By
From the * Department of Physiology and Neuroscience, and Department of Clinical Pharmacology,
Lund University Hospital, 221 85 Lund, Sweden; § Department of Inflammation Pharmacology,
Astra Draco, 221 00 Lund, Sweden;
Microbiology and Tumor Biology Center, Karolinska Institute,
171 77 Stockholm, Sweden; ¶ Howard Hughes Medical Institute, Department of Microbiology and
Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232; and ** Department of Clinical Immunology and Transfusion Medicine, Uppsala University Hospital,
751 85 Uppsala, Sweden
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Abstract |
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The earliest contact between antigen and the innate immune system is thought to direct the
subsequent antigen-specific T cell response. We hypothesized that cells of the innate immune
system, such as natural killer (NK) cells, NK1.1+ T cells (NKT cells), and /
T cells, may regulate the development of allergic airway disease. We demonstrate here that depletion of
NK1.1+ cells (NK cells and NKT cells) before immunization inhibits pulmonary eosinophil
and CD3+ T cell infiltration as well as increased levels of interleukin (IL)-4, IL-5, and IL-12 in
bronchoalveolar lavage fluid in a murine model of allergic asthma. Moreover, systemic allergen-specific immunoglobulin (Ig)E and IgG2a levels and the number of IL-4 and interferon
-producing splenic cells were diminished in mice depleted of NK1.1+ cells before the priming regime. Depletion of NK1.1+ cells during the challenge period only did not influence pulmonary eosinophilic inflammation. CD1d1 mutant mice, deficient in NKT cells but with normal NK cells, developed lung tissue eosinophilia and allergen-specific IgE levels not different
from those observed in wild-type mice. Mice deficient in
/
T cells showed a mild attenuation of lung tissue eosinophilia in this model. Taken together, these findings suggest a critical
role of NK cells, but not of NKT cells, for the development of allergen-induced airway inflammation, and that this effect of NK cells is exerted during the immunization. If translatable to
humans, these data suggest that NK cells may be critically important for deciding whether allergic eosinophilic airway disease will develop. These observations are also compatible with a
pathogenic role for the increased NK cell activity observed in human asthma.
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Introduction |
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Airway mucosal inflammation in allergic asthma is thought
to be dependent on T lymphocytes producing proinflammatory cytokines (1, 2). The production of IL-4 and
IL-5 by these cells is considered to be pivotal for the recruitment of eosinophils to the airways, a hallmark of asthma (3,
4). The development of T cell effectors from naive T cells
may depend on many factors, including the nature of antigen presentation and the local cytokine milieu during the
period of T cell priming (5, 6). It has been suggested that
rapid generation of key cytokines by cells of the innate immunity regulates the subsequent antigen-specific T cell response (7). Thus, cells such as NK cells, NK1.1+ T cells
(NKT cells),1 and /
T cells have been given tentative
roles in determining the nature of the acquired immune response. This possibility has been discussed particularly with
regard to host response to infection (8, 9).
NK cells are recognized as an important component in
immune responses against a number of pathogens (10, 11).
Their cytokine production rather than cytolytic activity
contributes to the resistance against infectious agents (10,
11). NK cells can rapidly produce IFN-. However, these
cells may also produce a variety of other immunoregulatory
mediators, including TGF-
, TNF-
, TNF-
, GM-CSF, macrophage inflammatory protein (MIP)-1
, IL-1, IL-2,
IL-3, IL-5, IL-8, and IL-10 (10). In addition to NK
cells, mouse NK1.1+ cells comprise a small population of
cells that coexpress NK1.1 and TCR, i.e., NKT cells (13-
15). A human counterpart of the mouse NKT cell, coexpressing IL-4 and IFN-
, has recently been identified (16).
Little is known about the role of NK cells in induction
of immune responses to allergens. Bogen et al. (17) have
demonstrated that the earliest detectable response to subcutaneous administration of a protein antigen (OVA) in adjuvant was the appearance of IFN--producing NK1.1+ cells
at the site of immunization. NK cells are normally present in considerable numbers in human lung interstitium, suggesting involvement of these cells in pulmonary immunity
(18). Furthermore, it has been demonstrated that patients
with asthma show increased numbers of NK cells and
stronger NK activity in peripheral blood than normal
healthy blood donors (19). NK cell activity may also be
increased after bronchial allergen challenge in asthmatic subjects (22). However, the focus in these previous clinical studies has been on the possible association of asthma with
a reduced risk for tumor disease development.
/
T cells represent another lymphocyte population
that can produce various cytokines early in an immune response. A recent study has shown that
/
T cells differentially produce IFN-
and IL-4 in response to Th1- and
Th2-inducing pathogens (23). However, the role of
/
T
cells in allergic diseases such as asthma is still controversial.
McMenamin et al. (24) have shown that CD8+
/
T cells
in mice may specifically downregulate IgE responses to soluble OVA. Recently, Zuany-Amorim et al. (25) reported that
/
T cells are required for inducing allergen-specific
IgE and IgG1 responses and Th2-mediated airway inflammation in a mouse model of asthma.
We hypothesized that innate immunity, particularly NK
cells, NKT cells, and /
T cells, may be involved in the
processes that cause eosinophilic airway inflammation in
immunized and allergen-exposed mice. Specifically, this
study asks whether mice that are depleted of NK1.1+ cells,
i.e., NK cells and NKT cells (26), and whether mice that
genetically lack NKT cells (27) or
/
T cells (28), develop
pulmonary inflammation in an established model of allergic asthma (29, 30).
