Mice Lacking NK Cells Develop an Efficient
Th1 Response and Control Cutaneous
Leishmania major Infection
This information is current as
of June 18, 2017.
Abhay R. Satoskar, Luisa M. Stamm, Xingmin Zhang, Anjali
A. Satoskar, Mitsuhiro Okano, Cox Terhorst, John R. David
and Baoping Wang
J Immunol 1999; 162:6747-6754; ;
http://www.jimmunol.org/content/162/11/6747
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References
Mice Lacking NK Cells Develop an Efficient Th1 Response and
Control Cutaneous Leishmania major Infection1
Abhay R. Satoskar,2* Luisa M. Stamm,* Xingmin Zhang,† Anjali A. Satoskar,‡
Mitsuhiro Okano,* Cox Terhorst,† John R. David,* and Baoping Wang†
T
he 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-g (2– 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-, CD3gde-, 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- g (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-g
production in resistant C3H/HeN mice and renders them susceptible to cutaneous L. major infection, suggesting that NK cells are
*Department of Immunology and Infectious Diseases, Harvard School of Public
Health, Boston MA 02115; and †Department of Medicine, Division of Immunology,
and ‡Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA
02115
Received for publication December 1, 1998. Accepted for publication March
17, 1999.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
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: [email protected]
Copyright © 1999 by The American Association of Immunologists
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
Tge26 mice were maintained through sib breeding in the animal facility of
the Beth Israel Deaconess Medical Center (Boston, MA) (13, 14). Although the tge26 transgenic founder was a (C57BL/6 3 CBA/J)F2, all
tge26 mice used in this study were H-2k. Mice lacking NK cells (NK2T1)
were generated by transplanting fetal liver or bone marrow cells from the
(C57BL/6 3 CBA/J)F1 mice into neonatal tge26 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 3 CBA/J)F1 (H-2b/k) generated through breeding
of C57BL/6 3 CBA/J. The other type was tge26 mice reconstituted with
(C57BL/6 3 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 NK2T1 mice. These
immunocompetent mice were termed as NK1T1(tge26Y) mice. Five
weeks after the transplantation, NK1T1(tge26Y) mice develop functionally competent NK and T cells, and their levels are comparable to those
observed in wild-type (C57BL/6 3 CBA/J)F1 mice. Furthermore, NK1T1
(tge26Y) and NK2T1 mice have similar levels of CD41 and CD81 T cells.
Of note, all the NK1T1, NK1T1(tge26Y), and NK2T1 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 3 106 L. major stationary-phase promastigotes. Disease progression
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.
0022-1767/99/$02.00
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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 tge 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 (NK2T1) developed an
efficient Th1-like response, produced significant amounts of IL-12 and IFN-g, and controlled cutaneous L. major infection. Administration of neutralizing IL-12 Abs to NK2T1 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 NK2T1 mice following L.major
infection. The Journal of Immunology, 1999, 162: 6747– 6754.
6748
NK CELL-DEFICIENT MICE CONTROL CUTANEOUS LEISHMANIASIS
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 3 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
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-2k3b) 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 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 mg/ml L. major Ag (prepared from
stationary-phase promastigotes by six cycles of freezing at 270°C and
thawing at 37°C) or 1 mg/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-g (reagents
purchased from PharMingen; detection limit, 20 pg/ml) by capture ELISA
as described previously (15).
Histopathology
Infected foot pads from L. major-infected NK2T1, NK1T1, and NK2T2
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-mm 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).
NK2T1 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 MannWhitney U prime test.
4
Abbreviation used in this paper: YAC, yeast artificial chromosome.
NK2T1 mice have functionally normal CD41 and CD81 T cells
but lack cytotoxic NK cells
Recently, we demonstrated that transplantation of wild-type bone
marrow or fetal liver cells into neonatal tge26 mice results in normal development of T cells, but poor NK cell development.3 All
the neonatal transplanted tge26 mice used in this study had a normal number of CD41 and CD81 T cells as confirmed by flow
cytometry. The NK1.11CD32TCR-ab2, 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 NK2T1 mice also showed markedly diminished levels
of NK1.11CD32TCR-ab2 cells than those from NK1T1 mice
(1–2% in NK1T1 mice and background levels (,0.3%) in
NK2T1 mice). In contrast, NK1.11TCR-ab1 T cells, which represent 0.4 –1% of wild-type splenocytes, were present in the neonatal transplanted mice (Fig. 1B). The selective NK cell-deficient
mice were termed as NK2T1 mice. Both CD41 and CD81 T cells
from NK2T1 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 cellsensitive YAC-1 cells, however, was generally nondetectable or
,10% of wild-type control levels (Fig. 1E). When young tge26
mice were reconstituted with F1 bone marrow or fetal liver cells at
2–3 wk of age (tge26Y), these mice had comparable wild-type (F1)
T (Fig. 2A) and NK cell (2.5 6 0.63%, 1.3 6 0.05%, and 0.3 6
0.1% of NK 1.11TCR-ab2 cells in NK1T1, NK1T1(tge26Y),
and NK2T1 mice, respectively) levels. Furthermore, NK cells
from NK1T1(tge26Y) mice were functionally as competent as
those from NK1T1 mice (Fig. 2B). Therefore, these mice were
termed as NK1T1(tge26Y). Since they have identical background
as the NK2T1 mice, the NK1T1(tge26Y) mice were also used as
NK cell competent controls, in addition to the wild-type mice
(NK1T1).
