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B/Rel Signaling in the Type 1 But Not Type 2 T Cell-Dependent Immune Response In Vivo1
,
Divisions of
*
Allergy, Pulmonary, and Critical Care Medicine and
Rheumatology, Department of Medicine,
Department of Microbiology and Immunology, and
§
Department of Pathology, Vanderbilt University Medical School, Nashville, TN 37232; and
¶
Pulmonary Division, Department of Medicine, Brigham and Women’s Hospital, Boston, MA 02115.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-dominant) or type 2 (IL-4-, IL-5-dominant) patterns. To
investigate mechanisms that connect extracellular stimuli with the
regulation of effector T cell function, we have measured immune
responses of transgenic mice whose NF-
B/Rel signaling pathway is
inhibited in T cells. Surprisingly, these mice developed type 2 T
cell-dependent responses (IgE and eosinophil recruitment) in a model of
allergic pulmonary inflammation. In contrast, type 1 T cell responses
were severely impaired, as evidenced by markedly diminished
delayed-type hypersensitivity responses, IFN- production, and
Ag-specific IgG2a levels. Taken together, these data indicate that
inhibition of NF-B can lead to preferential impairment of type 1 as
compared with type 2 T cell-dependent responses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B/Rel family (1, 2, 3). These
data have raised the possibility that NF-B/Rel signaling is a key
element in the modulation of T cell-dependent immunity. Specific
features linking signal transduction with transcriptional regulation in
this pathway have revealed it as an attractive target for therapeutic
intervention. In quiescent cells, trans-activating members
of the NF-B/Rel family of transcription factors are sequestered in
the cytoplasm by inhibitory molecules such as the inhibitor of B 
(IB)3 (3, 4). Signal transduction pathways from many classes of receptor,
including the TCR, converge on IB kinase complex(es)
(4, 5, 6). IB is subsequently phosphorylated by these
kinases and degraded by the 26S proteasome, leading to translocation of
NF-B to the nucleus, where it regulates gene transcription
(1, 2, 3, 4). NF-B induction is associated with
proinflammatory stimuli that affect both lymphocytes and nonlymphoid
cells, but it is not clear which immune responses are affected after in
vivo interruption of IB degradation specifically in T cells.
Subsequent to antigenic stimulation, T cells acquire distinct patterns
of function during immune responses (7, 8, 9). The function
of these effector T cells is determined in part by their cytokine
production profiles. Type 1 T cell effectors activate macrophages and
serve as mediators of cell-mediated immune responses such as
delayed-type hypersensitivity (DTH), in part through production of
IFN- and TNF-ß (8, 9, 10, 11). In contrast, type 2 effector
T cells induce IgE- and eosinophil-mediated reactions through
production of IL-4, IL-5, and IL-13 (8, 9, 12, 13). These
patterns of differential cytokine expression are controlled through
transcriptional mechanisms in T cell clones and primary effector cells
(14). Thus, a central question has been to identify
transcription factors that are involved in differential regulation of
type 1 and type 2 T cell responses in vivo. In this regard, Stat6,
GATA-3, c-Maf, and NF-ATc all appear to make T cell-intrinsic
contributions to the strength of type 2 T cell-dependent responses and
to Th2 development (15, 16, 17), whereas only Stat4 and IFN
regulatory factor-1 in T cells have been associated with type 1 T
cell-dependent responses or Th1 effectors (18, 19, 20).
NF-B induction can be correlated with the regulation of multiple
gene products involved in inflammatory responses (3, 4, 21, 22). Moreover, Ag receptor cross-linking led to IB
degradation and nuclear induction of NF-B in Th1 but not Th2 clones,
thus suggesting that type 1 (inflammatory) T cells might prove more
dependent on NF-B than their type 2 counterparts (23, 24). However, it has been unclear whether the function of
NF-B/Rel proteins in T lymphocytes could preferentially affect type
1 as compared with type 2 T cell-dependent responses in vivo.
