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B2 Is Required for Optimal CD40-Induced IL-12 Production but Dispensable for Th1 Cell Differentiation1
* Department of Pathobiology, University of Pennsylvania, Philadelphia, PA 19104; and
Medical Research Council Center for Immune Regulation, School of Medicine, University of Birmingham, Edgbaston, Birmingham, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B is a ubiquitously expressed transcription factor involved
in the regulation of innate and adaptive immunity. As part of studies
to define the role of various NF-B family members in Th cell
development and maintenance, we infected NF-B2-/- and
control mice with Leishmania major and followed disease
progression. NF-B2-/- mice on a normally resistant
background develop chronic nonhealing lesions associated with
uncontrolled parasite replication and a failure to develop an IFN-
response. We show that there are no intrinsic defects in Th cell
differentiation in the absence of NF-B2. Indeed,
NF-B2-/- T cells are able to develop a Th1 phenotype
and protect recombination-activating gene-/- mice from
progressive cutaneous leishmaniasis. We demonstrate instead that the
susceptibility of NF-B2-/- mice to L.
major is the result of an IL-12 deficiency, and we provide
evidence for a specific impairment in CD40-induced IL-12 production by
macrophages lacking this transcription factor. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B/Rel eukaryotic transcription factors are crucial regulators of
the immune response through their ability to induce the expression of
genes for a wide range of cytokines, chemokines, adhesion molecules,
and acute phase response proteins (1, 2). The NF-B
family is composed of five different members, NF-B1, NF-B2, RelA,
RelB, and c-Rel, which are maintained as homo- and heterodimeric
complexes within the cytoplasm until the cell is stimulated to induce
their nuclear translocation (3). In the cytoplasm, NF-B
complexes are bound to one of several inhibitory proteins of the IB
family or the preprocessed NF-B precursors, p105 and p100. These
serve to sequester the NF-B/Rel complexes in the cytoplasm by
masking their nuclear localization signal (1, 3). Once
cells are stimulated by factors that induce NF-B activity (including
cytokines such as TNF-
and IL-1, bacterial and viral products, TCR
stimulation, and Ag recognition), a signal transduction cascade is
initiated that results in the phosphorylation, ubiquitination, and
degradation of IB. This allows the translocation of NF-B to the
nucleus, where it can bind to B DNA motifs and induce gene
transcription (1, 4).
Until the recent generation of mice deficient in individual NF-B
components, few studies distinguished between NF-B family members.
Now, however, infection of various NF-B-/-
mice with a variety of pathogens has uncovered unique roles for each
family member (5, 6, 7, 8). For example, NF-B1-deficient mice
are resistant to infection with Toxoplasma gondii, whereas
mice lacking RelB and NF-B2 demonstrate enhanced susceptibility to
this parasite (9, 10). Lymphocytic choriomeningitis virus
infection of RelB- and NF-B2-deficient mice reveals a distinction
between these two family members in mediating resistance to viruses.
While NF-B2 is dispensable for protective immunity to lymphocytic
choriomeningitis virus, the absence of RelB results in susceptibility
to disease (6, 11). Current research is now focused on
defining the mechanisms underlying the distinct phenotypes observed in
NF-B-/- mice infected with different
pathogens.
Resistance to infection often hinges on the appropriate
differentiation of CD4+ T cells into Th1 or Th2
subsets. Th1 cells mediate the development of cell-mediated
inflammatory responses against intracellular microorganisms, while Th2
cells are required for resistance to extracellular pathogens
(12). Molecular studies indicate that NF-B can play a
potential role at many levels in the development of both types of
immune response. There are putative B binding sites in the promoters
of numerous cytokine genes, including IL-9, IL-12, and IFN-, and
NF-B is important in the activation and proliferation of T
lymphocytes (13, 14, 15, 16). A general requirement for the
NF-B family in Th1-dependent delayed-type hypersensitivity responses
was illustrated using transgenic mice whose T cells fail to activate
the NF-B/Rel signaling pathway (17). Most recently,
NF-B1 was shown to be essential for Th2 cell development
(18). However, the role of the closely related family
member, NF-B2, in Th cell differentiation has not been investigated.
NF-B2 is structurally homologous to NF-B1 in both its DNA binding
and inhibitory domains, but is largely restricted to cells
of hemopoietic origin (1, 19). Mice deficient in the
nfb2 gene lack follicular dendritic cells and have a
dramatic reduction in the B cell compartments of all peripheral
lymphoid organs (8, 20).
