Mice Lacking NK Cells Develop an Efficient Th1 Response and Control Cutaneous Leishmania major Infection

Abhay R. Satoskar, Luisa M. Stamm, Xingmin Zhang, Anjali A. Satoskar, Mitsuhiro Okano, Cox Terhorst, John R. David and Baoping Wang
J Immunol June 1, 1999, 162 (11) 6747-6754;

Abstract

NK cells are believed to play a critical role in the development of immunity against Leishmania major. We recently found that transplantation of wild-type bone marrow cells into neonatal tgε 26 mice, which are deficient in T and NK cells, resulted in normal T cell development, but no or poor NK cell development. Using this novel model we analyzed the role of NK cells in the development of Th1 response and control of cutaneous L. major infection. Mice selectively lacking NK cells (NK−T+) developed an efficient Th1-like response, produced significant amounts of IL-12 and IFN-γ, and controlled cutaneous L. major infection. Administration of neutralizing IL-12 Abs to NK−T+ mice during L. major infection resulted in exacerbation of the disease. These results demonstrate that NK cells are not critical for development of protective immunity against L. major. Furthermore, they indicate that IL-12 can induce development of Th1 response independent of NK cells in NK−T+ mice following L.major infection.

The leishmaniases comprising a number of diseases caused by the intracellular protozoan parasite Leishmania have a wide spectrum of clinical manifestations (1). While susceptible BALB/c mice develop large nonhealing lesions following L. major infection, most other mouse strains, including C3H, CBA/J, and C57BL/6, are resistant and develop small lesions that heal spontaneously. It is widely accepted that protective immunity against cutaneous L. major infection is associated with the development of a Th1-like response and the production of cytokines such as IL-12, IL-2, and IFN-γ (2, 3, 4), whereas susceptibility to L. major is associated with the development of a Th2-like response and the production of cytokines such as IL-4 and IL-10 (5).

NK cells are a subpopulation of bone marrow-derived large, granular lymphocytes that lack T cell- and B cell-specific subset markers (TCR-, CD4-, CD8-, CD3γδε-, and Ig-), but express some specific markers, such as NK 1.1 and ASGM1 (6). NK cells have been shown to play a critical role in innate immunity against a variety of viruses, bacteria, fungi, and parasites (7). The protective role of NK cells has been attributed to their ability to secrete immunoregulatory cytokines, such as IFN- γ (8), lyse host cells infected with the intracellular pathogens, and directly inhibit growth of microorganisms (9, 10).

Previous studies have demonstrated that depletion of NK cells using anti-asialo GM1 antiserum significantly reduces early IFN-γ production in resistant C3H/HeN mice and renders them susceptible to cutaneous L. major infection, suggesting that NK cells are involved in host defense against this parasite (11). Furthermore, a recent study indicated that NK cells are involved in protection and healing of cutaneous leishmaniasis in humans (12). Therefore, we examined the development of Th1 response and growth of L. major in mice specifically lacking NK cells. Our results show that NK cells are not essential for the development of Th1 response and immunity to L. major infection in these mice.

Materials and Methods

Mice

Tgε26 mice were maintained through sib breeding in the animal facility of the Beth Israel Deaconess Medical Center (Boston, MA) (13, 14). Although the tgε26 transgenic founder was a (C57BL/6 × CBA/J)F2, all tgε26 mice used in this study were H-2k. Mice lacking NK cells (NK−T+) were generated by transplanting fetal liver or bone marrow cells from the (C57BL/6 × CBA/J)F1 mice into neonatal tgε26 mice, as described recently.3 Age- and sex-matched wild-type CBA/J (H-2k), C57BL/6 (H-2b), and BALB/c (H-2d) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Two types of immunocompetent mice with functionally competent NK and T cells were used as wild-type controls in this study. One type was (C57BL/6 × CBA/J)F1 (H-2b/k) generated through breeding of C57BL/6 × CBA/J. The other type was tgε26 mice reconstituted with (C57BL/6 × CBA/J)F1 (H-2b/k) bone marrow or fetal liver cells at 2–3 wk of age, instead of neonatally as in the generation of NK−T+ mice. These immunocompetent mice were termed as NK+T+(tgε26Y) mice. Five weeks after the transplantation, NK+T+(tgε26Y) mice develop functionally competent NK and T cells, and their levels are comparable to those observed in wild-type (C57BL/6 × CBA/J)F1 mice. Furthermore, NK+T+ (tgε26Y) and NK−T+ mice have similar levels of CD4+ and CD8+ T cells. Of note, all the NK+T+, NK+T+(tgε26Y), and NK−T+ mice used in this study were analyzed by flow cytometry of PBL before the infection, and the lymph node and spleen cells upon sacrificing animals to confirm the lack or presence of NK cells and the presence of T cells.