To investigate the impact of impaired IB degradation on T cell
function in vivo, we generated transgenic mice in which NF-B
induction is inhibited specifically in T cells by a mutated form of
IB (25). This mutant, termed IB(
N), is
resistant to signal-induced degradation and thus functions as a
trans-dominant inhibitor of NF-B/Rel induction
(23, 24, 25). Expression of the transgene was targeted
specifically to the T lineage using a combination of the T
cell-specific CD2 locus control region and the lck-proximal
promoter (22, 26, 27). The CD4+ T
cell population in these transgenic mice was nearly normal, while the
CD8+ T cell population was diminished to about
one-quarter its normal size (23). In vitro
characterizations of these cells demonstrated enhanced susceptibility
of T cells to apoptosis after TCR ligation, defects in IL-2 production
(23), and decreases in proliferation that were
incompletely reversed by exogenous IL-2 (23). Moreover,
mobility shift analyses demonstrate that a low but reproducible level
of these transcription factors can be detected in nuclei from activated
thymocytes and T cells of IB(N) transgenic mice, despite a
substantial diminution of nuclear B/Rel proteins (23).
Of note, such a state of incomplete blockade to the nuclear induction
of NF-B has been reported after tolerance induction both in B and T
lymphocytes (28, 29), suggesting that inhibition of
NF-B might create the equivalent of tolerance. To determine the
effect of partial NF-B inhibition on immune responses in vivo, we
have used models characteristic of type 1 (DTH) and type 2 (allergic
lung disease) T cell-dependent reactions to one Ag, OVA, and measured
immune responses of IB(N) mice.
The DTH response is a classic example of T cell mediated immunity
dependent on IL-12 induction of the type 1 immune response, leading in
turn to T cell-derived IFN- and further macrophage activation
(9, 10, 11, 30, 31). We show that the DTH response, IFN-
production, and induction of Ag-specific IgG2a were all dramatically
impaired in IB(N) transgenic mice compared with wild-type
littermates. Type 2 immune responses are crucial in allergic responses
to Ags and involve the production of IL-5 and IL-4 in association with
eosinophil and mast cell activation and preferential switching to IgE
production. In sharp contrast to the dramatic inhibition of type 1
responses, the IB(N) transgenic mice maintained a normal
Ag-specific IgE response and mounted an inflammatory response in a
model of allergic pulmonary inflammation. Taken together, our findings
demonstrate a differential requirement for NF-B/Rel signaling in
type 1 T cell-mediated immune responses relative to the type 2 T
cell-mediated reaction.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IB(N) transgenic mice, in which expression of a
FLAG-tagged trans-dominant inhibitor of NF-B/Rel
transcription factors is targeted specifically to the T lineage using
the proximal lck promoter and cointegration of a CD2 locus
control region, have been described previously (23). These
mice were generated from F1 zygotes derived by
mating (DBA/2 x C57BL/6) breeders (low responders for Th2
development). As previously reported, tissue Northern blot analyses and
immunoblots using T cell-depleted B cells failed to detect significant
expression of IB(N) outside of T lineage cells
(23). In addition, immunoblotting of extracts from T
cell-depleted splenocytes did not detect the FLAG-tagged IB(N)
protein (J. Chen, personal communication). The original lines,
backcrossed three generations to C57BL/6, were backcrossed to BALB/c
(susceptible to airway hyperreactivity induction), as indicated in the
figure legends. In brief, for experiments quantifying the induction of
allergy and airway hyperreactivity, data are presented on
F1 progeny generated by mating
B6-IB(N)-transgenic mice with BALB/c. Similar results have
been obtained using progeny at the sixth sequential backcross to
BALB/c. For experiments on cytokine production and DTH,
IB(N)-transgenic mice and wild-type littermates were the
progeny resulting from three sequential backcrosses to BALB/c. HNT TCR
transgenic mice extensively backcrossed to BALB/c (32)
were obtained from D. Lo (Scripps Research Institute, La Jolla, CA).
Adoptive transfer experiments were performed using donor cells from
mice (IB(N) transgenics and wild-type littermates) at the
fifth backcross generation and BALB/c recipients. Breeding stock were
purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were
maintained in specific pathogen-free conditions using microisolator
cages and were used at 6–8 wk of age in accordance with the applicable
regulations after institutional approval.
DTH protocol
An emulsion consisting of equal volumes of OVA 100 µg/20 µl
in sterile PBS and CFA H37 Ra (Difco, Detroit, MI) was made by rapid
mixing between two glass syringes connected by a three-way stopcock.
