T-Cell Responses during Trypanosoma brucei Infections in Mice Deficient in Inducible Nitric Oxide Synthase

Mice.

iNOS-deficient mice were generated as described previously (32). Disruption of the murine iNOS gene was achieved by homologous recombination in 129sv embryonic stem cells. The recombinant allele was passed through the germ line following the mating of the embryonic stem cell chimeras with MF-1 mice (Harlan Ltd., Oxon, United Kingdom). The homozygous and heterozygous mice thus generated were backcrossed with MF-1 mice for three generations. All the mice used were from the matings of littermates and should therefore have had a similar genetic background. Peritoneal cells from mutant mice did not produce iNOS protein following activation by IFN-γ and lipopolysaccharide in vitro as judged by Western blotting. They also did not produce detectable amounts of NO after being cultured for 48 h with IFN-γ and lipopolysaccharide. By 72 h, however, a low level of nitrite was detectable in the culture supernatant of cells from the iNOS-deficient mice. This may reflect either the accumulation of nitrite produced by constitutive NOS (14) or the induction of constitutive NOS (1, 17). iNOS-Deficient mice remained competent for other aspects of macrophage function (14). Female mutant mice and their heterozygous littermates were used when 8 to 10 weeks old. Because only mice with an outbred background were available, all assays were conducted on individual mice to take into account the potential genetic variability.

Trypanosomes.

To initiate experimental infections, groups of five adult female mice were inoculated with 10⁴ trypanosomes/mouse via an intraperitoneal route with the cloned pleomorphic trypanosome line GUTat (Glasgow University Trypanozoon antigen type) 7.2. This line produces resolving infections in BALB/c mice in which more than 95% of the population expresses the same variable antigen type (VAT), GUTat 7.2, during the first wave of parasitemia (15). Two other VATs used in this study, ILTat (ILRAD Trypanozoon antigen type) 1.3 and 1.61, are not expressed during the first 15 days of infection of the GUTat 7.2 line. In rabbits (which are less susceptible to infection than mice) these two VATs are detected at approximately days 20 through 30 of infection (15, 27a). The parasitemia of each mouse was monitored daily by removing 2 μl of blood and diluting it in 0.85% NH₄Cl in phosphate-buffered saline (PBS), and parasites were counted by using an Improved Neubauer hemocytometer.

To obtain parasites for ex vivo proliferation and cytokine assays, cloned trypanosome lines that stably express GUTat 7.2, ILTat 1.3, or ILTat 1.61 were grown in adult female CFLP mice (Harlan Olac) and purified from infected mouse blood (9). Trypanosomes were suspended in 0.1 M PBS (pH 7.4) and counted. They were then incubated at room temperature for 20 min in 4% paraformaldehyde in PBS, washed three times in PBS by centrifugation at 900 × g for 5 min each time, and left overnight in 0.1 M NH₄Cl in PBS to neutralize NH₂ groups. The trypanosomes were centrifuged, resuspended in PBS at a concentration of 10⁷/ml, and stored at 4°C for up to 4 weeks. Populations were checked for VAT homogeneity by immunofluorescence on air-dried smears fixed in 70% ethanol by using VAT-specific antibodies as previously described (29).

Isolation of mononuclear splenocytes.

Spleens were removed from individual mice, and mononuclear splenocytes were isolated. Briefly, spleens were disrupted through sterile tea strainers into RPMI 1640 medium and passed through 100-μm-pore-size monofilament nylon filters to obtain single cell suspensions. Cymelarsan (Rhone-Merieux, Lyon, France) was added to each suspension (including controls) at 50 μg/ml to kill contaminating trypanosomes, and suspensions from infected mice were monitored by microscopy until lysis of trypanosomes was observed (usually 10 to 30 min). The splenocytes were centrifuged, and loose pellets were resuspended, layered onto cushions of Nycoprep (Nycomed Ltd.), and centrifuged for 15 min at 700 × g. The mononuclear interface layer was removed and centrifuged, and the pellet was resuspended in 2 ml of medium. The cells were counted, their viability was checked by trypan blue exclusion, and the cells were resuspended at the required density. The centrifugation steps after Cymelarsan treatment removed dead parasites from these preparations, as judged by phase-contrast microscopy.