Parasites and infection protocols

L. major.

LV39 was maintained by serial passage of amastigotes inoculated s.c. into the shaven rumps of BALB/c mice. Amastigotes isolated from the lesions of infected mice were grown to stationary-phase promastigotes as described previously (15). Mice were injected in the hind footpad with 2 × 106 L. major stationary-phase promastigotes. Disease progression was monitored by measuring the increase in thickness of the infected footpad using a dial-gauge micrometer (Mitutoyo, Kanagawa, Japan) at weekly intervals up to 10 wk after infection and comparing this to the thickness of the contralateral uninfected footpad.

Quantitation of parasite loads

Parasite burdens in the infected footpad were determined by homogenizing footpads of individual mice and carrying out limiting dilution analysis as described previously (15). The results were expressed as reciprocal log parasite titers.

Ab ELISA

Peripheral blood was collected at 2-wk intervals from tail snips of all experimental animals infected with L. major. Blood was centrifuged at 200 × g, and serum was collected to determine titers of Th1-associated IgG2a and Th2-associated IgG1 Leishmania-specific Abs by ELISA as described before (15).

Flow cytometric analysis

The lymph node cells, spleen cells, and PBL were analyzed by three-color flow cytometry as described previously (15). Briefly, 0.5–1 × 106 cells in 50 μl were incubated with prestaining buffer (PBS, 4% BSA, 0.5% sodium azide, 15% mixture of normal hamster, normal rat, and normal mouse sera, anti-Fc receptor Ab) for 5 min. The cells were then stained with biotinylated Ab for 30 min, washed once, followed by staining with a mixture of streptavidivin-RED670 (0.4 μl/sample; Life Technologies, Rockville, MD), PE- and FITC- conjugated Abs (0.5 μg/sample) for 30 min. The cells were washed twice, fixed in 1% formaldehyde, and analyzed with a FACScan using CellQuest software (Becton Dickinson, Mountain View, CA). All procedures were performed on ice until analysis. The following Abs were used: 145-2C11 (CD3-ε, H57-597 (TCR-αβ), RM4-5 (CD4), 53-5.8 (CD8α), 53-2.1 (Thy 1.2), RA3-6B2 (B220), PK136 (NK1.1), and DX5 (all purchased from PharMingen, San Diego, CA).

Allogenic T cell proliferation and cytotoxicity assays

Allogenic T cell proliferation and cytotoxicity assays were performed by stimulating responsive spleen cells (H-2k or H-2k×b) with irradiated allogenic BALB/c (H-2d) spleen cells as described previously (14). NK cell cytotoxicity assay was performed using yeast artificial chromosome (YAC)4 cells as described before (13).

T cell proliferation and cytokine assays

The draining popliteal lymph nodes were removed from L. major-infected mice at week 10 postinfection, and T cell proliferation assays were performed as previously described (15). Briefly, 3 × 105 lymph node cells were added in triplicate to the wells of 96-well flat-bottom tissue culture plates and stimulated with either 20 μg/ml L. major Ag (prepared from stationary-phase promastigotes by six cycles of freezing at −70°C and thawing at 37°C) or 1 μg/ml of Con A. Culture supernatants from these assays were analyzed for production of IL-4 (reagents purchased from Endogen, Cambridge, MA; detection limit, 5 pg/ml) and IFN-γ (reagents purchased from PharMingen; detection limit, 20 pg/ml) by capture ELISA as described previously (15).

Histopathology

Infected foot pads from L. major-infected NK−T+, NK+T+, and NK−T− mice were excised and fixed in decalcifying solution F (Stephens Lab, Riverdale, NJ) for 7 days. The tissues were processed and embedded in paraffin, and 4- to 8-μm sections were cut. The sections were hydrated and stained by routine hematoxylin eosin staining.