Mice were anesthetized with methoxyflurane and then immunized by s.c.
injection with 200 µl emulsion divided among three sites, one on each
flank and a third at the base of the tail on day 0. One week later,
mice were challenged with OVA (100 µg/20 µl in sterile PBS) given
as a single injection into the left footpad. Footpad swelling was
measured with a caliper micrometer (Mitutoyo, Cole-Parmer Instrument,
Vernon Hills, IL) with baseline, t = 24 and
t = 48 h measurements recorded. Data are expressed
as the difference in mm between the left (challenged) footpad and the
right (control) footpad. Control mice were sham immunized with an
equivalent amount of sterile PBS and CFA and then challenged with
either PBS alone or OVA. Control data from both IB(N) and
wild-type mice were combined, as there was no difference between these
controls.
Cytokine analysis by ELISA
An emulsion consisting of equal volumes of CFA H37 Ra and Ag
(OVA (100 µg/50 µl in sterile PBS) or HNT peptide (150 µg/50
µl)) was prepared by rapid mixing between two glass syringes
connected by a three-way stopcock. Mice were anesthetized with
methoxyflurane and then immunized with 0.1-ml injection at the base of
the tail. Alternatively, mice received an i.p. injection of 0.1 ml (100
µg) of OVA complexed with 20 mg of Al(OH)3
(alum) on day 0. One week later, mice were sacrificed and draining
lymph nodes were harvested (inguinal and periaortic for OVA/CFA;
periaortic and mesenteric for OVA/alum). Single cell suspensions were
prepared and cultured in RPMI 1640 containing 10% FBS, as previously
described (23). The cells were cultured in 96-well plates
for 72 h at 2 x 106/ml (200 µl/well)
in medium alone, or with Ag (HNT peptide or OVA, as indicated).
Supernatants were collected and analyzed for IFN- and IL-4 by
sandwich ELISA using Ab pairs (PharMingen, Sorrentino, CA), according
to the manufacturer’s recommended procedures. The lower limits of
sensitivity in the ELISA were 10 pg/ml (IL-4) and 60 pg/ml (IFN-),
using mouse IL-4 and IFN- as standards (PharMingen).
Allergen sensitization protocol
Mice received an i.p. injection of 0.1 ml (10 µg) of OVA
(chicken OVA, grade V; Sigma, St. Louis, MO) complexed with 20 mg of
Al(OH)3 (alum) on day 0. On days 14 through 21,
groups of mice were placed in an acrylic box so as to breathe an
aerosol of OVA 1% w/v diluted in sterile PBS using an ULTRA-NEB 99
nebulizer (DeVILBISS, Somerset, PA) for 40 min each day. To minimize
environmental variations between groups, transgene positive and
negative littermates were maintained as cagemates and sensitized
together. Changes in lung resistance caused by the bronchoconstrictor
methacholine were measured, as described previously (33, 34). Control data from both IB(N) and wild-type mice
were combined, as there was no difference between these controls.
Bronchoalveolar lavage
Twenty-four hours following the final aerosol inhalation, the animals were anesthetized with pentobarbital. After placement of a tracheostomy tube, bronchoalveolar lavage (BAL) was performed by instilling 600 µl of 5% BSA diluted in normal saline and then withdrawing the fluid with gentle suction via the syringe. The typical BAL fluid return was 300–400 µl. White blood cells were counted on a hemocytometer, while cytologic examination was performed on cytospin preparations fixed and stained using Diff Quick (American Scientific Products, McGaw Park, IL). Differential counts were based on counts of 100 cells using standard morphologic criteria to classify the cells as eosinophils, lymphocytes, or other mononuclear leukocytes (alveolar macrophages and monocytes). Counts were performed by a single observer who was blinded to the study group.
Lung histopathology
After BAL, the left lung was excised and placed directly in 10% phosphate-buffered Formalin, paraffin embedded, cut in 6-mm sections, mounted, and stained using hematoxylin and eosin for routine histology and Luna stains to specifically evaluate eosinophils. Quantification of eosinophils was performed using the Luna-stained slides by evaluating the three most inflamed arteries in each section. The total number of cells, and the percentage of eosinophils among these cells, were counted and a mean value of the percentage of eosinophils was calculated based on six vessels per group.
