of BALB/c and C57BL/6 mice infected with T. congolense. Northern blots of total cellular RNA from the pooled spleens or livers of groups of four to five mice infected from 0 to 8 days were probed with 32P-labeled IFN-{gamma}, TNF-{alpha}, and {gamma}-actin cDNAs. The results are presented graphically as the ratios of the IFN-{gamma} and TNF-{alpha} to {gamma}-actin signals to accommodate any lane-to-lane variations in RNA loading. The results presented are from one of two similar experiments.

 
Splenocytes from infected BALB/c mice produce higher levels of IL-4 and IFN-{gamma} than spleen cells from infected C57BL/6 mice

Splenocytes from infected, highly susceptible BALB/c and relatively resistant C57BL/6 mice were cultured in vitro for 48 h either alone or in the presence of Con A, and the levels of IL-4 and IFN-{gamma} in the supernatants were determined using a sandwich ELISA. On each day tested, both unstimulated (Fig. 3Go) and Con A-induced secretion of these cytokines (data not shown) were significantly higher in cultures of BALB/c cells than in those from C57BL/6 mice. The production of IL-4 by unstimulated BALB/c splenocytes first became apparent on day 4, peaked on day 6, and decreased by day 8 postinfection (Fig. 3GoA), but unstimulated C57BL/6 splenocytes produced no detectable IL-4 throughout the infection period tested. There was a gradual decline in Con A-induced production of IL-4 following infection in BALB/c mice, but by day 7 postinfection, the trend was reversed and Con A-driven IL-4 production increased markedly and remained high until the death of the mice (data not shown). In contrast, Con A-driven production of IL-4 by splenocytes from C57BL/6 mice remained either undetectable or were significantly lower (p < 0.001) than those from BALB/c mice (data not shown). The production of IFN-{gamma} was significantly higher in BALB/c cultures than in the C57BL/6 cultures. By day 4 postinfection, IFN-{gamma} production by unstimulated splenocytes from BALB/c mice was very high and remained so throughout the remainder of the infection (Fig. 3GoB). In contrast, the splenocytes from resistant C57BL/6 mice only produced significant amounts of IFN-{gamma} on days 5 and 6, and by days 7 and 8 postinfection, the production of this cytokine had largely waned (Fig. 3GoB). There was a strong correlation between the rise in parasitemia and the secretion of IFN-{gamma} in culture by unstimulated splenocytes of infected BALB/c mice (r = 0.89, p < 0.01). Because of the potentially contradictory coproduction of high levels of IFN-{gamma} as well as IL-4 and IL-10 (26) in the BALB/c mice, we wished to confirm the IFN-{gamma} ELISA results using a functional assay for this cytokine. We observed similar patterns of splenocyte IFN-{gamma} production using this assay, and thereby also confirmed that the immunologically detectable IFN-{gamma} in both BALB/c and C57BL/6 mice was functionally active (Fig. 3GoC). We were unable to detect TNF-{alpha} production by ELISA or bioassay in culture fluids of either the unstimulated or Con A-stimulated splenocytes from infected BALB/c or C57BL/6 mice after 48 h in culture.



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  		FIGURE 3. Secretion of IL-4 (A) and IFN-{gamma} (B), as measured by ELISA or functional assay (IFN-{gamma} only; frame C), in cultures of unstimulated splenocytes from BALB/c and C57BL/6 mice infected with T. congolense. Splenocytes from mice infected for varying periods of time were cultured for 48 h, and the supernatant fluids were assayed for IL-4 and IFN-{gamma}, as described in Materials and Methods. Data are presented as mean ± SE. The results presented are from one of three similar experiments.

 
IFN-{gamma} mediates the suppression of mitogen-driven splenocyte proliferation in T. congolense-infected BALB/c mice

A prominent feature of African trypanosomiasis is suppression of lymphocyte reactivity to mitogens and Ags (4, 5, 6), and the degree of immunosuppression correlates negatively with the resistance exhibited by different strains of mice (11). Because it has been shown that IFN-{gamma} is involved in the down-regulation of T cell responses in T. brucei brucei (19) and other parasitic infections (36, 37) in mice, we wished to determine whether the increased production of IFN-{gamma} in T. congolense-infected BALB/c mice was involved in the suppression of their T cell responses to Con A (see below and 26 .