Anti-IL-12 neutralizing Ab treatment

Rat anti-mouse IL-12 (p40/p70) (clone: C17.8) neutralizing mAb was kindly provided by Dr. T. Veldman (Genetics Institute, Cambridge, MA). NK−T+ mice were treated by i.p. administration of 0.5 mg anti-IL-12 neutralizing Ab or control Ab 1 day before L. major infection and weekly dose of 0.5 mg/mouse thereafter until 7 wk.

Statistical analyses

Student’s unpaired t test was used to determine significance of values obtained. Differences in Ab endpoint titers were determined using Mann-Whitney U prime test.

Results

NK−T+ mice have functionally normal CD4+ and CD8+ T cells but lack cytotoxic NK cells

Recently, we demonstrated that transplantation of wild-type bone marrow or fetal liver cells into neonatal tgε26 mice results in normal development of T cells, but poor NK cell development.3 All the neonatal transplanted tgε26 mice used in this study had a normal number of CD4+ and CD8+ T cells as confirmed by flow cytometry. The NK1.1+CD3−TCR-αβ−, which represent 2–4% of wild-type splenocytes, however, were markedly diminished in the neonatal transplanted mice (Fig. 1⇓, A and B). Peripheral lymph nodes from NK−T+ mice also showed markedly diminished levels of NK1.1+CD3−TCR-αβ− cells than those from NK+T+ mice (1–2% in NK+T+ mice and background levels (<0.3%) in NK−T+ mice). In contrast, NK1.1+TCR-αβ+ T cells, which represent 0.4–1% of wild-type splenocytes, were present in the neonatal transplanted mice (Fig. 1⇓B). The selective NK cell-deficient mice were termed as NK−T+ mice. Both CD4+ and CD8+ T cells from NK−T+ mice were functionally competent as assessed by MLR and CTL assays, respectively (Fig. 1⇓, C and D). NK cell lytic function, as measured by splenocyte cytotoxicity against NK cell-sensitive YAC-1 cells, however, was generally nondetectable or <10% of wild-type control levels (Fig. 1⇓E). When young tgε26 mice were reconstituted with F1 bone marrow or fetal liver cells at 2–3 wk of age (tgε26Y), these mice had comparable wild-type (F1) T (Fig. 2⇓A) and NK cell (2.5 ± 0.63%, 1.3 ± 0.05%, and 0.3 ± 0.1% of NK 1.1+TCR-αβ− cells in NK+T+, NK+T+(tgε26Y), and NK−T+ mice, respectively) levels. Furthermore, NK cells from NK+T+(tgε26Y) mice were functionally as competent as those from NK+T+ mice (Fig. 2⇓B). Therefore, these mice were termed as NK+T+(tgε26Y). Since they have identical background as the NK−T+ mice, the NK+T+(tgε26Y) mice were also used as NK cell competent controls, in addition to the wild-type mice (NK+T+).

FIGURE 1.
FIGURE 1.

NK−T+ mice lack NK1.1+CD3−TCR-αβ− cells but have normal T cell development. A, CD4 and CD8 profile of lymph node cells from a wild-type (NK+T+) and an NK−T+ mouse. B, NK1.1 and TCR-αβ profile of spleen cells, indicating that NK−T+ mice have markedly diminished NK1.1+αβ− cells, but have a relatively normal number of NK 1.1+αβ+ cells. C, NK−T+ mice have functionally normal CD4+ T cells as assessed by MLR assay. D, NK−T+ mice have functionally normal CD8+ T cells as assessed by CTL assay. E, NK−T+ cell mice lack NK cell cytotoxicity against YAC-1 cells. Data for C–E represents the analysis of the spleen cells obtained from a single NK+T+ and two NK−T+ mice.

FIGURE 2.
FIGURE 2.