Ig assays by ELISA
Before sacrifice, sera were collected from sensitized and control mice, then analyzed by isotype-specific ELISA to determine levels of total and OVA-specific Abs. Briefly, ELISA plates (Corning Glass, Corning, NY) were incubated overnight at 4°C with 50 µl of capture Ag solution (20 µg/ml OVA for OVA-specific Ab measurements; sheep anti-mouse IgE (Serotec, Oxford, U.K.) or anti-mouse IgG2a (Southern Biotechnology, Birmingham, AL) for total isotype determinations). After discarding coating solutions, the plates were washed and blocked with 1% BSA in PBS (2 h at room temperature) and washed. Mouse serum or standard Abs diluted in PBS containing 1% BSA were added to each well (50 µl). Antiserum against OVA, mouse IgE (affinity purified from mouse serum immunized with DNP; Sigma), or mouse IgG2a were used as standards for OVA-specific, total IgE, or total IgG2a ELISA, respectively. Plates were then incubated overnight at 4°C, washed, and incubated with detection Abs (rat monoclonal anti-mouse IgE-biotin (BioSource International, Camarillo, CA) or anti-mouse IgG2a-AP). IgE plates were washed and incubated with avidin-HRP (Zymed, San Francisco, CA) and washed, and HRP activity was determined with a tetra-methylbenzidine (Sigma) developing solution (1% TMB in DMSO, 0.001 M sodium acetate, and 0.45% H2O2 final concentration). Alkaline phosphatase activity was determined with phosphatase substrate tablets (Sigma). Both were assessed during the linear phase of the reaction using an ELISA reader (340 ATCC; SLT-Lab Instruments, Crailsheim, Germany) at 450 nm (IgE-HRP) or 420 nm (IgG2a-AP) and DeltaSoft 3 analytical software (BioMetallics, Princeton, NJ). Each sample was tested in duplicate, and the mean value was recorded.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B(N) transgenic mice
Our previous studies suggested that incomplete inhibition of
NF-B in mature T cells may be sufficient to impair T cell function
under in vitro conditions (23). In light of data
implicating NF-B activation in inflammatory processes, we first
tested whether the IB(N) transgene leads to any impaired type
1 T cell-dependent response by using an assay of cell-mediated
immunity. DTH is a T cell-dependent immune response that is manifested
as an inflammatory recall response reaching peak intensity 24–48 h
after antigenic challenge. The DTH response is dependent on the
development of CD4+ T effector cells due to their
interaction with APC resulting in T cell proliferation and the release
of cytokines associated with type 1 but not type 2 T cell help
(9, 10, 11, 30, 31, 35). Indeed, adoptively transferred type 1
T cells are sufficient to lead to DTH in recipient mice after Ag
challenge, whereas normally a priming step followed by Ag challenge
would be required (9, 10, 11). As shown in Fig. 1
A, sham-sensitized mice
developed no footpad swelling after Ag rechallenge. Unlike these
nonsensitized controls, OVA-sensitized wild-type mice developed
significant swelling in the footpad into which Ag was injected. In
sharp contrast, the IB(N) transgenic mice had no significant
increase in their rechallenged footpads and markedly reduced footpad
swelling when compared with wild-type mice. These data indicate that
NF-B induction in T cells is required for type 1 T cell-dependent
inflammation.