Flow cytometry analyses.

From each spleen, aliquots of 10⁶ mononuclear splenocytes were made, resuspended in PBS with 5% fetal calf serum and 0.05% azide in 50-μl volumes, and double-labelled with phycoerythrin (PE)-conjugated anti-CD4 plus fluorescein isothiocyanate (FITC)-conjugated anti-CD8, PE-conjugated anti-CD4 plus FITC-conjugated anti-CD25α chain, or PE-conjugated anti-CD8 plus FITC-conjugated anti-CD25α chain (Pharmingen) at the manufacturer’s recommended concentrations for 1 h on ice. The cells were washed twice in PBS-fetal calf serum and resuspended in 0.1 M Tris–0.9% NH₄Cl for 10 min to lyse any contaminating erythrocytes. The cells were pelleted by brief centrifugation, resuspended in PBS (pH 7.0), and analyzed with a FACScan flow cytometer (Becton Dickinson).

Proliferation assays.

The isolated mononuclear splenocytes were plated out at 2 × 10⁶ cells/well in 200-μl aliquots in flat-bottomed 96-well microtiter plates with either medium alone, concanavalin A (ConA) (8 μg/well; Sigma), or paraformaldehyde-fixed trypanosomes (2 × 10⁶/ml) expressing GUTat 7.2, ILTat 1.3, or ILTat 1.61. The cells were incubated at 37°C in 5% CO₂ for 48 h and radiolabelled with 1 μCi of [³H]thymidine per well for the last 16 h before being harvested; results were read on a Betaplate counter. The proliferative responses were determined by [³H]thymidine incorporation, and results are expressed as the mean counts per minute (± 2 standard errors [SE]) of four wells.

Cytokine assays.

The cells were dispensed into 24-well plates at 4 × 10⁶ cells/well in 1.25-ml volumes and incubated with either medium alone, ConA (8 μg/well), or paraformaldehyde-fixed trypanosomes (2 × 10⁶/ml) expressing GUTat 7.2, ILTat 1.3, or ILTat 1.61. The cells were incubated at 37°C in 5% CO₂, supernatants were harvested at 72 h and microcentrifuged for 30 s to remove cells, and the cell-free supernatants were stored at −20°C until analysis by enzyme-linked immunosorbent assay for IFN-γ, IL-2, and IL-5 with commercial capture and detection antibodies (Pharmingen).

Plasma nitrate levels.

The levels of nitrite and nitrate present in the plasma from individual mice were measured by the reduction of nitrate to nitrite and by the Griess reaction as previously described (7, 12).

Statistical analyses.

Parasitemia data were analyzed by repeated measures analysis of variance with log-transformed data, and levels of IFN-γ and plasma nitrate were analyzed by Mann-Whitney U tests.

Comparison of parasitemia levels in iNOS-deficient and control mice.

Levels of GUTat 7.2 parasitemia in iNOS-deficient and control mice increased exponentially at similar rates and reached similar densities at the peak of parasitemia on the same day postinfection (Fig. 1). Parasitemic decline after the parasitemic peak was different between the two groups (F_{1, 6} = 43.0, P < 0.001), however, with parasitemias in the iNOS-deficient mice decreasing more rapidly. From days 8 to 11 of infection there was an approximately 1-order-of-magnitude difference in levels of parasitemia between iNOS-deficient and control mice. These levels of parasitemia in the heterozygous MF-1 mice were higher than those previously described for BALB/c mice (30); therefore, these experiments were terminated on day 11 of infection, and several components of the immune system were assayed.

Cytometric analyses of mononuclear splenocytes.