NK+T+(tgε26Y) mice develop Th1 response and control L. major infection as efficiently as NK+T+ mice. A, CD4 and CD8 profile of lymph node cells from a wild-type (NK+T+) and NK+T+(tge26Y) mice. B, NK+T+(tge26Y) mice have normal NK cell cytotoxicity against YAC-1 cells similar to that observed in NK+T+ mice. C, Course of L. major infection in NK+T+, NK+T+(tgε26Y), NK−T+, and NK−T− mice. Disease progression was monitored by measuring increase in the thickness of infected footpad, as described before. D, In vitro LmAg-induced IFN-γ production by the lymph node cells. Data expressed as mean ± SE. Asterisk indicates statistically significant difference between two groups. ND, not detectable.

NK−T+ mice control cutaneous L. major infection

Following infection with L. major, NK−T+, NK+T+, and NK+T+(tgε26Y) mice developed lesions, which resolved spontaneously within 60–70 days (Figs. 2⇑C and 3A). The course of L. major infection was similar in NK−T+, NK+T+, and NK+T+(tgε26Y) mice (Figs. 2⇑C and 3A). In contrast, concomitantly infected tgε26 (NK−T−) and BALB/c mice developed large nonhealing lesions and did not control the infection (Figs. 2⇑C and 3A). The lesion grew significantly faster in NK−T− mice than in BALB/c mice (Fig. 3⇓A). Examination of the histopathology of the infected footpads from NK−T− and BALB/c mice revealed ulceration and extensive s.c. tissue destruction with a diffuse inflammatory infiltrate consisting of heavily parasitized macrophages, eosinophils, and neutrophils (Fig. 3⇓C). On the other hand, infected foot pads from NK−T+ and NK+T+ mice displayed inflammatory infiltrate comprised predominantly of lymphocytes and macrophages with few parasites (Fig. 3⇓, D and E). There were no significant differences in the parasite burdens in footpads of NK+T+ and NK−T+ mice. The lesions from L. major-infected NK−T− and BALB/c mice, however, contained significantly more parasites (at least 10 logs more) than NK−T+ and NK+T+ mice (Fig. 3⇓B). These results indicate that, although NK cells may play a role in innate immunity to L. major as reported in previous studies using SCID and RAG-2−/− mice, they suggest that NK cells are not essential for control of L. major infection when immunocompetent T cells are present.

FIGURE 3.
FIGURE 3.

NK−T+ mice control cutaneous L. major infection. A, Course of L. major infection following infection with 2 × 106 stationary-phase promastigotes in NK−T+, NK+T+, BALB/c, and tgε26 (NK−T−) mice. Progress of lesion growth was monitored by measuring the increase in thickness of the infected footpad and comparing this to the thickness of the contralateral uninfected footpad. NK−T− were sacrificed on day 30 after the infection, due to the development of large lesions. All other mice were monitored up to 60 days. B, Footpad parasite burdens in L. major-infected NK+T+, NK−T+, and NK−T− mice. Data expressed as log parasite titer ± SE. C–E, Hematoxylin-eosin stained skin lesions from L. major-infected NK−T−, NK+T+, and NK−T+ mice. Lesions from NK−T− mice showed ulceration and extensive tissue destruction with inflammatory infiltrate comprising of parasitized macrophages, neutrophils, and eosinophils. C and D, Similarly stained skin from the inoculation sites of NK+T+ and NK−T+ mice displayed a more preserved skin structure with lymphocytes and some macrophages with few intracellular parasites (original magnification, ×40). Results are representative of three experiments with four to five animals per group. Data expressed as mean ± SE.