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Materials and Methods |
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Animals and Study Design.
Male C57BL/6 mice (n = 127, 8-9 wk of age in experiment I and II, 4-6 mo of age in experiment IV) were purchased from Bomholtgaard (Table I). CD1d1 mutant (129/Sv × C57BL/6) mice and wild-type littermates (n = 20, 7 wk of age) were used in experiment III (27). TCR
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Immunization and Challenge.
We have used a protocol slightly modified from that developed by Brusselle and colleagues (29). On the first day of the experiment (day 0), all mice were actively immunized by injection of 10 µg i.p. chicken OVA (Grade III; Sigma), adsorbed to 1 mg of alum adjuvant. From days 14-15 (groups 1-4) or from days 14-20 (all other groups) after immunization, the mice were exposed daily to aerosolized saline (SAL) or OVA over a 30-min period by placing groups of 5-12 awake mice in an exposition chamber (Table I). The aerosols were generated into the chamber using a nebulizer (500 ml Inline Micronebulizer driven at 4 bar; Bird Co.). The concentration of OVA in the nebulizer was 1% wt/vol.Depletion of NK1.1+ Cells.
To deplete NK1.1+ cells in vivo, mice were injected starting 2 d before immunization with 100 µg i.p. of anti-NK1.1 mAb (26), and every 5 d thereafter with 25 µg i.p. of the anti-NK1.1 mAb until termination of the experiment. One group of mice was depleted of NK1.1+ cells in a similar manner, but the treatment regimen started 7 d before immunization (group 11). A third group of animals was also depleted of NK1.1+ cells, but the treatment started 1 d before first allergen aerosol challenge (group 13). Control mice were injected with a similar volume (0.2 ml) and dose of mouse IgG antibody (Sigma) as the appropriate isotype control. The efficacy of depletion of NK1.1+ cells was monitored by flow cytometric analysis of spleen cells at the end of the experimental period. Animals exhibiting >1.0% NK1.1+ cells in the spleen at the end of the experiment were excluded from the study (n = 5).Histochemistry.
Lung tissue specimens obtained 8, 24, and 30 h after the last OVA or SAL exposure were immersed overnight in Stefanini's fixative (2% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer, pH 7.2), rinsed repeatedly in buffer (Tyrode buffer supplemented with 10% sucrose), frozen in mounting medium (Tissue-Tek; Miles, Inc.), and stored atImmunohistochemistry.
Cryosections (10 µm) were fixed in cold acetone diluted 1:2 in distilled water for 30 s, followed by final fixation in cold acetone (100%) for 5 min. Subsequent incubations were carried out sequentially for 30 min, with 5 min in PBS between each step. Unspecific antibody binding was blocked by incubation with PBS containing 10% (vol/vol) normal rabbit serum for 5 min (X0902; Dako). Incubation with an mAb recognizing the mouse CD3 antigen (clone KT3; Serotec) was followed by rabbit anti-rat IgG antibody (Z494; Dako) diluted 1:50 in PBS containing 10% (vol/vol) normal rabbit serum. After a final incubation with a monoclonal rat alkaline phosphatase-anti-alkaline phosphatase reagent (D488; Dako), the alkaline phosphatase reaction was developed using BCIP/NBT/ INT mixed with Levamisole (K599 and X3021; Dako) for 10 min.Analysis of Cytokines in BAL Fluid.
All mice in experiments I and III and groups 12 and 13 in experiment II were subjected to BAL at 8 h after last aerosol exposure (Table I). After the animals had been anesthetized with an intraperitoneal injection of pentobarbital, a tracheal cannula was inserted via a midcervical incision and the airways were lavaged twice with 0.9 ml of PBS (Life Technologies). The BAL fluid (BALF) was immediately centrifuged (10 min, 4°C, 160 g), and the supernatant was rapidly frozen. Commercial ELISA kits were used to measure levels of IL-4 (Biosource International), IL-5 (Nycomed Amersham plc), IL-12 (Genzyme), and IFN-Measurement of OVA-specific IgE and IgG2a in Plasma.
Blood was drawn from all mice in experiments II-IV (Table I) by cardiac puncture and placed in EDTA tubes. After centrifugation, the plasma samples were rapidly frozen. OVA-specific IgE and IgG2a levels were determined by sandwich ELISA in 96-well ELISA plates. Sample wells were coated overnight with OVA grade III (100 µg/ml, 100 µl/well; Sigma). Wells were then blocked with 3% BSA in PBS (200 µl/well) at 37°C for 2 h. Diluted samples and standard (100 µl/well) were incubated overnight at 4°C. Plates were washed with 0.05% Tween 20/PBS and incubated with biotin-conjugated anti-mouse IgE (diluted 1:500) or IgG2a (diluted 1:100, 100 µl/well; PharMingen) at room temperature for 90 min. After another wash procedure, plates were incubated for 90 min at room temperature with 100 µl/well of ExtrAvidin-HRP (Sigma) diluted 1:600 in 1% BSA/PBS. Plates were developed with TMB Microwell Peroxidase Substrate (100 µl/well; Kirkegaard & Perry Labs). Plates were read at 450 nm. A plasma pool of OVA-immunized mice was used as internal laboratory standard. A 1:100 dilution of this pool was chosen as arbitrary unit.Enzyme-linked Immunospot Analysis of Cytokine-producing Spleen Cells.
The number of spleen cells producing IL-4 and IFN-Quantification and Statistics.