NK2T1 mice control cutaneous L. major infection
Following infection with L. major, NK2T1, NK1T1, and
NK1T1(tge26Y) mice developed lesions, which resolved spontaneously within 60 –70 days (Figs. 2C and 3A). The course of L.
major infection was similar in NK2T1, NK1T1, and
NK1T1(tge26Y) mice (Figs. 2C and 3A). In contrast, concomitantly infected tge26 (NK2T2) and BALB/c mice developed large
nonhealing lesions and did not control the infection (Figs. 2C and
3A). The lesion grew significantly faster in NK2T2 mice than in
BALB/c mice (Fig. 3A). Examination of the histopathology of the
infected footpads from NK2T2 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. 3C). On the other hand, infected
foot pads from NK2T1 and NK1T1 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 NK1T1
and NK2T1 mice. The lesions from L. major-infected NK2T2
and BALB/c mice, however, contained significantly more parasites
(at least 10 logs more) than NK2T1 and NK1T1 mice (Fig. 3B).
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-22/2 mice, they suggest that NK cells are not
essential for control of L. major infection when immunocompetent
T cells are present.
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The lymph node cells, spleen cells, and PBL were analyzed by three-color
flow cytometry as described previously (15). Briefly, 0.5–1 3 106 cells in
50 ml 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 ml/sample; Life Technologies, Rockville,
MD), PE- and FITC- conjugated Abs (0.5 mg/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-e, H57-597 (TCR-ab), RM4-5 (CD4), 53-5.8
(CD8a), 53-2.1 (Thy 1.2), RA3-6B2 (B220), PK136 (NK1.1), and DX5
(all purchased from PharMingen, San Diego, CA).
Results
The Journal of Immunology
6749
NK2T1 mice develop efficient Th1-like response following L.
major infection
NK cells have been shown to be a major source of IFN-g, a cytokine critical for development of the Th1 lymphocyte subset of
the CD41 T cell population in resistant mice following L. major
infection (11). Therefore, we compared IL-12 and IFN-g production by Leishmania Ag-stimulated draining lymph node cells from
L. major-infected NK2T1 and NK1T1 mice. On day 60 postinfection, the draining lymph node cells from L. major-infected
NK1T1 and NK2T1 mice contained a similar number of lymphocytes (1.02 6 0.13 3 107 and 1.35 6 0.4 3 107 in NK1T1 and
NK2T1 mice, respectively; p , 0.375). Furthermore, there were
no significant differences in proportions of B2201 (43.7 6 5.4%
and 41.6 6 1.9% in NK1T1 and NK2T1 mice, respectively),
CD41 (28.6 6 4.2% and 32.9 6 4.7% in NK1T1 and NK2T1
mice, respectively), and CD81 (10.1 6 1.6% and 11.1 6 1.1% in
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FIGURE 1. NK2T1 mice lack
NK1.11CD32TCR-ab2 cells but
have normal T cell development. A,
CD4 and CD8 profile of lymph node
cells from a wild-type (NK1T1) and
an NK2T1 mouse. B, NK1.1 and
TCR-ab profile of spleen cells, indicating that NK2T1 mice have markedly diminished NK1.11ab2 cells,
but have a relatively normal number
of NK 1.11ab1 cells. C, NK2T1
mice have functionally normal CD41
T cells as assessed by MLR assay. D,
NK2T1 mice have functionally normal CD81 T cells as assessed by
CTL assay. E, NK2T1 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 NK1T1 and two
NK2T1 mice.
