Effector cells of both nonhemopoietic and hemopoietic origin are required for interferon (IFN)-gamma- and tumor necrosis factor (TNF)-alpha-dependent host resistance to the intracellular pathogen, Toxoplasma gondii

Abstract

Although interferon (IFN)-gamma-activated, mononuclear phagocytes are considered to be the major effectors of resistance to intracellular pathogens, it is unclear how they control the growth of microorganisms that reside in nonhemopoietic cells. Pathogens within such cells may be killed by metabolites secreted by activated macrophages or, alternatively, directly controlled by cytokine-induced microbicidal mechanisms triggered within infected nonphagocytic cells. To distinguish between these two basic mechanisms of cell-mediated immunity, reciprocal bone marrow chimeras were constructed between wild-type and IFN-gamma receptor-deficient mice and their survival assessed following infection with Toxoplasma gondii, a protozoan parasite that invades both hemopoietic and nonhemopoietic cell lineages. Resistance to acute and persistent infection was displayed only by animals in which IFN-gamma receptors were expressed in both cellular compartments. Parallel chimera experiments performed with tumor necrosis factor (TNF) receptor-deficient mice also indicated a codependence on hemopoietic and nonhemopoietic lineages for optimal control of the parasite. In contrast, in mice chimeric for inducible nitric oxide synthase (iNOS), an enzyme associated with IFN-gamma-induced macrophage microbicidal activity, expression by cells of hemopoietic origin was sufficient for host resistance. Together, these findings suggest that, in concert with bone marrow-derived effectors, nonhemopoietic cells can directly mediate, in the absence of endogenous iNOS, IFN-gamma- and TNF-alpha-dependent host resistance to intracellular infection.

Figure 1

Schematic representation of the trans and cis models of cell-mediated effector function against T. gondii within nonhemopoietic cells (i.e., somatic cells). In the trans model, the protective lymphokines (IFN-γ/ TNF-α) activate (designated by stippling) the professional effector cell, the macrophage resulting in the synthesis and diffusion of toxic metabolites (e.g., NO) to neighboring somatic cells. These mediators suppress the growth of T. gondii within the somatic cell and protect it from lytic infection. In the cis model, the lymphokines directly activate the somatic cell to suppress intracellular T. gondii growth.

Figure 2

Assessment of hemopoietic reconstitution by donor-type progenitors in BM chimeric mice. Mice chimeric for iNOS gene deficiency were constructed by lethal irradiation and BM reconstitution as described in Materials and Methods. Unmanipulated iNOS KO and WT C57BL/6 mice were included as negative and positive controls, respectively. Spleen cells (A) and day 5 thioglycollate-elicited peritoneal cells (B) were stimulated with IFN-γ (100 U/ml) and STAg (10 μg/ml). Nitrite levels were measured in 24-h culture supernatants. Each bar represents the mean of 2–4 mice per group. All supernatants from unstimulated spleen and peritoneal exudate cells produced <5 μmol/liter of NO₂ ⁻, which also represented the threshold value for a positive signal in this assay.

Figure 2

Assessment of hemopoietic reconstitution by donor-type progenitors in BM chimeric mice. Mice chimeric for iNOS gene deficiency were constructed by lethal irradiation and BM reconstitution as described in Materials and Methods. Unmanipulated iNOS KO and WT C57BL/6 mice were included as negative and positive controls, respectively. Spleen cells (A) and day 5 thioglycollate-elicited peritoneal cells (B) were stimulated with IFN-γ (100 U/ml) and STAg (10 μg/ml). Nitrite levels were measured in 24-h culture supernatants. Each bar represents the mean of 2–4 mice per group. All supernatants from unstimulated spleen and peritoneal exudate cells produced <5 μmol/liter of NO₂ ⁻, which also represented the threshold value for a positive signal in this assay.

Figure 3

T. gondii–induced IL-12 p40 and IFN-γ production in IFN-γR–deficient mice. IFN-γR WT and KO mice (three per group) were either unimmunized (A and B) or immunized with 10⁶ lethally irradiated T. gondii tachyzoites (C). 2 wk later, spleen cells were harvested and their IL-12 p40 and IFN-γ responses to ConA (5 μg/ml) and STAg (10 μg/ml) assessed. A, IL-12 p40/naive spleen; KO, white bar; WT, hatched bar. B, IFN-γ/naive spleen; naive KO, white bar; naive WT, hatched bar. C, IFN-γ/immune spleen; immune KO, white bar; immune WT, hatched bar.

Figure 3

T. gondii–induced IL-12 p40 and IFN-γ production in IFN-γR–deficient mice. IFN-γR WT and KO mice (three per group) were either unimmunized (A and B) or immunized with 10⁶ lethally irradiated T. gondii tachyzoites (C). 2 wk later, spleen cells were harvested and their IL-12 p40 and IFN-γ responses to ConA (5 μg/ml) and STAg (10 μg/ml) assessed. A, IL-12 p40/naive spleen; KO, white bar; WT, hatched bar. B, IFN-γ/naive spleen; naive KO, white bar; naive WT, hatched bar. C, IFN-γ/immune spleen; immune KO, white bar; immune WT, hatched bar.

Figure 3

T. gondii–induced IL-12 p40 and IFN-γ production in IFN-γR–deficient mice. IFN-γR WT and KO mice (three per group) were either unimmunized (A and B) or immunized with 10⁶ lethally irradiated T. gondii tachyzoites (C). 2 wk later, spleen cells were harvested and their IL-12 p40 and IFN-γ responses to ConA (5 μg/ml) and STAg (10 μg/ml) assessed. A, IL-12 p40/naive spleen; KO, white bar; WT, hatched bar. B, IFN-γ/naive spleen; naive KO, white bar; naive WT, hatched bar. C, IFN-γ/immune spleen; immune KO, white bar; immune WT, hatched bar.