For evaluation of the number of eosinophils and CD3+ cells in pulmonary tissue, 40 randomly selected areas (0.04 mm2 each) in 1 lung section from each animal were examined. The number of eosinophils and CD3+ cells in the 40 areas was counted at a magnification of 400, and the mean was expressed as eosinophils or CD3+ cells per unit area. All quantifications were performed blind. Data are expressed as mean ± SEM unless otherwise indicated. To calculate significance levels between treatment groups, the Student's t test was used throughout the study. Values below detection limits were assigned the value of the detection limit. To achieve comparable SDs, ELISA values were transformed to logarithms before statistical analysis. Probabilities <0.05 were used as the generally accepted level of statistical significance for differences between mean values. ![]() |
Results |
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Histologic analysis of lungs taken 8 h after last (second) aerosol exposure from both IgG-treated and NK1.1+ cell-depleted mice receiving two OVA challenges (groups 3 and 4) exhibited a slight pulmonary eosinophilia (3.9 ± 1.0 and 3.8 ± 0.7 cells/unit area, respectively). Although a few small eosinophilic infiltrates were detected in lung tissue in the majority of these animals, the eosinophilia was not significantly increased compared with corresponding immunized and SAL-exposed animals (2.2 ± 0.4 and 2.3 ± 0.4 cells/unit area, respectively). To detect the emerging Th2 response in the lungs of these animals after allergen exposure, we measured IL-4 in BALF using ELISA. The levels of IL-4 in BALF were increased in immunized and OVA-challenged IgG-treated mice compared with corresponding immunized and SAL-exposed animals (142.7 ± 14.8 vs. 89.7 ± 19.7 pg/ml; P < 0.05). A tendency towards higher IL-4 levels in NK1.1+ cell-depleted animals compared with IgG-treated mice was demonstrated. Thus, IL-4 levels in BALF from OVA-challenged and NK1.1+ cell-depleted mice did not differ significantly from those in the corresponding SAL-exposed group (159.0 ± 25.2 vs. 131.4 ± 7.4 pg/ml).
Late Allergen-induced Airway Changes in Mice Depleted of NK1.1+ Cells before Immunization.Immunized and IgG-treated mice receiving seven OVA challenges exhibited at the 8 h time point a marked eosinophilia perivascularly and peribronchially in the lung (groups 7 and 9; Fig. 1, a and b, and Fig. 2 a). In contrast, corresponding mice depleted of NK1.1+ cells (groups 8 and 10) showed a clearly inhibited eosinophilia in lung tissue (Fig. 1, a and b). These mice exhibited a few scattered eosinophilic infiltrates, or a complete absence of pulmonary inflammation (Fig. 2 b). Eosinophilic infiltrates were not observed in lung tissue from SAL-exposed mice of either the IgG-treated or the NK1.1+ cell-depleted groups (groups 5 and 6; Fig. 1 a). The marked attenuation of lung tissue eosinophilia in mice depleted of NK1.1+ cells was still observed 30 h after the last OVA exposure (group 15; Fig. 1 b). At this time point, one outlier (determined by Q test) was obvious, without which the difference between IgG-treated and depleted animals had been statistically significant (P < 0.05). It is possible that this single animal (exhibiting the second most pronounced pulmonary eosinophilia in the experiment) was not successfully depleted of NK1.1+ cells after the first injection of mAb NK1.1, 2 d before immunization.
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The number of CD3+ cells in lung tissue from immunized and IgG-treated animals receiving seven OVA challenges was increased compared with corresponding immunized and SAL-exposed animals at the 8-h time point (12.0 ± 2.0 vs. 5.1 ± 0.4 cells/unit area; P < 0.05). In contrast, the number of CD3+ cells in lung tissue from immunized and NK1.1+ cell-depleted animals remained low after allergen challenges (6.7 ± 1.0 vs. 6.8 ± 1.4 cells/unit area in corresponding SAL-exposed animals).
Sections stained with hematoxylin and erythrosin or PAS further demonstrated that the pulmonary eosinophilia was accompanied by a dense infiltration of mononuclear cells and an evident increase in airway epithelial mucus cells in lungs of OVA-exposed IgG-treated mice. Also, these morphological changes were markedly reduced in OVA-challenged mice depleted of NK1.1+ cells.
To determine the type of immune response (Th1 and/or
Th2) being induced in the airways of immunized mice after
multiple allergen aerosol exposures, we measured cytokines
in BALF taken 8 h after last aerosol exposure. The levels
of IL-4 in BALF were similar in both IgG-treated and
NK1.1+ cell-depleted mice receiving seven OVA challenges compared with corresponding SAL-exposed animals
(Fig. 3). Thus, the increased levels demonstrated after two
OVA exposures in immunized IgG-treated mice (but not
in corresponding mice depleted of NK1.1+ cells) were not
detected under these more chronic conditions. BALF from
IgG-treated mice receiving seven allergen challenges contained measurable amounts of IL-5 (P < 0.05, compared
with SAL-exposed IgG-treated animals; Fig. 3). In contrast,
NK1.1+ cell-depleted animals failed to release IL-5 after
OVA exposure (Fig. 3). The levels of IL-12 in BALF increased after allergen exposure in IgG-treated animals (P < 0.001, compared with corresponding SAL-challenged animals; Fig. 3). In contrast, IL-12 levels in BALF from
NK1.1+ cell-depleted mice remained low in response to
allergen exposure, similar to the levels in corresponding
SAL-exposed animals. The levels of IFN- in BALF decreased after allergen challenge to values very near or below the detection limit of the assay in five out of seven
IgG-treated animals (Fig. 3). Interestingly, the two animals
exhibiting high IFN-
values showed undetectable IL-5
levels in BALF and no pulmonary eosinophilia. OVA challenge of mice depleted of NK1.1+ cells caused a moderate
decrease in levels of IFN-
(P < 0.05, compared with corresponding SAL-exposed mice; Fig. 3).