Spleen cells from infected BALB/c mice showed a progressive suppression of their proliferative responses to Con A stimulation, most markedly on days 7 to 9 postinfection (65 ± 10% suppression relative to uninfected controls on day 8). To assess the involvement of IFN-{gamma} in this suppression, we set up splenocyte cultures of uninfected and day 8-infected BALB/c mice. We added either anti-IFN-{gamma} or equivalent levels of isotype-matched control Abs to the cultures and again assessed the proliferative response of the cells to Con A challenge. As shown in Fig. 4Go, anti-IFN-{gamma} Ab had no significant effect on the Con A response of splenocytes from normal mice, but essentially abrogated the suppressive effects of T. congolense infection on the proliferation of splenocytes from infected mice. In contrast, isotype-matched control Ab had no detectable effects on the Con A response of splenocytes from uninfected or infected mice.



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  		FIGURE 4. Proliferative response of splenocytes from uninfected or 8-day-infected BALB/c mice. The cells were stimulated with Con A alone (hatched bars), Con A plus isotype-matched control Ab (dotted bars), or Con A plus anti-IFN-{gamma} Abs (solid bars). The proliferation of the unstimulated splenocytes from normal or infected mice in medium alone was less than 8%. Data are presented as mean ± SE. The results are from one of three similar experiments. *, p < 0.01.

 
Suppression of splenocyte proliferative responses to mitogens in mice infected with either T. brucei brucei (18) or T. brucei rhodesiense (17) has been reported to be partially mediated by NO produced by IFN-{gamma}-activated macrophages. However, Taylor et al. (7, 38) reported that NO-mediated immunosuppression does not operate in cattle infected with T. congolense, and Beschin et al. (39) reported that NO-mediated immunosuppression occurs only during the early stages of T. brucei brucei infection in mice. Con A-stimulated splenocytes from the relatively resistant C57BL/6 mice produced significantly (p < 0.05) more NO from days 2 to 5 postinfection than similarly treated BALB/c splenocytes (40). However, from day 7, when the proliferative responses of BALB/c splenocytes were highly suppressed relative to those of C57BL/6 (26), both mouse strains produced essentially similar amounts of NO (14.5 ± 2.3 versus 16.3 ± 1.6 µM, day 7; and 18.5 ± 3.3 versus 17.6 ± 2.1 µM, day 8 for BALB/c and C57BL/6 mice, respectively). To test whether NO in some way affects the suppression of the splenocyte proliferative response observed in T. congolense-infected mice, we repeated our proliferation assays, but included a specific NOS inhibitor (NGMMA) in the spleen cultures to block the production of NO. As shown in Table IGo, anti-IFN-{gamma} Abs again effectively reversed the suppression of Con A-driven proliferation of splenocytes from T. congolense-infected mice, but the addition of 500 µM NGMMA to the cultures, which inhibited NO production by 93.5 ± 9.2%, had no significant effect on the proliferative responses of these splenocytes.


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  		Table I. NO-independent suppression of Con A-induced proliferation of splenocytes from T. congolense-infected BALB/c mice

 
These results clearly show that IFN-{gamma} is involved in the suppression of splenocyte proliferative response associated with T. congolense infections, and suggest that this cytokine could be important in the disease-promoting immunosuppression observed in infected, genetically susceptible mice. They also demonstrate that, as in cattle (7, 38), NO does not mediate the suppression of splenocyte proliferative responses observed in T. congolense-infected mice.

In vivo anti-IFN-{gamma} administration early during infection reduces parasitemia and dramatically prolongs the survival period of genetically susceptible BALB/c mice

We next wished to determine whether neutralization of endogenous IFN-{gamma} in genetically susceptible BALB/c mice infected with T. congolense would influence the outcome of the disease. The reasons for this arose from: 1) our earlier observation that the plasma and splenocyte culture supernatants from infected BALB/c mice consistently contained significantly more IFN-{gamma} than those from their C57BL/6 counterpart mice; 2) the strong correlation of IFN-{gamma} expression with the rise in parasitemia in susceptible mice; 3) the anti-IFN-{gamma}-mediated reversal of suppression of the proliferative responses of splenocytes from infected BALB/c mice to Con A challenge; and 4) the observation that treatment of infected mice with the trypanocidal drug Berenil effectively abolished IFN-{gamma} expression in infected BALB/c mice.