To assess the role of NF-B2 in Th cell differentiation and
maintenance, we have used the Leishmania major model of
infection. Resistance to L. major depends on the production
of IL-12, required for CD4+ Th1 cell polarization
(21, 22, 23, 24). We found that
NF-B2-/- mice on a normally resistant
background developed chronic nonhealing lesions following infection
with L. major. Associated with this increased susceptibility
was the failure of NF-B2-/- mice to develop
a Th1 response and restrict parasite replication. We demonstrate that
this nonhealing phenotype was not the result of an intrinsic defect in
T cell function, as NF-B2-deficient T cells were able to protect
recombination-activating gene
(RAG)3-/-
mice from infection. Instead, NF-B2 was shown to play an important
role in the production of IL-12 by macrophages.
NF-B2-/- mice had impaired IL-12 levels
following L. major infection, and exogenous IL-12 conferred
protection against disease. Importantly, our studies demonstrated a
signal-specific requirement for NF-B2 in the expression of IL-12. We
showed that while LPS signaling was intact in the absence of NF-B2,
CD40-induced IL-12 production was dramatically impaired. A defect in
CD40 signaling may, in part, explain the susceptibility of
NF-B2-/- mice to L. major.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NF-B2-/- mice were backcrossed to
C57BL/6 (five generations) (20). Mice were typed for the
absence of the nfb2 gene using a PCR-based method, which
differentiates between the wild-type (WT) NF-B2 gene and the
targeted NF-B2-neo allele, as previously described (9).
Naive NF-B2-/- mice are healthy and do not
show signs of autoimmunity or developmental abnormalities
(20). Controls for our experiments included
NF-B2+/+ and
NF-B2+/- littermates of the
NF-B2-/- mice, as well as C57BL/6 mice. In
our studies, no differences were observed between these control groups.
Female mice were used between 6 and 8 wk of age.
Infections and in vivo IL-12 and anti-IL-12 treatment
L. major (MHOM/IL/80/Friedlin) parasites were cultured as promastigotes in Grace’s insect cell culture medium (Life Technologies, Grand Island, NY), containing 20% FCS (HyClone Laboratories, Logan, UT), 100 U/ml penicillin 6-potassium, 100 µg/ml streptomycin sulfate, and 2 mM L-glutamine. Stationary phase metacyclics were purified by negative selection with Arachis hypogae agglutinin, as previously described (25). For experimental infections, mice were infected in the hind footpad with 2 x 106 metacyclic parasites. IL-12-treated mice were injected intralesionally with 0.2 µg IL-12 (Genetics Institute, Cambridge, MA) six times over the first 2 wk of infection. Anti-IL-12-treated mice were injected i.p. with 1 mg anti-IL-12 (C17.8) once per week for the first 3 wk of infection. Lesion size was monitored weekly using a dial caliper and expressed as the difference between the infected and uninfected contralateral footpad. Parasite numbers were enumerated by plating homogenates of infected lesions in 10-fold serial dilutions in Grace’s insect culture medium starting with a 1/100 dilution. Each sample was plated in triplicate, and the mean of the negative log parasite titer was calculated after 5 days.
Histology
The footpads were removed from 2-, 4-, and 6-wk infected mice,
fixed in 10% neutral buffered formalin, and decalcified in
hydrochloric acid. Tissue was embedded in paraffin, and 4- to 6-µm
sections were cut. The sections were hydrated and stained with H&E.
Histopathologic evaluation of the NF-B2-/-
and NF-B2+/- mice was undertaken.
NO production and parasite killing
Bone marrow macrophages (BMM
) from
NF-B2-/- and
NF-B2+/- mice were derived from bone marrow
monocytes grown on petri dishes in complete tissue culture medium
(CTCM; DMEM containing 10% FCS, 100 U/ml penicillin, 100 µg/ml
streptomycin, 2 mM L-glutamine, 25 mM HEPES, and 5 x
10-5 M 2-ME), supplemented with 30% L929
cell-conditioned medium (26, 27). After 7 days of
incubation at 37°C, adherent cells were harvested from the plates
using disposable cell scrapers, washed twice, and suspended at 1
x 106 cells/ml in 5-ml polypropylene tubes.