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To assess the peripheral immune response to immunization and allergen challenge, we measured OVA-specific IgE and IgG2a levels in plasma using ELISA. Immunization and allergen exposure of IgG-treated animals induced both OVA-specific IgE and IgG2a (Fig. 4). In corresponding NK1.1+ cell- depleted animals, this induction of OVA-specific IgE and IgG2a was significantly suppressed (P < 0.01, P < 0.05, compared with immunized and OVA-challenged mice treated with IgG).
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To
estimate the systemic T cell cytokine response in spleen after immunization and OVA challenge, we used ELISPOT.
The number of IL-4-producing spleen cells (both unstimulated and OVA-stimulated cells) was overall lower in mice
depleted of NK1.1+ cells compared with IgG-treated animals (Fig. 5, a and b). The number of OVA-stimulated
IL-4-producing cells at the 8-h time point was significantly
lower in NK1.1+ cell-depleted mice compared with IgG-treated animals (P < 0.05). Similar patterns were obtained
for IFN--producing cells (Fig. 5, a and b). Significant differences were obtained at the 8- and 30-h time points between the number of unstimulated IFN-
-producing spleen cells from IgG-treated and NK1.1+ cell-depleted
animals (P < 0.05).
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To determine if NK1.1+
cells are required for the initiation of the immune response
at the time of immunization or for the onset of pulmonary
inflammation secondary to allergen challenge, mice were
depleted of NK1.1+ cells during the challenge period only
(group 13). Immunization and OVA exposure (seven
times) of mice depleted of NK1.1+ cells during the challenge period only led to the development of a pulmonary
eosinophil-rich inflammation similar to that observed in
corresponding IgG-treated animals (26.3 ± 3.9 and 19.5 ± 2.5 eosinophils/unit area, respectively). These groups of
mice also exhibited similar levels of IL-4 (70.9 ± 8.8 and
85.6 ± 20.9 pg/ml, respectively), IL-12 (1,524.5 ± 137.1 and 1,184.5 ± 202.1 pg/ml, respectively), and IFN- (levels < detection limit in both groups) in BALF. Furthermore, systemic levels of OVA-specific IgE were not altered
(4,132.3 ± 1,661.3 versus 6,281.0 ± 2,563.6 U/ml in the
corresponding IgG-treated group).
To confirm that no acute nonspecific effects (such as cytokine release) of depletion with
mAb NK1.1 caused the demonstrated effects on the immune response, mice were depleted of NK1.1+ cells from
day -7 before immunization (group 11). This group of
mice exhibited a reduction of lung tissue eosinophilia compared with mice treated with IgG from day 2 before immunization (group 9) (14.2 ± 2.4 vs. 25.9 ± 3.7 cells/unit
area; P < 0.05). Also, these mice exhibited suppressed levels of OVA-specific IgE compared with mice treated with
IgG from day -2 before immunization (group 9) (1,133.7 ± 428.8 vs. 6,971.1 ± 4,735.1 U/ml; P < 0.05). Depletion of NK1.1+ cells from day
7 before immunization also
suppressed OVA-specific IgG2a levels. In this group, all
mice had levels of IgG2a below detection limit (P < 0.05, compared with mice treated with IgG from day -2 before immunization).
To elucidate if the suppression of the allergic immune response seen after NK1.1+ cell depletion requires NK cells
or NKT cells, CD1d1 mutant mice, which selectively lack
NKT cells (group 17), were immunized and OVA challenged. Immunization and OVA exposure (seven times) of mice deficient in NKT cells led to the development of a
pulmonary eosinophil-rich inflammation similar to that observed in corresponding wild-type animals (Fig. 6). CD1d1
mutant and wild-type mice also exhibited similar levels
of OVA-specific IgE (4,342.9 ± 1,054.3 and 6,814.8 ± 2,702.9 U/ml, respectively). The numbers of IL-4- and
IFN--producing spleen cells in immunized and allergen-challenged CD1d1 mutant and wild-type mice were determined by the ELISPOT method. There was a tendency,
although statistically insignificant, of reduced numbers
of IL-4-producing unstimulated and OVA-stimulated spleen cells from CD1d1 mutant mice compared with
wild-type mice (5.4 ± 1.2 vs. 16.2 ± 6.0 SFCs/106 CD3+
unstimulated cells and 150.0 ± 27.1 vs. 418.2 ± 212.8 SFCs/106 CD3+ OVA-stimulated cells). The number of
unstimulated IFN-
-producing cells was increased in
CD1d1 mutant mice compared with wild-type mice (60.0 ± 17.2 vs. 15.4 ± 7.8 SFCs/106 CD3+ cells; P < 0.05).
There was also a tendency of increased numbers of OVA-stimulated IFN-
-producing cells from mutant mice compared with wild-type animals (134.4 ± 54.1 vs. 40.9 ± 11.9 SFCs/106 CD3+ cells).
