By
From the * Department of Internal Medicine, Pulmonary and Critical Care Section, the Section of
Immunobiology, and the § Department of Pathology, Yale University School of Medicine, New Haven,
Connecticut 06520
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Abstract |
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The molecular mechanisms that contribute to an eosinophil-rich airway inflammation in
asthma are unclear. A predominantly T helper 2 (Th2)-type cell response has been documented
in allergic asthma. Here we show that mice deficient in the p50 subunit of nuclear factor (NF)-
B are incapable of mounting eosinophilic airway inflammation compared with wild-type
mice. This deficiency was not due to a block in T cell priming or proliferation in the p50
/
mice, nor was it due to a defect in the expression of the cell adhesion molecules VCAM-1 and
ICAM-1 that are required for the extravasation of eosinophils into the airways. The major defects in the p50
/
mice were the lack of production of the Th2 cytokine interleukin 5 and the
chemokine eotaxin, which are crucial for proliferation and for differentiation and recruitment,
respectively, of eosinophils into the asthmatic airway. Additionally, the p50
/
mice were deficient in the production of the chemokines macrophage inflammatory protein (MIP)-1
and
MIP-1
that have been implicated in T cell recruitment to sites of inflammation. These results demonstrate a crucial role for NF-
B in vivo in the expression of important molecules that
have been implicated in the pathogenesis of asthma.
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Introduction |
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Asthma is a chronic inflammatory disease of the lower airways that causes airway hyperresponsiveness to a wide variety of specific and non-specific stimuli. Both the prevalence and severity of asthma is increasing at an alarming rate in developed countries. Bronchoalveolar lavage (BAL)1 and biopsy of patients with mild to moderate asthma has provided impressive evidence for complex airway inflammation in asthma (1). The most striking and consistent pathophysiology, at least in atopic asthma, is damage to the bronchial epithelium and infiltration of the bronchial wall and lumen by eosinophils (2, 4). Eosinophils are conspicuous in the airways of the majority of asthmatics, from the mildest to the most severe, and the number of eosinophils in the peripheral blood and airways correlates with hyperresponsiveness and clinical severity (2, 4). Eosinophils can generate an array of potent proinflammatory mediators, including the cytotoxic proteins preformed and stored in the eosinophil granules such as major basic protein and eosinophil cationic protein, eicosanoid compounds including leukotriene (LT)C4, LTD4, platelet activating factor, and prostaglandins (PG)E1 and E2, and finally proinflammatory cytokines.
It is now generally accepted that the eosinophil-rich inflammatory response that mediates airway tissue damage in atopic individuals arises from aberrant T cell-mediated immune responses to a range of inhaled allergens that are seemingly ignored by nonresponsive "normal" individuals (3, 8, 12). Cytokine expression profiles of allergen-specific T cell clones isolated from atopics are skewed toward a Th2-like profile compared with a Th1-like profile in nonatopics (for review see references 15, 16). The cytokine IL-5, produced by Th2 cells, is now known to be the central mediator in the regulation of eosinophilic inflammation in asthma (1, 17). IL-5 is intimately associated with several features of eosinophil biology; it not only regulates the proliferation, differentiation, and activation of eosinophils (20), it also provides an essential signal for the rapid mobilization of eosinophils from the bone marrow and cooperates with the chemokine eotaxin in the homing of the eosinophils to sites of allergic inflammation (23). Due to the proposed central role of eosinophils in asthma and allergic disease, there is considerable interest in the molecular mechanisms that mediate the expression of genes that promote eosinophilia such as IL-5 and eotaxin.
The transcription factor nuclear factor (NF)-B plays an
important role in regulating many inflammatory processes
(24). NF-
B proteins control the expression of multiple genes involved in immune responses such as those encoding proinflammatory cytokines and chemokines (24-
26, 28, 29). The NF-
B proteins belong to the Rel family
of proteins. So far, five members of the NF-
B family have
been described: c-Rel; NF-
B1 (p50); NF-
B2 (p52); RelA (p65); and RelB (24). The classic NF-
B complex is a heterodimer of two polypeptide subunits, p50 and
RelA (p65). Most members of the NF-
B family can form
homo- or heterodimers (except for RelB, which only
forms heterodimers with p50 or p52) that bind slightly different
B motifs (24). (p50)2 has been shown to act as a
dominant repressor of transcription from specific
B sites
(30). We have recently shown that (p50)2 behaves as a selective repressor of RANTES (regulated on activation,
normal T cell expressed and secreted) gene expression (33).
It appears that in vivo (p50)2 functions as an activator in
certain tissues such as the thymus but acts as a repressor in
others present in peripheral lymphoid organs and in macrophages, such as T cells (34).