NK−T+ mice develop efficient Th1-like response following L. major infection

NK cells have been shown to be a major source of IFN-γ, a cytokine critical for development of the Th1 lymphocyte subset of the CD4+ T cell population in resistant mice following L. major infection (11). Therefore, we compared IL-12 and IFN-γ production by Leishmania Ag-stimulated draining lymph node cells from L. major-infected NK−T+ and NK+T+ mice. On day 60 postinfection, the draining lymph node cells from L. major-infected NK+T+ and NK−T+ mice contained a similar number of lymphocytes (1.02 ± 0.13 × 107 and 1.35 ± 0.4 × 107 in NK+T+ and NK−T+ mice, respectively; p < 0.375). Furthermore, there were no significant differences in proportions of B220+ (43.7 ± 5.4% and 41.6 ± 1.9% in NK+T+ and NK−T+ mice, respectively), CD4+ (28.6 ± 4.2% and 32.9 ± 4.7% in NK+T+ and NK−T+ mice, respectively), and CD8+ (10.1 ± 1.6% and 11.1 ± 1.1% in NK+T+ and NK−T+ mice, respectively). NK 1.1+ cells, however, were markedly reduced in the lymph nodes from NK−T+ mice (2.1 ± 0.31% and 0.4 ± 0.06% in NK+T+ and NK−T+ mice, respectively). At this time, lymphocytes from NK+T+, NK−T+, and BALB/c mice displayed greater Ag-specific proliferative responses than those derived from NK−T− mice. At this time, LmAg-stimulated lymph node cells from both NK−T+ and NK+T+ mice produced IL-12 and IFN-γ, although IL-12 levels were significantly higher in NK−T+ mice (Fig. 4⇓, A and B; p < 0.05). Ag-stimulated lymph node cells from concomitantly infected BALB/c mice produced significant amounts of IL-4, but no IL-4 was detected in culture supernatants from NK−T+ and NK+T+ mice (Fig. 4⇓C). Neither IL-4 nor IFN-γ was detectable in the lymph node cell culture supernatants from NK−T− mice, which contained basal levels of IL-12. (Fig. 4⇓, A–C). All groups produced comparable levels of IL-10 (data not shown). Similarly, at an earlier time point on day 36 postinfection, both NK−T+ and NK+T+ mice produced IL-12 (0.114 ± 0.4 ng/ml and 1.3 ± 0.17 ng/ml in NK+T+ and NK−T+ mice, respectively) and IFN-γ (1.068 ± 0.6 ng/ml and 2.9 ± 0.25 ng/ml in NK+T+ and NK−T+ mice, respectively), but no IL-4. In additional experiments, NK+T+(tgε26Y) mice were also examined. NK+T+(tgε26Y) mice controlled L. majorinfection as efficiently as NK+T+ mice (Fig. 2⇑C). Furthermore, there was no significant difference in IFN-γ production by LmAg-stimulated lymph node cells from NK+T+ and NK+T+(tgε26Y) mice (Fig. 2⇑D).

FIGURE 4.
FIGURE 4.

NK−T+ mice develop a Th1-like response. In vitro LmAg-induced (20 μg/ml) IL-12 (A), IFN-γ (B), and IL-4 (C) production by the lymph node cells from NK+T+, NK−T+, and BALB/c mice measured on day 60 postinfection. NK−T− (tgε26) mice were sacrificed on day 30 postinfection for proliferation assays and cytokine production, due to the development of large lesions. Data expressed as mean ± SE. Asterisks indicate statistically significant differences between NK+T+ and NK−T+ mice. Similar results were observed in two independent experiments. ND, not detectable.

NK−T+ mice fail to produce Leishmania-specific IgG2a, despite development of Th1 response

Ab responses in L. major-infected NK−T+, NK+T+, NK−T−, and BALB/c mice were analyzed by measuring titers of Leishmania-specific Th1-dependent IgG2a and Th2-dependent IgG1 Abs on days 30, 45, and 60 postinfection. On day 30 and thereafter, NK+T+ and BALB/c mice developed significant levels of Leishmania-specific IgG1 and IgG2a, although BALB/c mice produced significantly more (Fig. 5⇓A). On the other hand, L. major-infected NK−T+ mice displayed high titers of Leishmania-specific IgG1, but failed to produce any measurable quantities of Leishmania-specific IgG2a throughout the course of infection (Fig. 5⇓B). Both NK+T+ and NK+T+(tgε26Y) mice produced significant titers of LmAg-specific IgG2a (2666.2 ± 533 and 16200 ± 12049 in NK+T+ and NK+T+(tgε26Y) mice, respectively). Similar results were observed on days 30 and 45 postinfection (data not shown).

FIGURE 5.
FIGURE 5.

L. major-specific IgG1 and IgG2a production in NK+T+ and NK−T+ mice on day 60 postinfection presented as reciprocal endpoint titers on a log scale. Similar results were observed in three independent experiments. NK−T− mice were sacrificed on day 30 postinfection, due to the development of large lesions. ND, not detectable.

FIGURE 6.
FIGURE 6.