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A moderate reduction, although statistically insignificant, of
lung tissue eosinophilia was observed in OVA-challenged
/
T cell-deficient animals compared with corresponding
wild-type mice (Fig. 7). Similarly, no significant difference
in systemic levels of OVA-specific IgE was observed between OVA-challenged
/
T cell-deficient animals and
corresponding wild-type mice (1,066.3 ± 314.5 and
2,567.4 ± 1,497.1 U/ml, respectively).
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Discussion |
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This study demonstrates that depletion of NK1.1+ cells
(NK cells and NKT cells) before the priming regime inhibits pulmonary eosinophil and CD3+ T cell infiltration as
well as increased levels of IL-4, IL-5, and IL-12 in BALF in
a murine model of allergic asthma. Consistent with the
suppression of allergic airway inflammation seen in mice depleted of NK1.1+ cells before immunization, i.e., during
the initiation of the acquired immune response, diminished
systemic levels of OVA-specific IgE and IgG2a and impaired T cell cytokine production in spleen were observed
in these mice. The demonstration of a marked eosinophil-rich inflammation in lung tissue of NKT cell-deficient
CD1d1 mutant mice strongly supports an important role of
NK cells, but not of NKT cells, for development of allergen-induced pulmonary inflammation. However, we cannot completely exclude the possibility that NK cells and
NKT cells somehow interact and that depletion of both cell types may be required for inhibition of the allergic immune response in this model. A recent study has shown
that allergen-specific IgE and IgG1 responses and allergic
airway inflammation, induced by repeated immunization
and intranasal allergen challenges, are reduced in /
T
cell-deficient mice (25). In our experimental model, allergen-induced changes in
/
T cell-deficient animals were
attenuated, but this effect was moderate compared with the marked suppression of the allergic airway inflammation
demonstrated in mice depleted of NK1.1+ cells. Together,
these data support the hypothesis that innate immunity regulates the acquired immune response in allergic inflammation. Given the caution required in any translation of findings in murine allergic models to human asthma (33), the
present observations suggest the possibility that human NK
cells may govern development of allergic eosinophilic airway disease.
The present protocol for immunization and allergen
aerosol challenge was adopted from Brusselle et al. (29). In
this mouse model of asthma, repeated daily exposure of immunized mice to aerosolized OVA leads to a CD4+ T cell-
dependent (29) and IL-4-dependent (29, 34) eosinophilic inflammation with increased secretory cell epithelial lining
(30), lung and splenic Th2 cytokine production (35; this
study), and systemic allergen-specific IgE production (29, 34, 35; this study), consistent with a Th2-associated immune response. However, indices not only of Th2 expansion but also of Th1 expansion are present in this model.
Thus, systemic OVA-specific IgG2a production and increased levels of IL-12 in BALF were demonstrated in immunized and repeatedly allergen-challenged mice. Also in
allergic asthma there may be a mixed Th2/Th1 response.
Although there is a predominance of IL-4- and IL-5-producing CD4+ T cells, a population of IFN--producing
CD4+ T cells has thus been demonstrated in BALF from
asthmatic subjects (1, 36).
It may be hypothesized that the suppressed allergic response in mice depleted of NK1.1+ cells is caused by deficient IL-4 production at the time of immunization. Studies
have failed to detect IL-4 production by NK cells (12). However, it has been hypothesized that NKT cells may be
an important source of IL-4 and, according to the paradigm
of Th1 and Th2 (37), for the development of Th2 immune
responses (38). Accordingly, in this study, spleens of
NKT cell-deficient CD1d1 mutant mice exhibited a tendency of reduced numbers of IL-4-producing CD3+ T
cells and an associated increase in IFN--producing CD3+
T cells. However, since CD1d1 mutant mice developed
lung tissue eosinophilia and allergen-specific IgE levels not
different from those observed in wild-type mice, we conclude that NKT cells and their swift production of IL-4 are
not critical for the allergic responses in this model. Also,
previous studies using
2-microglobulin-deficient animals
have shown that NKT cells are dispensable for allergen- induced Th2 responses in the airways (41, 42).
Our data on systemic levels of OVA-specific IgE and IgG2a in NK1.1+ cell-depleted mice are in contrast to those reported recently by Wang et al. (43). In their study, depletion of NK1.1+ cells altered neither OVA-specific IgE and IgG2a levels nor OVA-stimulated cytokine production by spleen cells in bulk culture in immunized mice (43). There are no obvious explanations for this discrepancy. However, since they delivered the mAb NK1.1 on the day of immunization, it can be speculated that the lack of immune suppression may in part have been caused by antibody-induced nonspecific production of cytokines during the initiation of the immune response. Thus, Asea et al. (44) demonstrated recently that a single delivery (50 µg i.v.) of mAb NK1.1 triggered a population of NKT cells to produce IL-4 within 90 min. This production returned to baseline levels by 24 h after antibody treatment. To confirm that no acute nonspecific effects (such as cytokine release) of depletion with mAb NK1.1 on day -2 caused the effects on immunization in this study, mice were depleted of NK1.1+ cells from day -7 before immunization. These mice also exhibited suppressed pulmonary eosinophilia and decreased systemic levels of IgE and IgG2a. In another control experiment, the efficacy of depletion of NK1.1+ cells from day -7 and from day -2 was monitored by flow cytometric analysis of spleen cells at the day of immunization. All antibody-treated mice (n = 10) exhibited <1% NK1.1+ cells in the spleen at this time point (our unpublished data).