Groups of BALB/c mice were injected i.p. with either purified monoclonal anti-IFN-{gamma} Abs, anti-IL-4 Abs (200 µg in 200–400 µl of PBS per injection), or PBS on days 0, 2, 4, and 6 postinfection with T. congolense. The parasitemia and survival of the mice were monitored daily. All of the PBS- and anti-IL-4-treated mice succumbed to their infections within 7 to 9 days, with each becoming moribund at about the time their circulating parasite loads peaked (Fig. 5Go, A and B). However, anti-IFN-{gamma}-treated BALB/c mice effectively adopted a trypanosome-resistant phenotype. Although anti-IFN-{gamma} treatments were stopped on day 6 postinfection, more than one-half of the mice successfully controlled their disease through three to four successive waves of parasitemia over the next 25 days, almost eliminating trypanosomes from their blood twice (i.e., days 13 and 32; Fig. 5GoA). Overall, the anti-IFN-{gamma}-treated animals had substantially fewer circulating parasites (30% decrease, p < 0.01), and a dramatically prolonged mean survival period (>325% prolonged, p < 0.003; Fig. 5GoB) relative to the PBS- or anti-IL-4-treated groups.



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  		FIGURE 5. Effects of anti-IFN-{gamma} Abs (IgG1) on parasitemia (A) and survival (B) of BALB/c mice infected with T. congolense. Groups of six BALB/c mice were injected i.p. with either PBS (open squares), anti-IL-4 Abs (IgG1, 200 µg; closed squares), or anti-IFN-{gamma} Abs (200 µg; open circles) on days 0, 2, 4, and 6 postinfection. Parasitemia was monitored daily by direct assessment of peripheral blood. The results are from one of two similar experiments.

 
In vivo neutralization of IFN-{gamma} dramatically reduces plasma IL-10 and spontaneous and Con A-induced secretion of IL-10 by splenocytes from T. congolense-infected BALB/c mice

We studied the effects of our in vivo anti-IFN-{gamma} immunotherapy on IL-10 and IFN-{gamma} production in T. congolense-infected BALB/c mice for two reasons. First, we have shown previously that anti-IL-10 Abs reverse trypanosomiasis-associated suppression of splenocyte proliferative responses to Con A and also reduce the circulating parasitemia and increase the survival periods of infected BALB/c mice (26). Second, although the biology of T. brucei infections in mice is substantially different from that of T. congolense, and anti-IFN-{gamma} Ab treatments in T. brucei-infected mice are not as effective as those observed herein, it was shown in the T. brucei system that anti-IFN-{gamma} Abs inhibited the release of an unidentified soluble factor that is associated with suppression of lymph node cell proliferation from parasite-pulsed macrophages (19). We recently have obtained evidence that bone marrow-derived macrophages from genetically susceptible but not resistant mice, when pulsed simultaneously with IFN-{gamma} and T. congolense lysates, increase their secretion of IL-10 by two- to fourfold (R. S. Kaushik, J. E. Uzonna, J. R. Gordon, and H. Tabel, manuscript in preparation).

Groups of mice were injected i.p. with either PBS or 200 µg of anti-IFN-{gamma} Abs on days 0, 2, and 4 postinfection. On day 7, the mice were killed and we determined the amounts of IFN-{gamma} and IL-10 produced by the splenocytes in vitro. We also measured the levels of IL-10 in the plasma of the infected mice. As shown in Fig. 6Go, anti-IFN-{gamma} Ab treatments reduced the secretion of IFN-{gamma} (Fig. 6GoA) and IL-10 (Fig. 6GoB) by unstimulated splenocytes and the Con A-induced secretion of IL-10 (Fig. 6GoC) by splenocytes from infected mice, as well as the plasma levels of IL-10 (Fig. 6GoD).



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  		FIGURE 6. Reduction of spontaneous and Con A-induced secretion of IL-10 by splenocytes and decrease of IL-10 levels in the plasma of T. congolense-infected BALB/c mice treated with anti-IFN-{gamma} Abs. Groups of four infected BALB/c mice were injected with either PBS alone or 200 µg of anti-IFN-{gamma} Abs, as described in Materials and Methods. On day 7 postinfection, the mice were killed to obtain plasma samples and to establish splenocyte cultures. The results depict unstimulated secretion of IFN-{gamma} (A) and IL-10 (B) and Con A-induced IL-10 secretion (C) by splenocytes, as well as plasma levels of IL-10 (D) in anti-IFN-{gamma}-treated mice. Data are presented as mean ± SE (some error bars are too small to be visible). *, p < 0.01; **, p < 0.001.