Studies of gene knockout animals indicate that the different Rel family proteins are not functionally redundant.
For example, knockout of the RelA (p65) gene in mice
causes embryonic lethality apparently due to extensive apoptosis in the liver (35). On the other hand, p50/
mice
develop normally and express normal mature B and T cell subsets (36). However, the B cells in these mice are severely limited in their functions. They do not proliferate in
response to bacterial LPS (37), and furthermore, p50
/
B
cells are defective in IgG3, IgE, and IgA class switching and display markedly reduced levels of germline CH
3 and CH
RNA but normal levels of germline CH
1 and CH
RNA
(37).
Although gene knockout studies have eliminated notions
of simple redundancy of the different NF-B proteins, little
is known about the specific role of these proteins in the
pathogenesis of different inflammation-associated disease
states in vivo. For example, whether the characteristic eosinophilic inflammation observed in asthma is an NF-
B-
dependent phenomenon was not known before this study.
We used p50
/
mice in a murine model of airway inflammation to investigate the role of NF-
B in the pathogenesis of eosinophilic airway inflammation, which, as we and
others have shown, is predominantly a CD4+ T cell-dominated process (18, 38). We show that the p50
/
mice
are devoid of eosinophilic airway inflammation upon antigenic stimulation. The lack of inflammation was not due to
insufficient CD4+ T cell priming. Also, IL-2 production,
which is believed to be an NF-
B-regulated process, was
unimpaired in the p50
/
mice and the cells proliferated as
efficiently as the wild-type cells in response to antigen. The
absence of inflammation also was not due to a lack of expression of vascular cell adhesion molecule (VCAM)-1 and
intercellular adhesion molecule (ICAM)-1, which have
been shown to be important for eosinophil homing. A major deficiency in the p50
/
mice was the absence of IL-5
production by the CD4+ T cells upon antigenic stimulation. Another difference between the two types of mice
was the lack of expression of the eosinophilic chemoattractant eotaxin at both the RNA and the protein level in the
lungs of p50
/
mice. The expression of the chemokine
genes macrophage inflammatory protein (MIP)-1
and
MIP-1
, which have been implicated in T cell chemotaxis,
was also deficient in the p50
/
mice. Taken together, this
is the first report that demonstrates an essential role for NF-
B in the regulation of important molecules that regulate
eosinophilic inflammation in vivo. Furthermore, our studies also demonstrate a dissociation between the expression of the IL-4 and IL-5 genes, both of which are expressed by
Th2-type cells.
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Materials and Methods |
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Mice.
The p50Sensitization and Challenge of Mice.
Mice were sensitized and challenged essentially as described by Kung et al. (42). Mice were sensitized with 10 µg of OVA (Sigma Chemical Co., St. Louis, MO) and 1 mg of alum (Resorptar; Intergen Co., New York, NY) intraperitoneally on days 0 and 5. Sham-immunized mice received alum alone. On day 12, mice were challenged by exposure to an aerosol of 1% OVA in PBS twice for 1 h each at an interval of 4 h. Inhalation was carried out in a plastic chamber (27 × 20 × 10 cm). The inhalation chamber that we use has an attachment to allow entry of the aerosol from an ultrasonic nebulizer (1-5 µM particles by manufacturer's specifications, Model no. NE-U07; Omron, Vernon Hills, IL). The other end of the box has two small holes for the maintenance of continuous airflow.BAL.
BAL was performed at 24 h after aerosol challenge. Mice were anesthetized and the lungs and heart were surgically exposed. The trachea were cannulated and the lungs were lavaged twice with 1-ml aliquots of PBS. The live cells (excluded trypan blue) recovered were counted in a hemocytometer. Cytospin preparations of BAL cells were stained with Diff-Quik (Baxter HealthCare Corp., Miami, FL) and cell differentials were enumerated based on morphology and staining profile.Lung Histology.
Lungs were prepared for histology by perfusing the animal through the right ventricle with PBS to remove all blood. Lungs were then inflated to constant pressure with 1.0 ml of fixative instilled through a tracheostomy tube as described previously (43). For staining with hematoxylin and eosin (H&E), lungs were fixed in 10% formalin and embedded in paraffin. 5-µM sections were mounted on slides and stained with H&E according to established procedures (41, 43). For immunohistochemistry, lungs were perfused and fixed in 0.001 M periodate/ 0.075 M lysine/1% paraformaldehyde (PLP) and processed essentially as described previously (44). In brief, lungs were fixed in PLP overnight at 4°C with the tracheostomy tube left in place while in PLP. The tissue was washed three times for 5 min each in 0.1 M cold phosphate buffer. The tissue was then treated successively with 10 and 20% sucrose (in phosphate buffer) for 20 min each. Lungs were inflated via the tracheostomy tube with 1 ml of 40% OCT diluted in PBS (tissue freezing medium; Electron Microscopy Sciences, Fort Washington, PA). Lungs were embedded in 100% OCT in cryomold and plunged into a 2-methylbutane bath precooled on dry ice. Tissues were stored atPreparation of Nuclear Extracts from Whole Lung and Electrophoretic Mobility Shift Assays.