The critical role of NK cells in this model of allergic
asthma is limited to the immunization phase, as depletion
of NK1.1+ cells during the challenge period only did not
influence the magnitude of pulmonary eosinophilia, levels
of OVA-specific IgE, and cytokines in BALF. Thus, it appears likely that NK cells influence the initial antigen presentation to T cells and/or the differentiation of naive T
cells into effector cells, but not the secondary activation of
antigen-specific T cells. Recently, Zhang et al. (45) reported that depletion of NK cells augments disease severity,
T cell proliferation, and production of Th1 cytokines in
experimental autoimmune encephalomyelitis, a prototype
Th1-mediated condition. These novel data are in contrast to the general paradigm that NK cells are required for optimal activation of Th1-like immune responses. It can be
speculated that depletion of NK cells has similar effects in
our experimental model, causing a switch from Th2 to
Th1. However, our data on systemic IgE and IgG2a levels,
BALF cytokine levels, and CD3+ splenic T cell cytokine
production fail to indicate this. The early appearance of
NK cells at the site of immunization (17) and their ability
to produce a variety of cytokines implicate these cells in
several critical steps in the development of the acquired immune response to allergens. One possibility is that NK cells
provide mediators that critically influence differentiation of
resting APCs to an activated phenotype with the T cell costimulatory capacity that is required for the exaggerated T
cell activation in allergic pulmonary inflammation (5, 46).
Indeed, both isoforms of B7 on APCs are upregulated by
IFN- (47), a cytokine which may derive in part from NK
cells. Other immunoregulatory functions of NK cells may
be attributed to their capacity to produce IL-5 (12). Indeed, Walker et al. are now demonstrating that IL-5 produced by NK cells may contribute to eosinophil infiltration in a mouse model of allergic peritonitis (48). Although it
appears likely that NK cells exert their effects by cytokine
production, it cannot be excluded that NK cells influence
the immunization phase by cytolytic mechanisms. It has
been shown that APCs, i.e., dendritic cells and macrophages, are highly susceptible to lysis by NK cells, which
thus might modulate the antigen presentation (49). Further
studies are warranted to elucidate details of cellular and
molecular interplays that may explain the present observations.
The present data suggest the possibility that the increased NK cell activity that has been demonstrated in asthma (19- 21) reflects a predisposition of individuals with high NK cell activity to develop exaggerated T cell responses to inhaled antigens and hence to be at risk of developing asthma. In epidemiological investigations, a predisposition to develop asthma has been strongly associated with elevated levels of serum IgE (50). Interestingly, a correlation between NK cell activity and total serum IgE levels has been observed in healthy subjects (51).
In conclusion, the present data indicate that NK cells may have a critical role in immunization and subsequent development of allergen-induced airway inflammation. These new data suggest the possibility of a pathogenic role for the increased number of NK cells observed in asthma and suggest that NK cell activity may be one of the factors governing the development of allergic eosinophilic airway disease.
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Footnotes |
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Address correspondence to Magnus Korsgren, Department of Physiology and Neuroscience, Neuroendocrine Cell Biology, E-blocket, University Hospital, 221 85 Lund, Sweden. Phone: 46-46-177714; Fax: 46-46-177720; E-mail: Magnus.Korsgren{at}mphy.lu.se
Received for publication 2 September 1998 and in revised form 13 October 1998.
This work was supported by the Swedish Medical Research Council (V1180, 06P-11813, 16X-12219, 8308, 4499), Vårdalstiftelsen, the Swedish Heart and Lung Foundation, the Swedish Cancer Society, and Astra Draco, Lund, Sweden.
Abbreviations used in this paper BAL, bronchoalveolar lavage; BALF, BAL fluid; ELISPOT, enzyme-linked immunospot; NKT cells, NK1.1+ T cells; SAL, saline; SFC, spot-forming cell.
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References |
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---|
1. | Robinson, D.S., Q. Hamid, S. Ying, A. Tsicopoulos, J. Barkans, A.M. Bentley, C. Corrigan, S.R. Durham, and A.B. Kay. 1992. Predominant TH2-like bronchoalveolar T-lymphocyte population in atopic asthma. N. Engl. J. Med. 326: 298-304 [Abstract]. |
2. | Watanabe, A., H. Mishima, P.M. Renzi, L.-J. Xu, Q. Hamid, and J.G. Martin. 1995. Transfer of allergic airway responses with antigen-primed CD4+ but not CD8+ T cells in brown Norway rats. J. Clin. Invest. 96: 1303-1310 [Medline]. |
3. | Lukacs, N.W., R.M. Stieter, S.W. Chensue, and S.L. Kunkel. 1994. Interleukin-4-dependent pulmonary eosinophil infiltration in a murine model of asthma. Am. J. Respir. Cell Mol. Biol. 10: 526-532 [Abstract]. |
4. | Foster, P.S., S.P. Hogan, A.J. Ramsay, K.I. Matthaei, and I.G. Young. 1996. Interleukin 5 deficiency abolishes eosinophilia, airways hyperreactivity, and lung damage in a mouse asthma model. J. Exp. Med. 183: 195-201 [Abstract]. |
5. |
Tsuyuki, S.,
J. Tsuyuki,
K. Einsle,
M. Kopf, and
A.J. Coyle.
1997.
Costimulation through B7-2 (CD86) is required for
the induction of a lung mucosal T helper cell 2 (TH2) immune response and altered airway responsiveness.
J. Exp.
Med.