 
Because it has been reported that the production of NO by splenic macrophages in T. brucei rhodesiense-infected mice was mediated by IFN-{gamma} (17) and that inhibition of NO synthesis abolished immunosuppression and enhanced the resistance of T. brucei brucei-infected mice (41), we also determined whether the beneficial effects of anti-IFN-{gamma} Abs observed in this study were mediated, in part, by inhibition of NO synthesis. Surprisingly, anti-IFN-{gamma} Ab treatment of T. congolense-infected BALB/c mice significantly enhanced the production of NO by splenocytes on day 7 relative to untreated controls (41.3 ± 1.3 µM versus 27.4 ± 0.8 µM, p < 0.002; for anti-IFN-{gamma}- and PBS-treated mice, respectively).

These results unequivocally link IFN-{gamma} activity in infected BALB/c mice with the expression of IL-10. The results imply that the protective effect of anti-IFN-{gamma} Abs could be associated with reduction of IL-10 secretion in this model. This observation is consistent with our previous report, wherein we demonstrated that IL-10 is a disease-enhancing cytokine in the context of experimental T. congolense infections in BALB/c mice (26).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study clearly shows that endogenously produced IFN-{gamma} plays a disease-enhancing role in experimental T. congolense infections in highly susceptible BALB/c mice. The numbers of parasites in the blood were reduced and the survival periods were increased by more than 300% following in vivo injection of neutralizing anti-IFN-{gamma} Abs into such animals, and anti-IFN-{gamma} Abs reversed the splenocyte suppression observed in vitro. Furthermore, Berenil cure of these animals rapidly cleared the parasitemia and led to a marked reduction in the plasma levels and secretion of IFN-{gamma} by splenocytes from infected mice.

Suppression of Ag- and mitogen-driven T cell proliferation in the spleen and lymph nodes is a prominent feature of African trypanosomiasis (4, 5, 6). This has been suggested as one of the mechanisms responsible for the observed increase in the susceptibility of trypanosome-bearing hosts to opportunistic infections (8). In mice experimentally infected with T. brucei brucei, albeit a significantly different parasite than T. congolense, the suppression of lymph node T cell proliferative responses is associated with endogenously secreted IFN-{gamma}, which mediates the decrease of IL-2R expression on CD4+ T cells. In that system, anti-IFN-{gamma} Abs abolished the in vitro lymphoid suppression via the restoration of IL-2R expression (19). However, IFN-{gamma} by itself was not enough to mediate this suppression. An unknown factor(s) released by T. brucei-pulsed macrophages also was required to fully implement this suppressive effect (19). NO released by IFN-{gamma}-activated macrophages has been shown to mediate immunosuppression in mice infected with T. brucei brucei (18, 41) and T. brucei rhodesiense (17). However, Taylor et al. (7, 38) showed that NO does not contribute to immunosuppression in T. congolense infections in cattle, and suggested that the expression of high levels of IL-10 mRNA transcripts in the spleens and lymph nodes in these animals might contribute to the observed immunosuppression in infected cattle. We have shown previously that endogenously produced IL-10 is associated with the suppression of splenocyte proliferative responses to Con A challenge in T. congolense-infected mice (26). The highly suppressed splenocytes of susceptible BALB/c mice secreted significantly higher levels of this cytokine than the spleen cells of the resistant C57BL/6 mice (26). In addition, anti-IL-10 Abs reversed this in vitro suppression of proliferation of the BALB/c splenocytes (26). We have evidence that IFN-{gamma} treatment of bone marrow-derived macrophages from highly susceptible BALB/c mice, combined with exposure to T. congolense lysates, causes two- to fourfold increases in their production of IL-10 (R. S. Kaushik, J. E. Uzonna, J. R. Gordon, and H. Tabel, manuscript in preparation). In line with this, we observed herein that in vivo injections of anti-IFN-{gamma} Abs markedly reduced the plasma levels of IL-10 and the secretion of IL-10 by splenocytes from T. congolense-infected BALB/c mice. IL-10 down-regulates the expression on APCs, of the costimulatory molecule B7, which is necessary for T cell activation (42, 43). Thus, it is quite likely that the immunosuppressive effect of IFN-{gamma} observed in T. congolense-infected mice could be mediated, at least in part, via IL-10 secreted by macrophages following their interactions with trypanosomes (or their products). However, because the effect of in vivo administration of anti-IL-10 Abs was not as dramatic as that obtained herein with anti-IFN-{gamma} Abs (26), our data also suggest that there may be some IL-10-independent effects of IFN-{gamma} on the overall resistance of BALB/c mice to T. congolense infection.