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The DTH response after rechallenge with protein Ag is dependent on
creation of a population of Th1 effector T cells, which produce IFN-
(30, 31). To determine whether the observed impairment in
an anti-OVA DTH response reflected a defect in type 1 cytokine
production by T cells, mice were immunized with Ag in vivo under
conditions similar to the DTH protocol. At the time in which DTH would
be elicited by footpad injections of OVA, draining lymph nodes were
isolated from these Ag-primed mice. Restimulation of cells from these
draining lymph nodes in vitro demonstrated a dramatic defect in IFN-
production (Fig. 1
B). In contrast, IL-4 production by these
draining lymph node cells was at or below the limit of detection (data
not shown), perhaps reflecting the low frequency of OVA-specific type 2
cells recruited during the inflammatory response based on CFA as an
adjuvant. To determine whether the defect in IFN- production was
intrinsic to the T lineage cells, we transferred naive TCR-transgenic
cells into BALB/c recipients before immunization with peptide
(36). The HNT TCR transgene generates a
CD4+ T cell population specific for an influenza
hemagglutinin-derived peptide presented by I-Ad
(32). After transfer of equal numbers of HNT T cells into
recipients and immunization with this peptide, the IFN- response of
restimulated draining lymph node cells was abrogated in those samples
whose NF-B/Rel signaling was inhibited by the IB(N)
transgene (Fig. 1C). These data demonstrate that the
inhibition of NF-B signaling impaired the development of
IFN--producing Ag-specific effector cells in vivo. This failure to
develop a type 1 T cell response is not attributable to a lack of
wild-type cells in vivo and instead reflects a T cell-intrinsic
requirement for NF-B during an inflammatory T cell-dependent
response.
Recruitment of inflammatory cells to the lungs of IB(N)
transgenic mice
Although the above data showed that inhibition of NF-B
abrogated the inflammatory type 1 T cell response, the outcome of type
2 T cell-dependent immune responses could not be determined under these
experimental conditions. Mouse models of allergic pulmonary disease
include a type 2 immune response (37, 38, 39). Such allergic
pulmonary inflammation is dependent on CD4+ T
lymphocytes, in part due to their production and release of effector
cytokines such as IL-4 and IL-5, which mediate IgE production and are
essential for eosinophil cell recruitment (36, 37, 38, 39, 40). To
investigate the role of the NF-B/Rel signaling in Ag-induced type 2
responses, mice were sensitized using OVA in alum, then rechallenged by
repeated inhalation of OVA. Histopathology was then performed on lungs
from wild-type and IB(N) transgenic mice after OVA
sensitization and airway challenge. Consistent with previous studies of
this model, wild-type mice developed a marked inflammatory response in
response to OVA, with evidence of large periarterial infiltrates
composed predominantly of eosinophils and a lesser component of
mononuclear cells (Fig. 2, A
and B; Table I). Moderate
perivenous infiltrates composed of lymphocytes and eosinophils were
also observed. Finally, there was moderate intraalveolar inflammation
composed of multinucleated giant cells and eosinophils with a sparse
admixture of neutrophils and lymphocytes. Strikingly, the lungs of
IB(N) transgenic mice sensitized and challenged with OVA
revealed perivenous infiltrates that were similar to those of the
wild-type mice (Fig. 2, C and D; Table I). The
periarterial infiltrates were slightly smaller when compared with
wild-type (1+ vs 2+,
respectively), but maintained a predominant eosinophilia (77% vs
62%), whereas intraalveolar inflammation was minimal. These data show
that despite a dramatic decrease in DTH, the T cell-dependent steps
leading to the perivascular recruitment of eosinophils were little
affected by the inhibition of NF-B in IB(N) mice.
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B(N)
mice sensitized and challenged with Ag was approximately one-third that
of their wild-type littermates treated in parallel, but substantially
higher than in BALF from nonsensitized controls (Fig. 3). There was no increase in the number
of lymphocytes recovered from the sensitized IB(N) mice as
compared with nonsensitized mice. In contrast, OVA sensitization and
inhalation led to a substantial increase in the number of eosinophils
recovered from IB(N) mice, although the recovery of
eosinophils in BALF was lower for IB(N) mice than for
wild-type littermates that had undergone allergy induction at the same
time. Taken together, these findings reveal that eosinophils migrated
to the air spaces and, despite a reduction in this migration, there was
substantial recruitment of eosinophils to the perivascular spaces.
Thus, the IB(N) transgenic mice were capable of developing an
inflammatory response that requires type 2 T cell function.