185:
1671-1679
|
6. |
Swain, S.L.,
A.D. Weinberg,
M. English, and
G. Huston.
1990.
IL-4 directs the development of Th2-like helper effectors.
J. Immunol.
145:
3796-3806
|
7. | Fearon, D.T., and R.M. Locksley. 1996. The instructive role of innate immunity in the acquired immune response. Science. 272: 50-54 [Abstract]. |
8. | Bendelac, A., and D.T. Fearon. 1997. Innate immunity. Innate pathways that control acquired immunity. Curr. Opin. Immunol. 9: 1-3 [Medline]. |
9. | Medzhitov, R., and C.A. Janeway Jr.. 1997. Innate immunity: impact on the adaptive immune response. Curr. Opin. Immunol. 9: 4-9 [Medline]. |
10. | Biron, C.A.. 1997. Activation and function of natural killer cell responses during viral infections. Curr. Opin. Immunol. 9: 24-34 [Medline]. |
11. | Scharton-Kersten, T.M., and A. Sher. 1997. Role of natural killer cells in innate resistance to protozoan infections. Curr. Opin. Immunol. 9: 44-51 [Medline]. |
12. |
Warren, H.S.,
B.F. Kinnear,
J.H. Phillips, and
L.L. Lanier.
1995.
Production of IL-5 by human NK cells and regulation
of IL-5 secretion by IL-4, IL-10, and IL-12.
J. Immunol.
154:
5144-5152
|
13. | Bix, M., and R.M. Locksley. 1995. Natural T cells. Cells that co-express NKRP-1 and TCR. J. Immunol. 155: 1020-1022 [Medline]. |
14. | Vicari, A.P., and A. Zlotnik. 1996. Mouse NK1.1+ T cells: a new family of T cells. Immunol. Today. 17: 71-76 [Medline]. |
15. | Bendelac, A., M.N. Rivera, S.-H. Park, and J.H. Roark. 1997. Mouse CD1-specific NK1 T cells: development, specificity, and function. Annu. Rev. Immunol. 15: 535-562 [Medline]. |
16. |
Prussin, C., and
B. Foster.
1997.
TCR V![]() ![]() |
17. |
Bogen, S.A.,
I. Fogelman, and
A.K. Abbas.
1993.
Analysis of
IL-2, IL-4, and IFN-![]() |
18. | Weissler, J.C., L.P. Nicod, M.F. Lipscomb, and G.B. Toews. 1987. Natural killer cell function in human lung is compartmentalized. Am. Rev. Respir. Dis. 135: 941-949 [Medline]. |
19. | Timonen, T., and B. Stenius-Aarniala. 1985. Natural killer cell activity in asthma. Clin. Exp. Immunol. 59: 85-90 [Medline]. |
20. | Jira, M., E. Antosova, V. Vondra, J. Strejcek, H. Mazakova, and J. Prazakova. 1988. Natural killer and interleukin-2 induced cytotoxicity in asthmatics. I. Effect of acute antigen-specific challenge. Allergy. 43: 294-298 [Medline]. |
21. |
Krejsek, J.,
B. Kral,
D. Vokurkova,
V. Derner,
M. Touskova,
Z. Parakova, and
O. Kopechy.
1998.
Decreased peripheral
blood ![]() ![]() |
22. | Vesterinen, E., and T. Timonen. 1988. Natural killer cell activity in specific and non-specific bronchial challenge. Ann. Allergy. 60: 247-249 [Medline]. |
23. |
Ferrick, D.A.,
M.D. Schrenzel,
T. Mulvania,
B. Hsieh,
W.G. Ferlin, and
H. Lepper.
1995.
Differential production of interferon-![]() ![]() ![]() |
24. |
McMenamin, C.,
C. Pimm,
M. McKersey, and
P.G. Holt.
1994.
Regulation of IgE responses to inhaled antigen in mice
by antigen-specific ![]() ![]() |
25. |
Zuany-Amorim, C.,
C. Ruffie,
S. Haile,
B.B. Vargaftig,
P. Pereira, and
M. Pretolani.
1998.
Requirement for ![]() ![]() |
26. | Koo, G.C., and J.R. Peppard. 1984. Establishment of monoclonal anti-Nk-1.1 antibody. Hybridoma. 3: 301-303 [Medline]. |
27. | Mendiratta, S.K., W.D. Martin, S. Hong, A. Boesteanu, S. Joyce, and L. Van Kaer. 1997. CD1d1 mutant mice are deficient in natural T cells that promptly produce IL-4. Immunity. 6: 469-477 [Medline]. |
28. |
Itohara, S.,
P. Mombaerts,
J. Lafaille,
J. Iacomini,
A. Nelson,
A.R. Clarke,
M.L. Hooper,
A. Farr, and
S. Tonegawa.
1993.
T cell receptor ![]() ![]() ![]() ![]() ![]() |
29. | Brusselle, G.J., J.C. Kips, J.H. Tavernier, J.G. Van der Heyden, C.A. Cuvelier, R.A. Pauwels, and H. Bluethmann. 1994. Attenuation of allergic airway inflammation in IL-4 deficient mice. Clin. Exp. Allergy. 24: 73-80 [Medline]. |
30. |
Korsgren, M.,
J.S. Erjefält,
O. Korsgren,
F. Sundler, and
C.G.A. Persson.
1997.
Allergic eosinophil-rich inflammation
develops in lungs and airways of B cell-deficient mice.
J.
Exp. Med.
