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*
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
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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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-
, 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. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(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-, CD3
-, 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- (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-
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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tg26 mice were maintained through sib breeding in the animal
facility of the Beth Israel Deaconess Medical Center (Boston, MA) (13, 14). Although the tg26 transgenic founder was a (C57BL/6 x
CBA/J)F2, all tg26 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 x
CBA/J)F1 mice into neonatal tg26 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 x CBA/J)F1
(H-2b/k) generated through breeding of C57BL/6 x
CBA/J. The other type was tg26 mice reconstituted with (C57BL/6
x 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+(tg26Y) mice. Five weeks
after the transplantation, NK+T+(tg26Y) mice
develop functionally competent NK and T cells, and their levels are
comparable to those observed in wild-type (C57BL/6 x
CBA/J)F1 mice. Furthermore, NK+T+
(tg26Y) and NK-T+ mice have similar levels
of CD4+ and CD8+ T cells. Of note, all the
NK+T+, NK+T+(tg26Y),
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 x 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 x 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 x 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-, H57-597 (TCR-
ß), RM4-5 (CD4), 53-5.8
(CD8), 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-2kxb) 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 x 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- (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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, we demonstrated that transplantation of wild-type bone
marrow or fetal liver cells into neonatal tg26 mice results in
normal development of T cells, but poor NK cell
development.3 All the neonatal transplanted tg26 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-ß-, 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-ß- 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-ß+ 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 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. 1E). When young tg26 mice were reconstituted with
F1 bone marrow or fetal liver cells at 2–3 wk of age
(tg26Y), these mice had comparable wild-type (F1) T
(Fig. 2A) and NK cell
(2.5 ± 0.63%, 1.3 ± 0.05%, and 0.3 ± 0.1% of NK
1.1+TCR-ß- cells in
NK+T+, NK+T+(tg26Y),
and NK-T+ mice, respectively) levels.
Furthermore, NK cells from NK+T+(tg26Y) mice
were functionally as competent as those from
NK+T+ mice (Fig. 2B). Therefore,
these mice were termed as NK+T+(tg26Y).
Since they have identical background as the
NK-T+ mice, the
NK+T+(tg26Y) mice were also used as NK cell
competent controls, in addition to the wild-type mice
(NK+T+).
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Following infection with L. major,
NK-T+, NK+T+, and
NK+T+(tg26Y) 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+(tg26Y) mice (Figs. 2C and
3A). In contrast, concomitantly infected tg26
(NK-T-) and BALB/c mice developed large
nonhealing lesions and did not control the infection (Figs. 2C and 3A). The lesion grew significantly faster
in NK-T- mice than in BALB/c mice (Fig. 3A). 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. 3C). 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. 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-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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NK cells have been shown to be a major source of IFN-, 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-
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 x
107 and 1.35 ± 0.4 x 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-, 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. 4C). Neither IL-4 nor IFN- 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-
(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+(tg26Y) mice were also examined.
NK+T+(tg26Y) mice controlled L. majorinfection as efficiently as NK+T+ mice
(Fig. 2C). Furthermore, there was no significant difference
in IFN- production by LmAg-stimulated lymph node cells from
NK+T+ and
NK+T+(tg26Y) mice (Fig. 2D).
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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. 5A). 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. 5B). Both NK+T+ and
NK+T+(tg26Y) mice produced significant
titers of LmAg-specific IgG2a (2666.2 ± 533 and 16200 ±
12049 in NK+T+ and
NK+T+(tg26Y) mice, respectively). Similar
results were observed on days 30 and 45 postinfection (data not shown).
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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- (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).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß- (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 ß and
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- following incubation with
heat-killed L. monocytogenes (18). Furthermore,
administration of neutralizing anti-IFN- mAb or NK cell
depletion before infection abolished macrophage activation in SCID
mice. Together, these data indicate that NK cell-derived IFN- 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- 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- 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-/ß) 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- 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 tg26 mice results in aberrant NK cell development, which is
caused by the generation of aberrant T cells that leads to high levels
of TNF- in vivo (33). This mechanism, however, could not account for
NK cell deficiency in the neonatal tg26 model described in this
study. First, in the neonatal tg26 model, NK cell deficiency is
independent of T cell development,3 and there was no
overproduction of TNF- 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 tg26 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-ß-/- mice
lacking T cells on genetically resistant background (15) and
RAG-2-/- mice that have innately high levels of
IFN- 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 tg26 mice
(NK-T-) are susceptible to L.
major and develop lesions significantly faster than similarly
infected RAG2-/- or TCR-ß-/-
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- 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- (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- 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- 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- 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- 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- 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-/ß), 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-
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-ß
(NKT cells) has been shown to produce IL-4 (43) as well as IFN-
(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- (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- initiates
Th1 development in these mice. A recent study, however, demonstrated
that RAG-2/IFN--/- (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- 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- 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.
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Footnotes |
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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:
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 for publication December 1, 1998. Accepted for publication March 17, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, interleukin 2, interleukin 4, and interleukin 10 by CD4+ T lymphocytes in vivo during healing and progressive murine leishmaniasis. Proc. Natl. Acad. Sci. USA 88:7011. interferon production by natural killer cells is important in defense against murine listeriosis. Infect. Immun. 59:1892.
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