NK1T1 and NK2T1 mice, respectively). NK 1.11 cells, however, were markedly reduced in the lymph nodes from NK2T1
mice (2.1 6 0.31% and 0.4 6 0.06% in NK1T1 and NK2T1
mice, respectively). At this time, lymphocytes from NK1T1,
NK2T1, and BALB/c mice displayed greater Ag-specific proliferative responses than those derived from NK2T2 mice. At this
time, LmAg-stimulated lymph node cells from both NK2T1 and
NK1T1 mice produced IL-12 and IFN-g, although IL-12 levels
were significantly higher in NK2T1 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 NK2T1 and
NK1T1 mice (Fig. 4C). Neither IL-4 nor IFN-g was detectable in
the lymph node cell culture supernatants from NK2T2 mice,
which contained basal levels of IL-12. (Fig. 4, A–C). All groups
produced comparable levels of IL-10 (data not shown). Similarly,
6750
NK CELL-DEFICIENT MICE CONTROL CUTANEOUS LEISHMANIASIS
at an earlier time point on day 36 postinfection, both NK2T1 and
NK1T1 mice produced IL-12 (0.114 6 0.4 ng/ml and 1.3 6 0.17
ng/ml in NK1T1 and NK2T1 mice, respectively) and IFN-g
(1.068 6 0.6 ng/ml and 2.9 6 0.25 ng/ml in NK1T1 and NK2T1
mice, respectively), but no IL-4. In additional experiments,
NK1T1(tge26Y) mice were also examined. NK1T1(tge26Y)
mice controlled L. major infection as efficiently as NK1T1 mice
(Fig. 2C). Furthermore, there was no significant difference in
IFN-g production by LmAg-stimulated lymph node cells from
NK1T1 and NK1T1(tge26Y) mice (Fig. 2D).
NK2T1 mice fail to produce Leishmania-specific IgG2a, despite
development of Th1 response
Ab responses in L. major-infected NK2T1, NK1T1, NK2T2, and
BALB/c mice were analyzed by measuring titers of Leishmaniaspecific Th1-dependent IgG2a and Th2-dependent IgG1 Abs on
days 30, 45, and 60 postinfection. On day 30 and thereafter,
NK1T1 and BALB/c mice developed significant levels of Leishmania-specific IgG1 and IgG2a, although BALB/c mice produced
significantly more (Fig. 5A). On the other hand, L. major-infected
NK2T1 mice displayed high titers of Leishmania-specific IgG1,
but failed to produce any measurable quantities of Leishmaniaspecific IgG2a throughout the course of infection (Fig. 5B). Both
NK1T1 and NK1T1(tge26Y) mice produced significant titers of
LmAg-specific IgG2a (2666.2 6 533 and 16200 6 12049 in
NK1T1 and NK1T1(tge26Y) mice, respectively). Similar results
were observed on days 30 and 45 postinfection (data not shown).
IL-12 is critical for development of Th1 response and controls
L. major infection in NK2T1 mice
To determine whether IL-12 is critical for development of Th1
response in NK2T1 mice during L. major infection, we treated L.
major-infected NK2T1 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 NK2T1 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 NK2T1 mice produced significantly higher
amounts of IFN-g (mean levels, 1.34 6 0.4 ng/ml) than those from
anti-IL-12-treated NK2T1 mice, which produced only basal levels
(,0.05 ng/ml; p , 0.05).
Discussion
The results presented here indicate that although NK1.11CD32TCRab2 (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.
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FIGURE 2. NK1T1(tge26Y) mice develop Th1 response and control L. major infection as efficiently as NK1T1 mice. A, CD4 and CD8 profile of lymph
node cells from a wild-type (NK1T1) and NK1T1(tge26Y) mice. B, NK1T1(tge26Y) mice have normal NK cell cytotoxicity against YAC-1 cells similar
to that observed in NK1T1 mice. C, Course of L. major infection in NK1T1, NK1T1(tge26Y), NK2T1, and NK2T2 mice. Disease progression was
monitored by measuring increase in the thickness of infected footpad, as described before. D, In vitro LmAg-induced IFN-g production by the lymph node
cells. Data expressed as mean 6 SE. Asterisk indicates statistically significant difference between two groups. ND, not detectable.
The Journal of Immunology
6751
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 ab and gd
T cells but have NK cells develop activated macrophages and partially control Listeria monocytogenes (17–19). Later, it was demonstrated that spleen cells from naive SCID and nude mice produced significant levels of IFN-g following incubation with heatkilled L. monocytogenes (18). Furthermore, administration of
neutralizing anti-IFN-g mAb or NK cell depletion before infection
abolished macrophage activation in SCID mice. Together, these
data indicate that NK cell-derived IFN-g 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 –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 cellderived IFN-g 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-g 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-a/b) 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 antiAsGM1 Ab significantly decreased early IFN-g 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 1.11CD32 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 NK2T1 mice lack NK cell cytotoxicity
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FIGURE 3. NK2T1 mice control cutaneous L. major infection. A, Course of L. major infection following infection with 2 3 106 stationary-phase
promastigotes in NK2T1, NK1T1, BALB/c, and tge26 (NK2T2) 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. NK2T2 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 NK1T1, NK2T1,
and NK2T2 mice. Data expressed as log parasite titer 6 SE. C–E, Hematoxylin-eosin stained skin lesions from L. major-infected NK2T2, NK1T1, and
NK2T1 mice. Lesions from NK2T2 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 NK1T1 and NK2T1 mice displayed a more
preserved skin structure with lymphocytes and some macrophages with few intracellular parasites (original magnification, 340). Results are representative
of three experiments with four to five animals per group. Data expressed as mean 6 SE.