Administration of IL-12 neutralizing Ab to NK−T+ mice inhibits Th1 development and exacerbates cutaneous L. major infection. Data expressed as mean lesion size ± SE.

IL-12 is critical for development of Th1 response and controls L. major infection in NK−T+ mice

To determine whether IL-12 is critical for development of Th1 response in NK−T+ mice during L. major infection, we treated L. major-infected NK−T+ with i.p. injections of IL-12 neutralizing Ab or control Ab 1 day before infection and weekly thereafter for 7 wk. Anti-IL-12 mAb-treated NK−T+ mice developed significantly larger lesions than control animals following L. major infection (Fig. 6). At wk 8 postinfection, Ag-stimulated lymph node cells from control NK−T+ mice produced significantly higher amounts of IFN-γ (mean levels, 1.34 ± 0.4 ng/ml) than those from anti-IL-12-treated NK−T+ mice, which produced only basal levels (<0.05 ng/ml; p < 0.05).

Discussion

The results presented here indicate that although NK1.1+CD3−TCR-αβ− (NK) cells play a role in innate immunity to L. major, they are not required for development of Th1-like response and control of L. major infection in resistant mice. Furthermore, they also demonstrate that in the absence of NK cells, IL-12 can directly induce development of a Th1 response during L. major infection.

NK cells have been demonstrated to be involved in the first line of defense against viruses, bacteria, and parasites (7, 16). The importance of NK cells in early antibacterial immunity has been demonstrated by a study showing that SCID mice that lack αβ and γδ T cells but have NK cells develop activated macrophages and partially control Listeria monocytogenes (17, 18, 19). Later, it was demonstrated that spleen cells from naive SCID and nude mice produced significant levels of IFN-γ following incubation with heat-killed L. monocytogenes (18). Furthermore, administration of neutralizing anti-IFN-γ mAb or NK cell depletion before infection abolished macrophage activation in SCID mice. Together, these data indicate that NK cell-derived IFN-γ is involved in vivo macrophage activation following L. monocytogenes infection (19). Similarly, many studies have demonstrated that NK cells also play a critical role in immunity against viruses such as murine CMV, Coxsackievirus B4, and influenza virus (20, 21, 22). This has been attributed to their cytolytic capacity and ability to produce type I IFNs that have antiviral activity (23).

Although some studies using NK cell-deficient beige mice had suggested that NK cells were required for the control of visceral leishmaniasis (24), others using SCID mice indicated that NK cell-derived IFN-γ was unlikely to participate in the early regulation of visceral leishmaniasis caused by L. donovani (25). Similarly, some studies in murine cutaneous leishmaniasis indicate that the NK cell-derived IFN-γ plays an important role in early resistance and development of a Th1 response following L. major infection (11, 26). Others, however, using beige mice have demonstrated that NK cells are not required for the control of cutaneous L. tropica infection, which supports our observations in the present study. These differences are probably due to the different experimental models of NK cell deficiency used and the differences in the experimental approaches. For example, although poly(I:C) activates NK cells and significantly reduces the parasite burdens in the early course of L. major infection in BALB/c mice (26), it also induces production of type I (IFN-α/β) IFN from NK cells, which has been shown to induce expression of nitric oxide synthase 2 (NOS2) in vivo and regulate innate immunity to L. major (27). Conversely, depletion of NK cells prior to L. major infection using anti-AsGM1 Ab significantly decreased early IFN-γ production and exacerbated the infection in resistant C3H/HeN, but had no effect on the ultimate disease outcome (11, 26).