To prepare nuclear extracts from lung tissue, the lungs were snap frozen in liquid N2 and pulverized. The following steps all were carried out at 4°C. The lung powder was homogenized in 5 ml of solution A (10 mM Hepes, pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.5 mM PMSF, and 0.6% NP-40) in a Dounce tissue homogenizer (Wheaton, Millville, NJ) and centrifuged at 800 g for 30 s to remove cellular debris. The supernatant was left on ice for 5 min and then centrifuged at 2000 g for 5 min. The pelleted nuclei were resuspended in 150-200 µl of solution B (20 mM Hepes, pH 7.9, 420 mM NaCl, 1.2 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM PMSF, 2 mM benzamidine, 25% glycerol, and a mixture of protease inhibitors (Boehringer Mannheim, Indianapolis, IN) for 20 min. The lysed nuclei were briefly centrifuged at 16,000 g. The supernatant was aliquoted, snap frozen in a dry ice/methanol bath and stored atRNA Isolation and Ribonuclease Protection Assays.
Total RNA was isolated from lung tissue using TRIzol Reagent (GIBCO BRL, Gaithersburg, MD) according to the manufacturer's instructions. Each whole fresh lung was homogenized in 4 ml of TRIzol reagent using Tissumizer (Tekmar Co., Cincinnati, OH) and centrifuged to remove cellular debris. The RNA pellet was resuspended in nuclease-free H2O. 10 µg of total lung RNA from each mouse was used in ribonuclease protection assay (RPA) using the RPA II Kit (Ambion, Austin, TX). The murine cytokine template set mCK-5 (PharMingen) was used to obtain radiolabeled antisense RNA probes for RANTES, eotaxin, MIP-1Cell Proliferation Assays.
Mice were sensitized as above by intraperitoneal injections of OVA plus alum on days 0 and 5. Spleens were harvested on day 12 and CD4+ T cells were prepared by negative selection using mAbs to CD8, class II MHC I-A, and anti-Ig-coated magnetic beads (Collaborative Research, Bedford, MA). Syngeneic T cell-depleted splenocytes were used as APCs that were prepared by negative selection using antibodies to CD4 (GK1.5), CD8, Thy-1, and treatment with rabbit complement and mitomycin C as described previously (41). 5 × 105 CD4+ T cells (pooled from two mice in each group) together with 5 × 105 APCs were cultured with OVA (0.1-100 µg/ml) in Bruff's medium supplemented with 5% FCS. Cultures were incubated for 72 h, supernatants were collected for cytokine analyses and cultures were pulsed with 1 µCi of methyl-[3H]thymidine/well. After incubation at 37°C for 24 h, triplicate wells were harvested onto glass filters and incorporated radioactivity was measured in a beta counter. The background was subtracted from the results.Cytokine Assays.
IL-2, IL-4, IL-5, and IFN-Reverse Transcription-PCR.
RNA isolated from the OVA-stimulated CD4+ T cells were used in reverse transcription (RT)- PCR. To synthesize cDNA, random primers (100 pmol) were annealed to 1 µg of total RNA by incubation at 68°C for 10 min. RT was carried out by adding dNTP, 8 U of an RNase inhibitor (RNasin), 0.2 mM dithiothreitol, and 200 U SuperScript II (GIBCO BRL) and buffer and by incubating at 37°C for 10 min, at 42°C for 50 min and finally at 94°C for 2 min. The cDNAs synthesized were used in PCR. PCR was performed using primer sets corresponding to the murine IL-4 and IL-5 genes. The sequences of the primer were: IL-4; 5'-CAT CGG CAT TTT GAA CGA GGT CA-3' (forward) and 5'-CTT ATC GAT GAA TCC AGG CAT CG-3' (reverse); IL-5; 5'-CTC ACC GAG CTC TGT TGA CAA G-3' (forward) and 5'-GAA CTC TTG CAG GTA ATC CAG G-3' (reverse). The reactions were assembled in HotStart tubes (Molecular Bio-Product, Inc., San Diego, CA) as follows: first the lower layer mix was added that included 10mM dNTP, 1× Pfu (Stratagene, La Jolla, CA) reaction buffer and primers (50 pmol). The mixture was heated to 90°C for 30 s and cooled down to room temperature, and then the upper layer that included cDNA (1-2 µl), 1.25 U of Pfu DNA polymerase (Stratagene) and 1× Pfu buffer was added. For PCR, the samples were first heated to 94°C for 2 min (for denaturation) and the PCR cycle conditions were: 94°C for 45 s, 64°C for 45 s, and 72°C for 1 min. The reactions were terminated by incubation at 72°C for 7 min to complete chain extensions. The products were analyzed by electrophoresis on 1% agarose gels. ![]() |
Results |
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To investigate the
effects of antigenic (OVA) stimulation on NF-B activation in the whole lung, wild-type and p50
/
mice were
sensitized and challenged with OVA (hereafter referred to
as OVA/OVA). 2 h after challenge with OVA, mice were
killed and nuclear extracts were prepared from the whole
lung. The nuclear extracts were used in EMSAs using an
oligonucleotide containing a consensus binding sequence
for NF-
B. As shown in Fig. 1, lane 1, nuclear extracts prepared from sham-sensitized and OVA-challenged (Alum/
OVA) mice did not display appreciable DNA-binding
activity. On the other hand, nuclear extracts prepared from
the lungs of mice sensitized and challenged with OVA
(OVA/OVA) formed three distinct complexes. In the case
of p50
/
mice, no DNA-protein complex was observed
with nuclear extracts from control mice (Alum/OVA).