Some aspects of immunosuppression in mice infected with T. brucei brucei (18) and T. brucei rhodesiense (17) have been reported to be mediated by NO produced by IFN-{gamma}-activated macrophages. However, Darji et al. (19) reported that NO does not mediate immunosuppression in mice infected with T. brucei brucei, while Beschin et al. (39) reported that NO does not mediate immunosuppression during the late stages of T. brucei brucei infections in mice. Similarly, Taylor et al. (7, 38) did not observe any NO-mediated immunosuppression in cattle infected with T. congolense. Our data clearly indicate that, as in cattle (7, 38), NO does not mediate immunosuppression in BALB/c mice infected with T. congolense. This is consistent with our observation that splenocytes from T. congolense-infected BALB/c and C57BL/6 mice produce similar amounts of NO on days 7 and 8 postinfection, despite the apparently higher production of IFN-{gamma} and greater immunosuppression in BALB/c than in C57BL/6 splenocytes (40). Moreover, treatment of infected BALB/c mice with anti-IFN-{gamma} Abs actually increased the production of NO by splenocytes from these mice relative to untreated control mice. While it may be surprising that such marked differences exist between T. congolense and T. brucei, especially in view of their close structural and molecular similarities, we suspect that, among other things, the inherently invasive nature of T. brucei as well as differences in some enzymes could be important distinguishing factors between the two murine models of trypanosomiasis.

High levels of IFN-{gamma} have been shown to inhibit B lymphocyte activation (44), and thus the high levels of IFN-{gamma} in the susceptible BALB/c mice might inhibit parasite-specific Ab synthesis in these animals, resulting in enhanced susceptibility. We have determined the serum levels of Abs to T. congolense among the two mouse strains during T. congolense infections and found that susceptible BALB/c mice were unable to mount IgG2a and IgG3 Ab responses (53). Berenil treatment of infected BALB/c mice was associated with enhanced synthesis of these Abs (53). We have not determined the levels of these Abs in the susceptible mice treated with anti-IFN-{gamma} Abs. Because Berenil treatment was also associated with reduction in IFN-{gamma} synthesis, it may be suggested that high levels of IFN-{gamma} may be responsible for the very low levels of these Ab isotypes in the BALB/c mice. But care has to be taken in interpreting this result, since we found that IL-12 synthesis was higher in the resistant than susceptible mice. IL-12 has been shown to initiate the switch from IgM to IgG2a and IgG3 in B cells (45). Thus, it is likely that the low levels of T. congolense-specific IgG2a and IgG3 Abs in the plasma of susceptible mice are due to their inability to synthesize IL-12 rather than IFN-{gamma}-mediated suppression of Ab synthesis.

In experimental T. brucei infections in mice, IFN-{gamma} has been claimed to have a direct growth-stimulatory effect on the parasites (20), and as such, the reductions in parasitemia and increases in survival periods following anti-IFN-{gamma} Ab administration were attributed to an inhibition of this stimulatory effect by the Abs (46). We have been unable to demonstrate such a growth-stimulatory effect of IFN-{gamma} on T. congolense (47). The reduction in parasitemia and the enhanced survival of anti-IFN-{gamma} Ab-treated mice reported in this work would seem more likely to be due to an indirect effect resulting, at least in part, from the reversal of immunosuppression, with a subsequent induction of effective immune response against the parasites. In this regard, we have characterized an unusual population of CD3+Thy-1.2+{alpha}ß-CD4+8- and CD3+Thy-1.2+{alpha}ß-CD4-8- regulatory cells in the spleens of highly susceptible BALB/c mice infected with T. congolense (54). These cells are induced by trypanosomal Ags to secrete high amounts of IL-10 and IFN-{gamma} and nonspecifically suppress T and B cell responses to mitogens and parasite-unrelated Ags (54).

We detected much more TNF-{alpha} in the plasma of the relatively resistant C57BL/6 mice than we did in the highly susceptible BALB/c mice, and this difference increased markedly as the infections progressed. The role of TNF-{alpha} in the pathogenesis of African trypanosomiasis is not yet entirely clear. It has been associated with a suppression of lymph node cell proliferative responses (5), but it has also been shown to be both trypanostatic and trypanolytic for T. brucei brucei and T. brucei rhodesiense in vivo and in vitro (22, 23, 24). The trypanolytic effect of TNF-{alpha} on T. brucei occurs via a novel lectin-like affinity receptor on the trypanosomes that is functionally and spatially distinct from the previously characterized mammalian TNF-{alpha} receptor binding sites (23). It is undetermined whether T. congolense has binding sites for TNF-{alpha}, but the fact that the biologically active form of this cytokine was present at much higher levels in the plasma of the more resistant mice at the time of control of the parasitemia (Fig. 1GoD) is consistent with its being associated with a more effective parasite control and enhanced resistance.