Systemic depletion of cells using Ab treatment is efficient but not absolute (28). Furthermore, in a recent study, NK cell depletion using anti-NK1.1 as well as anti-AsGM-1 Abs failed to alter the Th1/Th2 balance of Ag-driven cytokine synthesis (29). The ability of NK-depleted C3H/HeN mice to heal L. major infection, however, could be attributed to the repopulation of NK cells in these mice following cessation of anti-AsGM-1 treatment (11, 26). Finally, beige mice, which controlled L. tropica (30), exhibit normal numbers of NK 1.1+CD3− cells and normal NK cell cytotoxicity against viruses, although they have very low cytotoxicity against YAC-1 cells (31, 32). By our use of a novel model of specific murine NK cell deficiency, these possibilities have been excluded. Unlike beige mice, the NK−T+ mice lack NK cell cytotoxicity against lymphocytic choriomeningitis virus and YAC-1 cells.3 Therefore, these mice are truly deficient in NK cell lytic function, and the number of NK1.1+CD3− cells are either absent or markedly diminished in these mice.3 We have reported previously that transplantation of wild-type bone marrow into adult tgε26 mice results in aberrant NK cell development, which is caused by the generation of aberrant T cells that leads to high levels of TNF-α in vivo (33). This mechanism, however, could not account for NK cell deficiency in the neonatal tgε26 model described in this study. First, in the neonatal tgε26 model, NK cell deficiency is independent of T cell development,3 and there was no overproduction of TNF-α in sera from L. major-infected NK+T+ and NK−T+ mice (97 ± 28 pg/ml and 71 ± 40 pg/ml in NK+T+ and NK−T+ mice, respectively). Second, we demonstrated that the lack of cytotoxic NK cells in the neonatally transplanted tgε26 mice was due to the failure of transplanted hematopoietic stem cells to home to bone marrow, whereas the T cell development in the same mice was due to the migration of the hematopoietic stem cells during neonatal period to the thymus.3

We have previously demonstrated that TCR-αβ−/− mice lacking T cells on genetically resistant background (15) and RAG-2−/− mice that have innately high levels of IFN-γ and IL-12 control early lesion growth of L. major but later succumb to the disease (our unpublished observations). Our findings in the present study that tgε26 mice (NK−T−) are susceptible to L. major and develop lesions significantly faster than similarly infected RAG2−/− or TCR-αβ−/− mice (NK+T−) suggest that NK cells may be involved in controlling early resistance to L. major. However, the ability of NK−T+ mice to control cutaneous L. major infection as efficiently as wild-type (NK+T+) mice indicates that, in the presence of functional T cells, NK cells are not essential for the control of L. major lesion growth. Furthermore, they also indicate that NK cell-derived IFN-γ is not essential for the Th1 differentiation of CD4+ T cell following cutaneous L. major infection.

Acquired protective immunity against cutaneous L. major infection in genetically resistant mice is dependent on the ability to develop a CD4 Th1-type lymphocyte response and produce cytokines such as IL-2 and IFN-γ (2, 34). Many recent observations indicate that IL-12 plays a critical role in the development of a Th1-like response in resistant mice following L. major infection (3, 4, 35). Thus, for example, IL-12 administered simultaneously at the time of vaccination with Leishmania Ag facilitated Th1 development (35). Furthermore, in two independent studies, susceptible BALB/c mice treated with IL-12 during L. major infection developed a significant Th1-like response and healed (3, 4). In contrast, administration of IL-12 neutralizing Ab to resistant C3H/HeN mice rendered them susceptible to L. major (36). Similarly, genetically resistant mice lacking the IL-12 gene failed to mount a Th1-like response and developed large nonhealing lesions following L. major infection (37).

Neutralization of IL-12 in vivo has been shown to abrogate NK cell cytotoxicity and decrease early IFN-γ production by lymph node cells from L. major-infected C3H mice (36). This result suggested that the protective role of IL-12 was mediated by its ability to activate NK cells to produce IFN-γ critical for the subsequent development of the Th1 subset of the CD4+ T cells (36). On the other hand, a recent study demonstrated that IFN-γ derived from CD4+ T cells is sufficient to mediate Th1 development following L. major infection (38). In the present study, we found that L. major-infected NK−T+ mice produced significantly higher amounts of IL-12 and IFN-γ than similarly infected NK+T+ mice and efficiently controlled the infection. Conversely, administration of anti-IL-12 neutralizing Abs to NK−T+ mice during L. major infection significantly reduced IFN-γ production and resulted in the development of large rapidly growing lesions. Possibly, the increased IL-12 production in NK−T+ mice is due to the development of compensatory mechanisms or loss of NK cell-mediated negative feedback regulating IL-12 production by macrophages. In fact, recent studies have demonstrated that NK cell-derived type 1 IFN (IFN-α/β), which has been shown to regulate innate responses in L. major infection (27), can down-regulate IL-12 production (39). Nevertheless, taken together, these results suggest that in the absence of NK cells, IL-12 can directly induce Th1 development in vivo following L. major infection.