With extracts prepared from OVA/OVA mice, only the
fastest migrating complex (shown by an arrow) was detected. We used specific antibodies to determine the composition of the different polypeptide complexes. Formation
of complex I was affected by both anti-p50 and anti-p65
antibodies, suggesting that it contained the classic p50-p65
heterodimer (Fig. 1, lanes 6 and 10). Complex II formation
was affected only by the anti-p50 antibody (Fig. 1, lane 6).
Also, complex II comigrated with the complex formed
with recombinant p50, confirming that it contained (p50)2.
The intensity of the complex shown by an arrow was variable in different experiments. This complex was not supershifted by the anti-p50 or the anti-p65 antibody, nor by
antibodies against other Rel proteins (data not shown), and
most likely represents nonspecific binding. Oct-1-binding
activity, used as a loading control, was comparable in all the
lanes (data not shown). Thus, the EMSAs demonstrated the
presence of active NF-
B dimers containing p50 in the lungs
of antigen-challenged wild-type mice and their absence in
the lungs of similarly challenged p50
/
mice.
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To assess the effect of inhaled antigen on airway inflammation in the wild-type (+/+)
and p50/
(
/
) animals, mice were killed 24 h after
aerosol challenge with OVA and the lungs were examined
histologically. As shown in Fig. 2 a, wild-type mice displayed a prominent inflammatory response. Eosinophilic
inflammation was widespread with both perivascular and
peribronchiolar infiltration. On the other hand, the p50
/
mice had no eosinophilic inflammation in their airways
upon antigen challenge (Fig. 2 b). In some p50
/
mice, a
few patchy areas of a low degree of inflammation were noted in which eosinophils or neutrophils were barely detectable. The lungs of animals sham-sensitized with alum
only and challenged with OVA (Fig. 2, c and d, respectively) were totally devoid of any inflammation.
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The impressive difference in lung inflammation between
the wild-type and p50/
mice was also observed in the
BAL fluid (BALF) recovered from the animals. The total
cell count in the BALF recovered from the p50
/
mice
upon antigen challenge was 10-fold lower than that in the BALF derived from the wild-type mice (Fig. 3 A). After
antigen provocation, although there was an ~2,000-fold
increase in the number of eosinophils in the airways of the
wild-type mice (compared with sham-sensitized animals),
the net increase in eosinophils was only 10-fold in the airways of the p50
/
mice (Fig. 3 A). In addition to a
blunted eosinophil recruitment, neutrophil recruitment
was also markedly attenuated in the airways of the p50
/
mice. Also, the number of lymphocytes recovered from the
airways of the p50
/
mice was ~5-6-fold less than that
obtained from the wild-type mice (Fig. 3 A). The cells in
the BALF recovered from the p50
/
mice contained predominantly monocytes/macrophages similar to those observed in the BALF derived from control mice that were sham-sensitized and challenged with OVA (Fig. 3 A).
|
We also examined cytokine levels in the airways of these
mice. As shown in Fig. 3 B, the predominant cytokines
present in the BALF obtained from the wild-type mice
were of the Th2 type as was expected in this model. First,
with the exception of IL-2, all other cytokines were
present at lower levels in the airways of the p50/
mice as
was expected from the deficient airway inflammation in these mice after antigen challenge. Second, IL-4 was detectable in the BALF of p50
/
mice, albeit at lower levels
compared with those in the wild-type mice. The lower
level of IL-4 in the BALF of the p50
/
mice is probably
due to fewer lymphocytes in the airways of these mice, as
Th2 lymphocytes are the major source of this cytokine in
asthma and allergic inflammation. The other Th2-type cytokine, IL-5, was barely detectable in the BALF of the
p50
/
mice (Fig. 3 B). Thus, the production of IL-5 appeared to be more severely affected by the absence of the
p50 subunit of NF-
B than the production of IL-4.