Expressing cytometric data in percentage terms fails to take into account the gross splenomegaly caused by trypanosomiasis; for this reason, our data are expressed as cell numbers per spleen. As a result of infection, CD4⁺-cell numbers increased in iNOS-deficient mice, and CD8⁺-cell numbers decreased slightly in both groups (Fig. 2). These changes caused the ratios of CD4⁺ cells to CD8⁺ cells to change from 2:1 to 8:1 in iNOS-deficient mice and from 3.8:1 to 7.3:1 in control mice. Figure 2 also shows that there were approximately fourfold more activated CD4⁺ cells (expressing CD25) present in the spleens from iNOS-deficient infected mice than in spleens from infected control or uninfected mice. Activated CD8⁺ cells were detected only in mononuclear splenocyte populations (in low numbers, 2 × 10⁴/spleen) from infected iNOS-deficient mice.

Proliferative responses.

The ability of the T cells to proliferate in response to ConA was examined (Fig. 3). High levels of proliferation were observed in splenocytes from infected mice in the absence of any stimulant, which is in accord with our previous observations, and we attribute these responses to increased numbers of activated cells in infected mice (5). Nevertheless, enhanced proliferation in response to ConA was seen in control mice (2.9-fold higher) and, most notably, in the iNOS-deficient mice (4.5-fold higher) (Fig. 3). Trypanosome-specific proliferation in response to paraformaldehyde-fixed parasites expressing one of the three VATs was not observed (data not shown). We attribute the difference in ConA-induced proliferative responses between iNOS-deficient and control mice to the way in which they respond to infection rather than to any intrinsic difference between the mice. No such differences were found between uninfected iNOS-deficient and control mice with an MF-1 genetic background in a previous study (14). Also, negligible differences were found in a study of iNOS-knockout mice with a C57BL/6 genetic background (10).

Cytokine production.

Mononuclear splenocytes from chronically infected control and iNOS-deficient mice differed in the capacity to produce IFN-γ (Fig. 4). The control mice produced very little IFN-γ when stimulated with ConA or with any of the three paraformaldehyde-fixed VATs. However, IFN-γ production by mononuclear splenocytes from the iNOS-deficient mice was very different, with high levels produced in response to ConA (W = 15, P < 0.05) and trypanosomes expressing the homologous, but not heterologous, VAT (W = 15, P < 0.05). The heterologous VATs, ILTat 1.3 and 1.61, were both undetectable by immunofluorescence analysis on days 7 and 11 of infection (data not shown), indicating that splenocytes had no detectable exposure to these VATs in vivo. The three trypanosome lines used to stimulate the mononuclear cells are of the same genetic origin and thus differ antigenically only in their VAT expression. All antigens except the variant surface glycoproteins (VSGs) (the invariant antigens) were thus the same in all three paraformaldehyde-fixed preparations. If invariant antigens were strongly immunogenic in this assay, then equivalent levels of IFN-γ production would be expected, irrespective of which antigen preparation was used as a stimulant. A similar result would also be expected if the three VSGs shared epitopes. The data shown in Fig. 4 thus confirm that VSG is the main immunogen of T. brucei and indicate VAT-specific stimulation of IFN-γ production.

In both control and iNOS-deficient infected mice, IL-5 production was <1 U/ml when stimulated with medium, ConA, or any of the three VATs (data not shown). The only detectable IL-2 production was from cells stimulated with ConA. These levels, in both groups of mice, were <10 U/ml 24 h after stimulation in vitro (data not shown).

Plasma nitrite and nitrate.

In control and iNOS-deficient uninfected mice, the mean concentrations of nitrate (±2 SE) were 24.7 ± 4.1 and 18.8 ± 2.9 μM, respectively. In the infected control mice there was a 6.7-fold increase in plasma nitrate levels, to 165.0 ± 45.3 μM (W = 49, P = 0.023), and a 3.8-fold increase in the iNOS-deficient infected mice, to 70.8 ± 7.9 μM (W = 56, P = 0.011).