NK cells have been shown to induce Ab production from activated murine B cells (40) as well as resting human B cells (41). A recent study, however, has demonstrated that IL-12 enhances Ab responses and increase levels of IgG2a to T-independent polysaccharide vaccines in the absence of T and NK cells (42). Interestingly, despite the production of IL-12 and development of Th1-like response, L. major-infected NK−T+ mice failed to produce Th1-associated Leishmania-specific IgG2a Ab throughout the course of infection. Similarly, in our ongoing studies, NK−T+ produced significant amounts of IFN-γ but displayed baseline levels of Ag-specific IgG2a following immunization with keyhole limpet hemocyanin (KLH) or OVA in CFA, which induces Th1-biased response (our unpublished observations). Furthermore, there was no difference in levels of Th2-type cytokines IL-4 and IL-10 produced by Ag-stimulated spleen cells from these mice (our unpublished observations). This data has been reproduced in four independent studies conducted so far in our laboratory using KLH and OVA. These results suggest that NK cells may play a critical role in production of IgG2a against T-dependent Ags.

Recently, a population of CD4 T cells that express NK1.1 and TCR-αβ (NKT cells) has been shown to produce IL-4 (43) as well as IFN-γ (44). Although early studies had indicated that NKT cells may be the initial source of IL-4 that induces Th2 development (43), recent studies indicate that in a susceptible mouse, L. major induces rapid IL-4 production by CD4+ cells that are NK 1.1-negative (45). Recent studies have demonstrated that IL-12-stimulated NK1.1+ T cells produce high levels of IFN-γ (46, 47), as well as exhibit cytotoxicity against tumor cells (46). Furthermore, endogenous IL-12 has been shown to down-regulate IL-4-producing NK 1.1+ T cells in liver and improve protective immunity against listeriosis (48). NK−T+ mice used in the present study have a normal number of NK1.1+CD4+ T cells. Additionally, administration of anti-IL-12 Ab L. major-infected NK−T+ mice inhibited Th1 development and rendered them susceptible to infection. Therefore, one may speculate that NK 1.1+ T cell-derived IFN-γ initiates Th1 development in these mice. A recent study, however, demonstrated that RAG-2/IFN-γ−/− (double mutant) mice reconstituted with the wild-type CD4+ NK 1.1− T cells develop Th1 response and control cutaneous L. majorinfection (38). Taken together, these results indicate that neither NK cell-derived IFN-γ nor NK 1.1+CD4+ T cells are critical for the development of protective Th1 response following L. major infection.

In conclusion, L. major-infected NK−T+ mice on genetically resistant background develop an efficient Th1-like response as measured by significant IFN-γ production by the lymph node cells following stimulation with Leishmania Ag and control cutaneous L. major infection. Furthermore, administration of neutralizing anti-IL-12 Abs to NK−T+ mice during L. major infection inhibits development of Th1-like response and enhances cutaneous lesion growth.

Footnotes

  • ↵1 This work was supported by National Institutes of Health Grants A122532-13 (to J.R.D.), AI17651 (to C.T.), and HD35562-01(to B.W.). B.W. is the recipient of a Basil O’Connor Starter Scholar Research Award.

  • ↵2 Address correspondence and reprint requests to Dr. Abhay R. Satoskar, Department of Immunology and Infectious Diseases, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115. E-mail address: asatoska{at}hsph.harvard.edu

  • ↵3 B. Wang, K. Nguyen, X. Zhang, A. Nichogiannopoulou, S. J. Simpson, J. Guimond, B. A. Croy, J.-C. Gutierrez Ramos, G. A. Hollander, C. A. Biron, and C. Terhorst. 1999. Distinct homing of engrafted hematopoietic stem cells in neonatal mice differentially affects T lymphocyte and NK cell development. Submitted for publication.

  • ↵4 Abbreviation used in this paper: YAC, yeast artificial chromosome.

  • Received December 1, 1998.
  • Accepted March 17, 1999.

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The Journal of Immunology: 162 (11)
The Journal of Immunology
Vol. 162, Issue 11
1 Jun 1999
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