We investigated the basal level of eosinophils in the wild-type and the
p50/
mice. As shown in Table 1, the level of eosinophils
in the bone marrow and peripheral blood of p50
/
mice
was comparable to that in the controls.
|
We
investigated the expression of the cell adhesion molecules
VCAM-1 and ICAM-1 in the airways of wild-type and
p50/
mice by immunohistochemistry since both of these
molecules have been implicated in eosinophil extravasation
from the blood vessel into the airways (45). After OVA
challenge, the animals were killed and the lungs were frozen for immunohistochemistry. As shown in Fig. 4 A,
upon OVA challenge, strong expression of VCAM-1 was
evident on the vascular endothelium in both the wild-type and p50-deficient mice (Fig. 4 A, a and b, respectively). No
staining was observed in the lungs of the control animals
sensitized with alum and challenged with OVA (Fig. 4. A, c
and d). Also, no reaction was observed with the isotype-matched control antibody (Fig. 4 A, e and f, respectively).
The expression of ICAM-1 was also similar in the wild-type and p50
/
mice (Fig. 4 B). Strong constitutive
ICAM-1 expression on the alveolar epithelium and somewhat weaker expression on the vascular endothelium was
noted in both wild-type and p50-deficient mice (Fig. 4 B, c
and d, respectively) and there was no significant change in
the pattern of staining after OVA challenge (Fig. 4 B, a and
b). Again, no staining was observed with the control antibody (Fig. 4 B, e and f ).
|
Since chemokines have been
shown to be important for the recruitment of leukocytes to
sites of inflammation, we investigated the expression of key
chemokines that have been implicated in eosinophil
chemotaxis. Mice were killed at different time points after
OVA challenge, and RNA isolated from the lungs of these mice was subjected to RNase protection assays for the expression of different chemokine genes. The expression of
RANTES mRNA was found to be constitutive in the
lungs of both wild-type and p50/
mice and the expression remained unchanged after challenge with OVA (Fig. 5
A). In the lungs of p50+/+ mice, the expression of mRNAs
for eotaxin, MIP-1
, MIP-1
, IP-10, and MCP-1 was detectable early, at 2 h after OVA challenge, and the steady-state levels of these messages were higher at 4 h after challenge (data not shown). At 24-36 h after challenge, eotaxin and MIP-1
mRNAs continued to be expressed at a high
level in the wild-type mice. In the p50
/
mice a different
pattern of chemokine mRNA expression was detected. The expression of eotaxin or MIP-1
or MIP-1
mRNA
was barely detectable in these mice at all time points. However, the expression of IP-10 mRNA was stronger in the
p50
/
mice compared with that in the wild-type mice at
all time points tested. The expression of MCP-1 mRNA
was comparable in the wild-type and p50
/
mice at all
time points. Given the importance of eotaxin as an eosinophil chemoattractant, we have also tested the level of
eotaxin protein in the BALF in the OVA-challenged wild-type and p50
/
mice. As shown in Fig. 5 B, eotaxin protein was readily detected at 24 h in the BALF recovered
from the wild-type mice but was barely detectable in that
obtained from the p50
/
mice.
|
We next examined
whether the absence of airway inflammation in the p50-deficient mice in response to antigen challenge was due to a defect in the ability of CD4+ T cells to respond to antigen.
Wild-type and p50/
mice were immunized with OVA intraperitoneally and recall proliferative responses of spleen
CD4+ T cells were analyzed by [3H]thymidine incorporation.
CD4+ T cells from the p50
/
mice displayed a lower basal
incorporation (~50%) compared with those isolated from the
wild-type mice. However, they proliferated as efficiently as
the wild-type cells in response to antigen (Fig. 6 A).
|
Cytokine production was measured in culture supernatants and RNA was isolated from the OVA-restimulated
cells for analysis by RT-PCR. IL-2, IL-4, and IFN- were
detected in the supernatants of cells derived from both
wild-type and p50
/
mice (Fig. 6 B). Interestingly, CD4+
T cells from the p50
/
mice produced ~5-7-fold more
IL-2 than did cells from wild-type mice. The most striking
difference was the total absence of IL-5 production by p50-deficient T cells (Fig. 6 B). RT-PCR analysis of RNA
isolated from the OVA-stimulated CD4+ T cells revealed
IL-4 RNA expression in both wild-type and p50-deficient T cells (Fig. 6 C). The same RNA samples when analyzed
for IL-5 mRNA expression revealed expression in the
wild-type CD4+ T cells with barely detectable expression
in the p50-deficient T cells (Fig. 6 C).