185:
885-892
|
31. | Ten, R.M., L.R. Pease, D.J. McKean, M.P. Bell, and G.J. Gleich. 1989. Molecular cloning of the eosinophil peroxidase: evidence for the existence of a peroxidase multigene family. J. Exp. Med. 169: 1757-1769 [Abstract]. |
32. | Rönnelid, J., and L. Klareskog. 1997. A comparison between ELISPOT methods for the detection of cytokine producing cells: greater sensitivity and specificity using ELISA plates as compared to nitrocellulose membranes. J. Immunol. Methods. 200: 17-26 [Medline]. |
33. | Persson, C.G.A., J.S. Erjefält, M. Korsgren, and F. Sundler. 1997. The mouse trap. Trends Pharmacol. Sci. 18: 465-467 [Medline]. |
34. | Brusselle, G., J. Kips, G. Joos, H. Bluethmann, and R. Pauwels. 1995. Allergen-induced airway inflammation and bronchial responsiveness in wild-type and interleukin-4-deficient mice. Am. J. Respir. Cell Mol. Biol. 12: 254-259 [Abstract]. |
35. |
Lambrecht, B.N.,
B. Salomon,
D. Klatzmann, and
R.A. Pauwels.
1998.
Dendritic cells are required for the development
of chronic eosinophilic airway inflammation in response to
inhaled antigen in sensitized mice.
J. Immunol.
160:
4090-4097
|
36. | Krug, N., J. Madden, A.E. Redington, P. Lackie, R. Djukanovic, U. Schauer, S.T. Holgate, A.J. Frew, and P.H. Howarth. 1996. T-cell cytokine profile evaluated at the single cell level in BAL and blood in allergic asthma. Am. J. Respir. Cell Mol. Biol. 14: 319-326 [Abstract]. |
37. |
Mosmann, T.R.,
H. Cherwinski,
M.W. Bond,
M.A. Giedlin, and
R.L. Coffman.
1986.
Two types of murine helper T
cell clone. I. Definition according to profiles of lymphokine
activities and secreted proteins.
J. Immunol.
136:
2348-2357
|
38. | Yoshimoto, T., and W.E. Paul. 1994. CD4+, NK1.1+ T cells promptly produce interleukin 4 in response to in vivo challenge with anti-CD3. J. Exp. Med. 179: 1285-1295 [Abstract]. |
39. | Bendelac, A., O. Lantz, M.E. Quimby, J.E. Yewdell, J.R. Bennink, and R.R. Brutkiewicz. 1995. CD1 recognition by mouse NK1+ T lymphocytes. Science. 268: 863-865 [Medline]. |
40. | Bendelac, A., R.D. Hunziker, and O. Lantz. 1996. Increased interleukin 4 and immunoglobulin E production in transgenic mice overexpressing NK1 T cells. J. Exp. Med. 184: 1285-1293 [Abstract]. |
41. |
Zhang, Y.,
K.H. Rogers, and
D.B. Davis.
1996.
![]() |
42. |
Brown, D.R.,
D.J. Fowell,
D.B. Corry,
T.A. Wynn,
N.H. Moskowitz,
A.W. Cheever,
R.M. Locksley, and
S.L. Reiner.
1996.
![]() |
43. |
Wang, M.,
C.A. Ellison,
J.G. Gartner, and
K.T. Hayglass.
1998.
Natural killer cell depletion fails to influence initial
CD4 T cell commitment in vivo in exogenous antigen-stimulated cytokine and antibody responses.
J. Immunol.
160:
1098-1105
|
44. | Asea, A., and J. Stein-Streilein. 1998. Signalling through NK1.1 triggers NK cells to die but induces NK T cells to produce interleukin-4. Immunology. 93: 296-305 [Medline]. |
45. |
Zhang, B.,
T. Yamamura,
T. Kondo,
M. Fujiwara, and
T. Tabira.
1997.
Regulation of experimental autoimmune encephalomyelitis by natural killer (NK) cells.
J. Exp. Med.
186:
1677-1687
|
46. | Keane-Myers, A., W.C. Gause, P.S. Linsley, S.-H. Chen, and M. Wills-Karp. 1997. B7-CD28/CTLA-4 costimulatory pathways are required for the development of T helper cell 2-mediated allergic airway responses to inhaled antigens. J. Immunol. 158: 2042-2049 [Abstract]. |
47. |
Boehm, U.,
T. Klamp,
M. Groot, and
J.C. Howard.
1997.
Cellular responses to interferon-![]() |
48. |
Walker, C.,
J. Checkel,
S. Cammisuli,
P.J. Leibson, and
G.J. Gleich.
1998.
IL-5 production by NK cells contributes to
eosinophil infiltration in a mouse model of allergic inflammation.
J. Immunol.
161:
1962-1969
|
49. | Chambers, B.J., M. Salcedo, and H.-G. Ljunggren. 1996. Triggering of natural killer cells by the costimulatory molecule CD80 (B7-1). Immunity. 5: 311-317 [Medline]. |
50. | Burrows, B., F.D. Martinez, M. Halonen, R.A. Barbee, and M.G. Cline. 1989. Association of asthma with serum IgE levels and skin-test reactivity to allergens. N. Engl. J. Med. 320: 271-277 [Abstract]. |
51. | Kusaka, Y., K. Sato, Q. Zhang, A. Morita, T. Kasahara, and Y. Yanagihara. 1997. Association of natural killer cell activity with serum IgE. Int. Arch. Allergy Immunol. 112: 331-335 [Medline]. |