TNF-{alpha} mRNA accumulated to higher steady state levels in the livers than in the splenic tissues of all mice, and the hepatic TNF-{alpha} mRNA levels were higher on day 9 postinfection in the resistant C57BL/6 than in the susceptible BALB/c mice. This suggests that much of the TNF-{alpha} detected in the circulation could have been of hepatic origin, most probably Kupffer cell derived. Elimination of trypanosomes from the blood of infected mice is reportedly mediated primarily by Kupffer cells, the primary macrophage population of the liver (48). Macrophages are of course a major source of TNF-{alpha} (49), and soluble extracts from T. brucei have been reported to induce production of this cytokine by macrophages in vitro (22), as have the glycophosphatidylinositol moieties derived from the variant surface glycoprotein of T. brucei (50). IFN-{gamma}-primed peritoneal and bone marrow-derived macrophages from C57BL/6 mice, upon interaction with T. congolense lysate, secrete significantly higher amounts of TNF-{alpha} than similarly treated BALB/c mice macrophages (R. S. Kaushik, J. E. Uzonna, J. R. Gordon, and H. Tabel, manuscript in preparation).

The simultaneous presence of high levels of IL-4, IL-10, and IFN-{gamma} in the plasma (51) and splenocyte culture supernatants (26) from infected BALB/c mice is intriguing, considering the ostensibly dichotomous cross-regulatory effects of these cytokines on Th1 and Th2 cells. The significantly higher levels of IL-4 detected in the plasma and culture supernatants of splenocytes from infected BALB/c mice contrast sharply with the lack of or very low levels of IL-4 detected in the C57BL/6 mice. According to the contemporary immunologic theory, IL-4 is a major factor in driving the differentiation of uncommitted Th cells to the Th2 phenotype (reviewed in 52 , the phenotype presently considered to favor more effective immunologic control of extracellular parasites (15). This hypothesis might not necessarily apply to the control of African trypanosomes. We found that treatment of resistant and susceptible mice with mAbs to IL-4 during T. congolense infections did not alter their disease. However, we cannot exclude the possibility that, for some unknown reason, there was an inadequate neutralization of the cytokine by the Abs.

It has been suggested that a balance between IFN-{gamma} and TNF-{alpha} secretion in mice infected with T. brucei determines the course of parasitemia and the outcome of the disease (5). If so, it is feasible that such a balance could also affect the disease outcome during T. congolense infections in mice. Although we presently have no direct evidence for the beneficial effects of TNF-{alpha} in T. congolense-infected mice, the observation that only this cytokine was elevated in the plasma of the resistant mice points to an association between high levels of this cytokine and resistance. As mentioned, we have recently found that bone marrow-derived macrophages from the resistant mice, upon interaction with T. congolense lysate, produce higher amounts of TNF-{alpha} than those from the susceptible mice. High levels of IFN-{gamma} could stimulate BALB/c macrophages, following their interaction with trypanosomes, to release copious amounts of IL-10 (R. S. Kaushik, J. E. Uzonna, J. R. Gordon, and H. Tabel, manuscript in preparation). IL-10, in turn, would contribute to a down-regulation of immune responses (26) and TNF-{alpha} secretion by macrophages (42). Conversely, high levels of TNF-{alpha} could restrict the parasite growth by its static and lytic effects on the trypanosomes (22, 23, 24), thereby limiting the parasite load and allowing the host to mount an effective immune response against the parasites.


   Acknowledgments
 
We are grateful to Ying Zhang for outstanding technical assistance.


   Footnotes
 
1 This work was supported by a research grant from the Natural Sciences and Engineering Council of Canada (to H.T.) and the Medical Research Council of Canada (to J.R.G.). Back

2 Address correspondence and reprint requests to Dr. Henry Tabel, Department of Veterinary Microbiology, WCVM, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 5B4, Canada. E-mail address: Back

3 Abbreviations used in this paper: NO, nitric oxide; NGMMA, NG-monomethyl-L-arginine; NOS, nitric oxide synthase. Back

Received for publication March 27, 1998. Accepted for publication July 6, 1998.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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