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Discussion |
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The molecular mechanisms that regulate eosinophilic inflammation in asthma is an area of intense investigation in
many laboratories. We have recently shown that the transcription factor GATA-3 is expressed in Th2 but not Th1
cells and plays a critical role in IL-5 gene expression (46-
48). In this study, we have investigated the role of NF-B
in the elicitation of eosinophilic inflammation in a murine
model of allergic airway inflammation. We show that the
absence of NF-
B abolishes airway inflammation. This results from the absence of IL-5 and eotaxin gene expression
in the p50
/
mice, both of which are key mediators of
eosinophilic inflammation in the airways. We also show
markedly attenuated lymphocyte recruitment in the lungs
of p50
/
mice that, in part, may be due to deficient expression of the chemokine genes MIP-1
and MIP-1
in
the p50
/
mice. These results provide the first in vivo evidence that NF-
B is required for the development of eosinophilic inflammation in response to inhaled allergens.
Airway inflammation in asthma and allergic disease is a
complex phenomenon driven predominantly by Th2-type
cells. The inflammation is characterized by the recruitment
of leukocytes, predominantly eosinophils, and their subsequent migration from the vasculature into the tissue where
they cause severe damage to the bronchiolar epithelium (2,
4). Th2, epithelial, and endothelial cells play major
roles in this cascade by secreting cytokines and chemokines and by expressing cell surface adhesion molecules. Since
multiple in vitro studies have implicated NF-B in the expression of many of these molecules (24, 28, 29), we
examined the role of this transcription factor in eosinophilic inflammation after antigen provocation. We show
that NF-
B plays a critical regulatory role in promoting
eosinophilia in vivo.
The phenotype of the p50/
mice with respect to inflammation was in sharp contrast to the recently reported
phenotype of animals lacking the precursor p105 but expressing p50 (34). p50 is generated by proteolytic removal
of the COOH terminus of a precursor protein p105. The
murine nfkb1 gene produces two transcripts, a longer 4.0-kb transcript that encodes the full-length precursor p105 and a shorter 2.6-kb mRNA that encodes the I
B
protein
that is identical to the COOH terminus of p105 that is
cleaved off to generate mature p50 (49, 50). Mice targeted
to lack p105 but containing p50 showed multiple abnormalities, including inflammation in the lungs and liver
composed largely of lymphocytes, increased susceptibility
to opportunistic infections, splenomegaly, lymph node
enlargement, and lymphoid hyperplasia (34). Since the
COOH terminus of p105 has an inhibitory effect on p50
homodimer activity, mice lacking p105 had an enhanced
p50 homodimer activity (34). Although excess (p50)2
present in p105
/
mice resulted in decreased cytokine
production (IL-2 and IL-4) by stimulated T cells isolated
from these mice, T cell-dependent immune responses
overall were not seriously impaired in the p105
/
mice
(34). In contrast, we demonstrate a severe impairment of T
cell-dependent eosinophilic inflammation in the p50
/
mice. It should be noted that although the p105
/
mice
have excess (p50)2 together with, potentially, a small increase in p50-p65 heterodimers, the p50
/
mice are devoid of both (p50)2 and p50-p65 dimers. Thus, the net effect in the p50
/
mice is a combination of deficient p50
homo- and heterodimeric activity.
The absence of eosinophilic inflammation in the p50/
mice did not result from a deficiency in the expression of
the cell adhesion molecules VCAM-1 and ICAM-1, which
have been shown to be important for the extravasation of
eosinophils into the lungs in models of airway inflammation. Many in vitro studies have implicated NF-
B in the
transcriptional regulation of VCAM-1 (51) and ICAM-1 (53) genes in endothelial cells. Therefore, it was surprising that p50 deficiency did not abolish the expression of
these molecules since these mice lack not only (p50)2 but
also the p50-p65 heterodimer, which is the predominant
NF-
B transactivator. In addition to the p50-p65 heterodimer, (p65)2 has been shown also to be a potent transactivator, although it is found in much lower abundance than the p50-p65 heterodimer (30). Indeed, a few studies propose a more important role for the (p65)2 rather than the
p50-p65 heterodimer in IL-1 or TNF-induced VCAM-1
(56, 57) and ICAM-1 (58) gene expression. Interestingly, in
one study, IL-4-induced VCAM-1 gene expression was
shown to involve an NF-
B-independent mechanism (59).