6752
NK CELL-DEFICIENT MICE CONTROL CUTANEOUS LEISHMANIASIS
FIGURE 4. NK2T1 mice develop a Th1-like response. In vitro LmAginduced (20 mg/ml) IL-12 (A), IFN-g (B), and IL-4 (C) production by the
lymph node cells from NK1T1, NK2T1, and BALB/c mice measured on
day 60 postinfection. NK2T2 (tge26) mice were sacrificed on day 30
postinfection for proliferation assays and cytokine production, due to the
development of large lesions. Data expressed as mean 6 SE. Asterisks
indicate statistically significant differences between NK1T1 and NK2T1
mice. Similar results were observed in two independent experiments. ND,
not detectable.
against lymphocytic choriomeningitis virus and YAC-1 cells.3
Therefore, these mice are truly deficient in NK cell lytic function,
and the number of NK1.11CD32 cells are either absent or markedly diminished in these mice.3 We have reported previously that
transplantation of wild-type bone marrow into adult tge26 mice
results in aberrant NK cell development, which is caused by the
generation of aberrant T cells that leads to high levels of TNF-a in
vivo (33). This mechanism, however, could not account for NK
cell deficiency in the neonatal tge26 model described in this study.
First, in the neonatal tge26 model, NK cell deficiency is independent of T cell development,3 and there was no overproduction of
TNF-a in sera from L. major-infected NK1T1 and NK2T1 mice
(97 6 28 pg/ml and 71 6 40 pg/ml in NK1T1 and NK2T1 mice,
respectively). Second, we demonstrated that the lack of cytotoxic
NK cells in the neonatally transplanted tge26 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-ab2/2 mice lacking T cells on genetically resistant background (15) and RAG-22/2
mice that have innately high levels of IFN-g 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 tge26 mice (NK2T2) are susceptible to L. major and develop
lesions significantly faster than similarly infected RAG22/2 or
TCR-ab2/2 mice (NK1T2) suggest that NK cells may be involved in controlling early resistance to L. major. However, the
ability of NK2T1 mice to control cutaneous L. major infection as
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FIGURE 5. L. major-specific IgG1 and IgG2a production in NK1T1
and NK2T1 mice on day 60 postinfection presented as reciprocal endpoint
titers on a log scale. Similar results were observed in three independent
experiments. NK2T2 mice were sacrificed on day 30 postinfection, due to
the development of large lesions. ND, not detectable.
The Journal of Immunology
FIGURE 6. Administration of IL-12 neutralizing Ab to NK2T1 mice
inhibits Th1 development and exacerbates cutaneous L. major infection.
Data expressed as mean lesion size 6 SE.
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 NK2T1 mice failed to produce Th1associated Leishmania-specific IgG2a Ab throughout the course of
infection. Similarly, in our ongoing studies, NK2T1 produced significant amounts of IFN-g but displayed baseline levels of Agspecific 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-ab (NKT cells) has been shown to produce IL-4 (43) as well
as IFN-g (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 CD41 cells that are NK 1.1negative (45). Recent studies have demonstrated that IL-12-stimulated NK1.11 T cells produce high levels of IFN-g (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 1.11 T cells in liver and improve protective immunity
against listeriosis (48). NK2T1 mice used in the present study
have a normal number of NK1.11CD41 T cells. Additionally, administration of anti-IL-12 Ab L. major-infected NK2T1 mice inhibited Th1 development and rendered them susceptible to infection. Therefore, one may speculate that NK 1.11 T cell-derived
IFN-g initiates Th1 development in these mice. A recent study,
however, demonstrated that RAG-2/IFN-g2/2 (double mutant)
mice reconstituted with the wild-type CD41 NK 1.12 T cells develop Th1 response and control cutaneous L. major infection (38).
Taken together, these results indicate that neither NK cell-derived
IFN-g nor NK 1.11CD41 T cells are critical for the development
of protective Th1 response following L. major infection.
In conclusion, L. major-infected NK2T1 mice on genetically
resistant background develop an efficient Th1-like response as
measured by significant IFN-g production by the lymph node cells
following stimulation with Leishmania Ag and control cutaneous
L. major infection. Furthermore, administration of neutralizing antiIL-12 Abs to NK2T1 mice during L. major infection inhibits development of Th1-like response and enhances cutaneous lesion
growth.
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