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B(N) transgenic mice
The above data suggested that the inhibition of NF-B signaling
in T cells had a greater effect on type 1 T cell responses as compared
with a type 2 response. The development of allergic pulmonary
inflammation in mice, as well as atopy in humans, is linked to the
production of Ag-specific IgE (42, 43). The generation of
Ag-specific IgE requires interaction between T cells and B cells and
the production of IL-4, which in turn leads to Ab class switching from
IgM to IgE (45, 46). In contrast, the production of IgG2a
is augmented by the type 1 T cell-derived cytokine IFN-. Previous
studies have shown that mice sensitized with OVA in alum and
subsequently subjected to inhaled Ag challenge develop both
OVA-specific IgE and IgG2a (47). Therefore, we measured
the effect of the IB(N) transgene on the humoral response to
allergen exposure in vivo. Remarkably, the serum levels of OVA-specific
IgE Abs were higher in sensitized IB(N) mice than their
cosensitized littermates, although the increase did not achieve
statistical significance (Fig. 4A). In sharp contrast, the
level of OVA-specific IgG2a after sensitization was markedly reduced in
the transgenic mice compared with wild-type littermates (Fig. 4B). The observed difference was specific for Ag-specific Ab
levels, because the steady state levels of Abs were essentially normal
(Fig. 4, C and D). Taken together, these findings
indicated that the defective Ab response is due to decreased T cell
help, and the help required for Ag-driven Ig class switching to IgG2a
was deficient, whereas the Th2-dependent process of Ag-specific IgE
production was not (23, 48). To test whether this finding
could be correlated with Ag-specific IL-4 production in IB(N)
transgenic mice, we performed immunizations similar to the OVA-priming
step of the allergic lung disease model and measured cytokine
production after restimulation in vitro. Whereas OVA-stimulated IFN-
production by lymph node cells from IB(N) mice was diminished
compared with immunized littermate controls, IL-4 production was
actually enhanced despite the transgenic inhibitor of NF-B (Fig. 5). We conclude that the type 2 T
cell-dependent response can develop despite profound inhibition of the
NF-B/Rel signaling pathway in T cells, whereas the type 1 T
cell-dependent response during inflammation depends on NF-B.
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B(N) mice
Ag-dependent allergic inflammation of mouse lungs depends on the
type 2 T cell-dependent response, and many of its features can be
mimicked when large numbers of activated allergen-specific Th2 effector
cells are adoptively transferred before a prolonged course of allergen
inhalation (49, 50). However, this process and an
accompanying component of airway hyperreactivity to bronchoconstrictors
may require additional T cell-dependent function(s), perhaps including
type 1 T cells (41). Specifically, similar transfer
experiments showed that Th1 cells potentiated eosinophil recruitment
and allergic manifestations. Moreover, recent experiments using
physiologic numbers of cells demonstrated that airway hyperreactivity
could develop despite a lack of both IL-4 and IL-5 function and in the
absence of significant eosinophil recruitment (51). These
findings and others raise the possibility that inhibition of T
cell-dependent functions other than eosinophil recruitment and type 2
help might attenuate airway hyperreactivity. Accordingly, we measured
the bronchoconstrictor response after OVA sensitization of
IB(N) transgenic mice and their littermates. As shown in Fig. 6, IB(N) mice developed airway
reactivity greater than nonsensitized controls, consistent with other
evidence of type 2 responses (
Figs. 2–5). Of note, however,
perivascular eosinophilia had been slightly diminished (Table I), and
alveolar eosinophil recruitment (interstitial inflammation and
bronchoalveolar eosinophil count) was also diminished in IB(N)
mice compared with sensitized controls (Table I and Fig. 3), despite
intact IL-4 and IgE production (Figs. 4 and 5). Consistent with this
evidence, the inhibition of NF-B in T cells resulted in a less
pronounced airway hyperreactivity of IB(N) mice when compared
with littermates. As with other lines of evidence (41, 51, 52, 53, 54, 55, 56), these data suggest that the magnitude of airway
reactivity may be regulated through T cell populations in addition to
type 2 T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Activation of the NF-B/Rel transcription factor family in
nonlymphoid cells (e.g., monocytes and endothelial cells) has been
associated with inflammation (3, 4). In this study, we
have investigated the role of this signal-dependent transcription
factor during immune responses in vivo. Using transgenic mice whose
NF-B/Rel signaling pathway is inhibited in T cells, we have measured
both allergic pulmonary inflammation (type 2) and DTH (type 1) induced
by the Ag OVA. We found that inhibition of NF-B/Rel signaling in T
cells had little effect on the recruitment of eosinophils to the
perivascular and peribronchial areas of allergen-challenged lungs, and
the Ag-specific IgE response in transgenic animals was unimpaired. In
contrast, IB(N) mice had severely impaired DTH responses and
diminished induction of Ag-specific IgG2a. These results provide strong
evidence for a critical role for T cell NF-B/Rel signaling in the
development of type 1 immune responses. Consistent with these
observations, transgenic mice expressing a mutant IBß exhibited
decreased contact hypersensitivity, although the effect on IFN- or
other cytokine production was not determined (60).