Our studies suggest that the expression of VCAM-1 and ICAM-1 in this allergen model of airway inflammation is
not critically dependent on the p50-p65 heterodimer.
The lungs of the p50/
mice were indistinguishable
from the lungs of either sham-immunized or unchallenged
animals and did not display an easily discernible increase in
eosinophil, neutrophil, or lymphocyte infiltration after antigen challenge. Leukocyte infiltration into sites of inflammation is now known to involve cytokine-inducible chemoattractants called chemokines. Chemokines that have been
implicated in the recruitment of activated CD4+ T cells to
inflammatory sites include RANTES, MIP-1
, MIP-1
(60), and IP-10 (the murine homologue is Crg-2; reference
61). In this model we and others have detected a low level
constitutive expression of RANTES mRNA that did not
increase after antigen challenge (62). The induction of
MIP-1
and MIP-1
mRNA was abolished in the p50
/
mice, suggesting that the induction of these chemokines in
vivo is an NF-
B-dependent process, at least in this model.
The lack of expression of the MIP-1
and MIP-1
genes
may be partly responsible for the reduced number of lymphocytes in the lungs of p50-deficient mice. In contrast to
lack of induction of eotaxin, MIP-1
and MIP-1
mRNA
in the p50-deficient animals, the steady-state level of IP-10
mRNA was greater in the lungs of antigen-challenged
p50
/
mice compared with that in the lungs of similarly
challenged wild-type mice. We have not determined the
cellular source of IP-10 in the lungs of these mice, but the
likely candidates are activated macrophages and/or endothelial cells. CXCR3, the receptor for IP-10, has been
shown to be predominantly expressed by human Th1 cells
(63). If the same is true in mice, then despite increased expression of IP-10 in p50
/
mice, this chemokine was ineffective in the recruitment of Th2-type cells to the lung due
to the absence of the cognate receptor on the Th2 cells.
Recently, a subset of human Th2 cells were shown to express the chemokine receptor CCR3 (63), the best described ligand for which is eotaxin. CCR3 is also highly
expressed on the cell surface of both murine and human
eosinophils (67) and accounts for the specific chemotactic effects of eotaxin on eosinophils. Although a role for
eotaxin in Th2 cell chemotaxis has not been demonstrated,
it is suggested that eotaxin may control the trafficking of a
subset of Th2s that stimulate growth and activation of
colocalized eosinophils and basophils (65, 66, 70). Thus,
the absence of key chemokines that have been shown to be
important for the recruitment of activated CD4+ T cells
probably explains the reduced T cell infiltration into the
lungs of antigen-challenged p50
/
mice.
An important difference between the wild-type mice
and the p50/
mice was the lack of eotaxin and IL-5 gene
expression in the p50-deficient animals. Eotaxin has been
shown to be a specific chemoattractant for eosinophils in
different species including the guinea pig, mouse, and human (71). The in vivo specificity and potency of eotaxin for eosinophil chemotaxis were demonstrated in studies showing eosinophil recruitment into tissue either after instillation of eotaxin into the airways or after injection
into the skin (73). Eotaxin is produced in large quantities by the type I cells in the alveolar epithelium, although
other cells such as endothelial cells and the infiltrating eosinophils are also known to produce eotaxin (73). In vivo,
the production of eotaxin has been shown to be dependent
on T cells (62). Using the OVA model of airway inflammation, MacLean et al. have shown a profound inhibition of
eotaxin but not RANTES or MIP-1
production in mice
treated with anti-CD3 antibody (62). The molecular basis for this effect is unclear. However, it is possible that eotaxin production upon allergen challenge is induced by a T cell-
derived cytokine, most likely a Th2 cytokine. Since, in addition to eotaxin, the p50
/
mice also do not produce IL-5,
it is possible that IL-5 is a necessary stimulus for the induction of eotaxin gene expression in this model of airway inflammation. A nonmutually exclusive explanation for the
lack of eotaxin gene transcription in p50
/
mice is that
eotaxin gene expression is directly regulated by NF-
B. Indeed, the 5' flanking region of the eotaxin gene contains NF-
B sites, although their functional significance has yet
to be determined (78). It is now believed that eosinophil
chemotaxis involves cooperation between IL-5 and eotaxin
as shown in the guinea pig and mouse (23, 76, 79, 80).
IL-5 and eotaxin are both produced in the lung, the major
source of IL-5 being Th2 cells. IL-5 secreted by Th2 cells is
transported by the blood to the bone marrow. A major role
of IL-5 at this step is to trigger a rapid mobilization of eosinophils and progenitors from the bone marrow into the
blood since this process is inhibited upon treatment with
the anti-IL-5 antibody TRFK5 (79). The role of eotaxin is
to recruit the eosinophils from the vasculature. Thus, given the important role of IL-5 and eotaxin in eosinophil recruitment to the lungs in allergic inflammation, the combined deficiency of both of these factors in the p50-deficient animals could explain the total abrogation of airway
eosinophilia in these mice.