Figure 4

Survival of IFN-γR BM chimeric mice infected with (A) T. gondii (ME49 strain; n = 4–7/group) or (B) L. monocytogenes (EGD strain; n = 4/group). IFN-γR–WT and –KO mice (both on a 129/Sv background) were used as partners for chimera construction. 2 mo after reconstitution, chimeric and unmanipulated control mice were infected with either (A) 20 T. gondii cysts or (B) an LD₅₀ dose of L. monocytogenes. Data presented are representative of four experiments performed. In each experiment, all IFN-γR KO→ KO (○), KO→ WT (□), and WT→ KO (▪) chimeric mice succumbed acutely to T. gondii infection, whereas only IFN-γR KO→ KO and KO→ WT died after L. monocytogenes infection. In both infections, IFN-γR KO→ KO chimeric mice succumbed acutely, as did control IFN-γR KO mice, whereas IFN-γR WT→ WT (•) and control WT mice survived >60 d (not shown).

Figure 4

Survival of IFN-γR BM chimeric mice infected with (A) T. gondii (ME49 strain; n = 4–7/group) or (B) L. monocytogenes (EGD strain; n = 4/group). IFN-γR–WT and –KO mice (both on a 129/Sv background) were used as partners for chimera construction. 2 mo after reconstitution, chimeric and unmanipulated control mice were infected with either (A) 20 T. gondii cysts or (B) an LD₅₀ dose of L. monocytogenes. Data presented are representative of four experiments performed. In each experiment, all IFN-γR KO→ KO (○), KO→ WT (□), and WT→ KO (▪) chimeric mice succumbed acutely to T. gondii infection, whereas only IFN-γR KO→ KO and KO→ WT died after L. monocytogenes infection. In both infections, IFN-γR KO→ KO chimeric mice succumbed acutely, as did control IFN-γR KO mice, whereas IFN-γR WT→ WT (•) and control WT mice survived >60 d (not shown).

Figure 5

Survival of T. gondii–infected and drug-treated IFN-γR BM chimeric mice. IFN-γR WT and KO mice (both on a 129/Sv background) were used as partners for chimera construction. Groups (n = 5/ group) of nonirradiated controls and irradiated BM reconstituted mice were infected with 20 T. gondii cysts. Bactrim-containing drinking water was administered on day 3 and withdrawn on day 20 postinfection. The data shown are from a single experiment performed. ○, KO→ KO; □, KO→ WT; ▴, WT→ KO; •, WT→ WT.

Figure 6

Survival of T. gondii–infected TNFR BM chimeric mice. TNFR p55/p75 KO and C57BL/6 × 129 F1 WT hybrids were used as partners for chimera construction. Nonirradiated and 8-wk reconstituted chimeric mice (n = 4/group) were infected with 20 T. gondii cysts and their survival monitored daily. Nonirradiated TNFR p55/p75 KO mice succumbed at the same time as TNFR KO→ KO chimeras, whereas both control WT and WT→ WT chimeric mice survived >60 d (data not shown). Data shown are representative of two independent experiments. Mean survival time of TNFR chimeric mice (KO→ WT and WT→ KO) was significantly different from that of KO→ KO mice (P < 0.01 in both experiments done) but not different from each other (P = 0.16 and 0.06). ○, KO→ KO; □, KO→ WT; ▴, WT→ KO; •, WT→ WT.

Figure 7

Survival of T. gondii– infected iNOS KO BM chimeric mice. C57BL/6 and iNOS-deficient mice were used as WT and KO partners for the construction of the chimeras. BM chimeric (A) and nonirradiated (B) control mice (n = 5/group) were infected with 20 T. gondii cysts. Results shown are representative of three independent BM chimera experiments. In all three cases, survival time of iNOS KO→ WT chimeras was not statistically different from that of either iNOS KO→ KO sham control mice (P > 0.3) or nonirradiated iNOS KO mice (P > 0.3; data not shown). Similarly in all three experiments, mean survival times were not different between WT→ iNOS KO and WT→ WT chimeras (P > 0.2). However, the latter groups of mice survived significantly longer than mice with iNOS-deficient BM (P < 0.001). The explanation for the reproducible early death of the WT→ WT chimeras with respect to infected, nonirradiated C57BL/6 mice (which, as expected, survived >60 d) is unclear (see above). (A) ○, KO→ KO; □, KO→ WT; ▴, WT→ KO; •, WT→ WT. (B) ○, KO; •, WT.

Figure 7

Survival of T. gondii– infected iNOS KO BM chimeric mice. C57BL/6 and iNOS-deficient mice were used as WT and KO partners for the construction of the chimeras. BM chimeric (A) and nonirradiated (B) control mice (n = 5/group) were infected with 20 T. gondii cysts. Results shown are representative of three independent BM chimera experiments. In all three cases, survival time of iNOS KO→ WT chimeras was not statistically different from that of either iNOS KO→ KO sham control mice (P > 0.3) or nonirradiated iNOS KO mice (P > 0.3; data not shown). Similarly in all three experiments, mean survival times were not different between WT→ iNOS KO and WT→ WT chimeras (P > 0.2). However, the latter groups of mice survived significantly longer than mice with iNOS-deficient BM (P < 0.001). The explanation for the reproducible early death of the WT→ WT chimeras with respect to infected, nonirradiated C57BL/6 mice (which, as expected, survived >60 d) is unclear (see above). (A) ○, KO→ KO; □, KO→ WT; ▴, WT→ KO; •, WT→ WT. (B) ○, KO; •, WT.