IB(N) mice are protected against autoimmune diabetes (S.
Stanley, J. W. Thomas, and M. Boothby, unpublished observations)
and collagen-induced arthritis (61), an effect associated
with a profound decrease in Ag-induced IFN- production. This
decrease in type 1 T cell effector function may arise due to the role
of NF-B in IL-18 signal transduction (62), enhanced
apoptosis of effector Th1 cells (23, 63), direct
regulation of the IFN- promoter by NF-B (64), or
some combination of these mechanisms. In addition, we have observed a
decrease in the IL-2 responsiveness and Stat5 induction in T cells with
impaired NF-B signaling, despite normal expression of IL-2R subunits
(23 ; A. L. Mora, et al., manuscript submitted). Such
a decrease in IL-2 responsiveness may also contribute to the dependence
of Th1 development on NF-B, inasmuch as there is evidence suggesting
that the Th1 response in vivo is enhanced by IL-2R
(65). As suggested by differences between Th1 and Th2
clones in their induction of NF-B by TCR ligation in vitro
(21, 22), however, our results further indicate that type
2 responses arising from naive T cells in vivo are far less dependent
on NF-B/Rel signaling than their type 1 T cell-dependent
counterparts. Our in vitro studies also indicate that the proliferative
response of T cells to IL-4 in vitro and induction of the
IL-4R-chain are regulated by NF-B (A. L. Mora, et al.,
manuscript submitted). Of note, however, activation of the two IL-4
signal transduction pathways implicated to date in Th2 development,
Stat6 and insulin receptor substrate-2, was decreased only to about
50% of control levels (A. L. Mora, et al., manuscript submitted).
Taken together with the present data on type 2 responses in vivo, these
observations suggest that Th2 development can be normal, despite
decreased expression of IL-4R, particularly when the concurrent Th1
response is attenuated so that less IFN- is present (Figs. 1 and 5).
Apart from the use of trans-dominant inhibitors, the
immunologic function of the NF-B/Rel family has been studied by
inactivation of its specific members through homologous
recombination/gene targeting. In considering the overall role of
NF-B in T cell function, it is important to compare the results
presented in this study with the available data on in vivo models of
immune responses and inflammatory disease in B/Rel knockout mice.
There are two important differences between such knockout mice and
animals expressing a trans-dominant inhibitor that are
intrinsic to these comparisons. First, in vivo studies of inflammation
in knockout animals typically involved a perturbation present in all
cells. Effects on T cell function during disease evolution may reflect
a requirement for NF-B in the APC population in vivo (35, 57). In contrast, expression of the IB(N) transgene has
only been detectable in T lineage cells, perhaps reflecting the
combined lineage specificity of the lck-proximal promoter
and the CD2 locus control region. Moreover, trans-dominant
inhibition requires a sufficient level of mutant IB to block most
NF-B binding to endogenous IB. Accordingly, it is likely that
substantial inhibition of NF-B is achieved only in T cells. Our cell
transfer results demonstrate that the inhibition of NF-B in T cells
creates a cell-intrinsic defect in Th1 development, because all other
cells in the recipient mouse have normal NF-B/Rel signaling. Second,
the biochemical effects of trans-dominant IB transgenes
differ from those achieved by targeted inactivation. Thus, knockout
mice selectively lack expression of a single member of the NF-B/Rel
family. Because IBs interact with multiple forms of NF-B/Rel
dimers, there is little evidence of such a selective effect under
conditions of physiologic regulation. In contrast, the IB(N)
transgene leads to diminished nuclear levels of both inducible
trans-activators in the NF-B/Rel family, RelA and c-Rel
(23). However, this inhibition is incomplete: trace levels
of complexes containing these proteins can be detected in nuclear
extracts after T cell activation. The impact of these differences
includes the finding that mice expressing this
trans-dominant inhibitor of NF-B/Rel signaling experience
increased apoptosis after T cell activation, whereas
NF-B1-deficient, c-Rel-deficient, and RelA-deficient T cells
apparently do not (23, 66, 67, 68, 69). For several of the
knockout lines, little information on T cell effector function is
available. The embryonic lethality of RelA (p65) deficiency has limited
investigation of the immunologic consequences of this knockout
(67, 68). RelB-deficient mice have impaired development
and function of dendritic cells, a crucial APC population, but
apparently no cell-intrinsic defect of T lymphocytes (70).