In this study, we also show that the absence of NF-B
influences T cell cytokine gene expression. Different in
vitro studies have suggested a role for NF-
B in the expression of the IL-2 gene. Although the p50-p65 heterodimer has been shown to interact with an NF-
B site in
the IL-2 promoter, (p50)2 has been shown to function as a
repressor of IL-2 gene expression (31). In our studies, not
only was IL-2 produced by the p50-deficient cells, the
amount of IL-2 secreted by these cells was ~5-7-fold
greater than that produced by the wild-type T cells. This
suggests, first, that (p65)2 or other Rel dimers can substitute
for the classic NF-
B dimer for transactivation of the IL-2
gene in this model. Second, our studies support the data of
Kang et al. (31) that an essential turn-off signal for IL-2
gene transcription is (p50)2 since the p50
/
mice produced
considerably more IL-2 than did the wild-type mice. Conversely, in p105
/
mice, which have excess (p50)2, IL-2
gene expression was found to be three- to sixfold lower
than that in wild-type mice (34). Although no difference
was noted between the wild-type and p50-deficient T cells
with respect to the net fold-stimulation of the cells upon
antigen challenge, the basal [3H]thymidine incorporation
by the p50-deficient cells was ~50% of that of the wild-type cells. This may be due to a lower basal expression of
the IL-2 or the IL-2R
gene (whose expression is also believed to be under NF-
B control) in the absence of the classic NF-
B dimer. We show that the absence of p50
profoundly inhibits IL-5 gene expression in CD4+ T cells.
Presently, it is unclear whether this is due to a primary or
secondary effect of NF-
B on IL-5 gene expression.
An important outcome of this study is the differential effect of NF-B on IL-4 and IL-5 gene expression. There is
increasing interest in the dissociation of IL-4 and IL-5 gene
expression in T cells in various disease situations and in T
cell lineages (for review see references 81, 82). For example, in intrinsic asthmatics, IL-5 but not IL-4 production is
elevated (83). In contrast, in leukemic Sezary cells, IL-4 has
been shown to be upregulated with concomitant decrease
in IL-5 production (84). Again, in a study of human T cells
using intracellular staining techniques, IL-4 and IL-5 were
found to be predominantly made by different cells (85).
There is no evidence to date that supports different lineage
of cells for IL-4 and IL-5 production. However, it is reasonable to speculate that once cells have been committed to
the Th2 lineage, different microenvironmental factors activate different sets of transcription factors such as NF-
B,
GATA-3 (46, 47, 86, 87), c-maf (88), and NF-AT (89),
which have differential effects on IL-4 and IL-5 gene expression. We have recently shown that ectopically expressed GATA-3 is sufficient for IL-5 but not IL-4 gene
expression (48). Thus, our studies provide clear evidence
that although IL-4 and IL-5 are often coordinately expressed, the absence of transcription factors such as NF-
B can cause selective impairment of IL-5 but not IL-4 gene
expression. The attenuation of both IL-5 and eotaxin gene
expression in p50
/
mice establishes a central role for NF-
B in airway eosinophilia in allergic inflammation.
![]() |
Footnotes |
---|
Address correspondence to Prabir Ray, Department of Internal Medicine, Pulmonary and Critical Care Section, Yale University School of Medicine, 333 Cedar St., LCI 105, New Haven, CT 06520. Phone: 203-785-3620; Fax: 203-785-3826; E-mail: prabir.ray{at}yale.edu
Received for publication 8 July 1998 and in revised form 24 August 1998.
This paper is dedicated to the memory of our beloved mentor and friend Dr. Jyotirmoy Das of Indian Institute of Chemical Biology, India.The authors would like to thank Irena Schvayetsky for excellent technical assistance with immunohistochemistry.
This work was supported by the National Institutes of Health grants HL52014 and HL60207 (to P. Ray), AI31137 and HL56843 (to A. Ray), P50-HL56389 (to A. Ray and R. Homer), and KO8-HL03308 (to L. Cohn).
Abbreviations used in this paper BAL, bronchoalveolar lavage; BALF, BAL fluid; EMSA, electrophoretic mobility shift assay; H&E, hematoxylin and eosin; ICAM, intercellular adhesion molecule; MCP, monocyte chemotactic protein; MIP, macrophage inflammatory protein; NF, nuclear factor; PLP, paraformaldehyde; RANTES, regulated on activation, normal T cell expressed and secreted; RT, reverse transcription; VCAM, vascular cell adhesion molecule.
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