Inactivation of c-Rel leads to a broad impairment of Ag-specific Ab
responses, but little is known about the susceptibility of such mice to
inflammatory or allergic diseases (66, 69).
In this regard, susceptibility to allergic lung disease has been
reported for the NF-B1 (p50) -/- model. p50 knockout (KO) mice
probably have impaired Th cell function (65), although the
relative contributions of defective APC function and T cell-intrinsic
defects are not clear. Unlike our results (Fig. 4), B lymphoid cells
from NF-B1KO mice exhibit a selective defect
in their ability to switch Ab isotype to IgE (54). Such
mice were protected against eosinophilic airway inflammation after OVA
sensitization (68), due either to the lack of
allergen-specific IgE (42, 54) or to a decrease in IL-5,
macrophage-inflammatory protein-1, and macrophage-inflammatory
protein-1ß production (71), or to other defects in
nonhemopoietic cell types. The profound decrease in perivascular
eosinophils observed in NF-B1KO mice after
induction of allergic lung disease (71) presents a
striking contrast with the preservation of this recruitment step in
IB(N) mice (Fig. 2). We speculate that recruitment of
eosinophils into air spaces sampled by BAL must involve at least two
steps. The first, transendothelial migration, may require a low level
of p50, which can translocate to the nuclei of IB(N) but not
NF-B1KO mice. The second step would be
migration from perivascular sites into air spaces and is inhibited by
IB(N) expression, perhaps due to the decrease in cytokine or
chemokine production by type 1 T cells. It is equally intriguing to
note that airway hyperreactivity developed in IB(N) mice and
indicators of type 2 T cell function were undiminished (Figs. 4, 5, and 6), yet airway hyperreactivity was decreased. T cells make a
contribution to airway hyperresponsiveness that can be achieved despite
neutralization of both IL-4 and IL-5 (51). The adoptive
transfer of supraphysiologic numbers of activated allergen-specific Th2
cells can prime airway hyperreactivity in immunologically intact
recipient mice (49, 50, 72). However, these mice were
subjected to prolonged antigenic stimulation after transfer and may
have generated pulmonary Th1 cells as a consequence: under physiologic
conditions, allergic sensitization activates a substantial population
of allergen-reactive Th1 cells (53, 54, 55 ; reviewed in Ref.
52). Interestingly, other studies employing adoptive
transfers of Th1 or Th2 cells generated in vitro suggest that
collaboration between these subsets is also important for eosinophil
immigration (41, 73). These findings raise a question as
to whether the decreased airway hyperreactivity and migration of
eosinophils into air spaces in IB(N) mice may be due to their
attenuated type 1 T cell function. Although these hypotheses remain to
be tested in each model, the observed differences between
NF-B1KO and IB(N) mice in their
allergic pathophysiology are probably due to differences in the
cellular and biochemical patterns of perturbation in the
NF-B1KO and IB(N) models.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Mark Boothby, Department of Microbiology and Immunology, AA-4214B Medical Center North, Vanderbilt University Medical School, Nashville, TN 37232-2363. E-mail address:
3 Abbreviations used in this paper: IB, inhibitor of B; BAL, bronchoalveolar lavage; BALF, BAL fluid; DTH, delayed-type hypersensitivity; HNT, anti-HA TCR.
4 Mora A., J. Youn, A. D. Keegan, and M. Boothby. NF-B/Rel participation in the lymphokine-dependent proliferation of T lymphoid cells. Submitted for publication.
Received for publication May 14, 1999. Accepted for publication August 19, 1999.
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