Cells were primed with 10 U/ml IFN- for 4 h and infected in
suspension cultures with stationary phase L. major
promastigotes at a 2:1 parasite:cell ratio. Infection was allowed to
proceed for 2 h, and the cells were washed to remove excess
parasites, as previously described (28). Cells were then
stimulated with 10 U/ml IFN- in the presence or absence of 100 ng/ml
LPS (L6143; Sigma-Aldrich, St. Louis, MO) and incubated for 72 h
at 34°C. Supernatants were collected and assayed for the presence of
reactive nitrogen intermediates using the Greiss assay, as
previously described (29). The number of parasites per 100
macrophages was enumerated from cytospins of infected cells.
Cytokine analysis
At 2, 4, and 8 wk postinfection, single cell suspensions were
prepared using splenocytes from infected mice. Cells were resuspended
in CTCM and cultured in 24-well tissue culture plates at 4 x
106 cells/ml. Cells were stimulated with 50
µg/ml soluble leishmanial Ag (SLA) alone or in the presence of 10
ng/ml IL-12 (Genetics Institute), and cultured with 2.5 µg/ml
anti-IL-4R mAb (M1; generously provided by F. Finkleman, University
of Cincinnati, Cincinnati, OH, and Immunex, Seattle, WA). SLA was
prepared as previously described (30). Supernatants were
harvested following a 72-h incubation at 37°C, and IFN- and IL-4
levels were measured using two-site ELISAs, as previously described
(31). IL-12 p40 levels were measured at 4 and 6 wk
postinfection, using mAb C17.8 and biotinylated mAb C15.6 (prepared
from hypbridoma supernatants originally provided by G. Trinchieri,
Wistar Institute, Philadelphia, PA).
To assess in vitro T cell differentiation,
NF-B2-/- and
NF-B2+/+ splenocytes from naive mice were
stimulated with 1 µg/ml plate-bound anti-CD3 (145-2C11) and
anti-CD28 (BD PharMingen, San Diego, CA) under Th1 (10 µg/ml
anti-IL-4 (11B11) and 10 ng/ml IL-12), Th2 (40 ng/ml IL-4 and 50
µg/ml anti-IFN- (XMG6)), or neutral conditions (without
cytokines or anti-cytokine mAbs). After 7 days, the cells were
harvested, washed, and restimulated under neutral conditions for an
additional 3 days. Supernatants were harvested, and IFN- and IL-4
levels were measured by ELISA.
For the in vitro detection of IL-12 p40, BMM were cultured in
flat-bottom 96-well plates at 2 x 105
cells/well. Cells were primed for 4 h with 10 U/ml murine rIFN-
and stimulated with 1, 5, and 10 g/ml anti-CD40 (clone IC10; R&D
Systems, Minneapolis, MN), or 100 ng/ml LPS. Supernatants were
harvested after 72 h at 37°C, and IL-12 p40 levels were measured
by ELISA. For the in vivo detection of IL-12, each mouse was treated
i.p. with 0.3 mg LPS (L4641; Sigma-Aldrich) or 0.5 mg anti-CD40.
Mice were tail bled at 3 and 24 h to measure serum IL-12 p40
levels in response to LPS and anti-CD40, respectively.
Cytofluorometric analysis
Splenocytes were harvested from naive mice, and single cell suspensions were prepared. To assess cell division, splenocytes were stained with the dye CFSE (Molecular Probes, Eugene, OR), as previously described (32). Briefly, cells were washed and suspended in PBS at a concentration of 4 x 106 cells/ml and incubated with an equal volume of CFSE for 5 min at room temperature. Labeling was quenched by adding 10 ml FCS and washing twice in CTCM. Splenocytes were cultured in 96-well U-bottom plates at a concentration of 2 x 105 cells/well, with 1 µg/ml anti-CD3 under neutral or Th1 conditions.
After 4 days, the cells were harvested and intracellular staining was
performed, as previously described (33). Briefly, the
splenocytes were stimulated with 50 ng/ml PMA, 500 ng/ml ionomycin, and
10 µg/ml brefeldin A for 4 h. The cells were then washed,
incubated with 10 µg anti-FcRIII/IIR Ab (2.4G2) and 10 µg
rat IgG (Sigma-Aldrich) for 5 min to inhibit nonspecific binding of
Abs, surface stained with anti-CD4-CyChrome (BD PharMingen), and
fixed overnight with 1% paraformaldehyde. Cells were then
permeabilized with 0.1% saponin in FACS buffer (PBS containing 0.1%
BSA and 0.1% sodium azide) for intracellular staining with
anti-IFN--PE or an isotype-matched control Ab (BD
PharMingen). Cells were washed and analyzed using a FACSCalibur
cytometer (BD Biosciences, San Jose, CA). Numbers of proliferating
cells producing IFN- were quantitated using CellQuest software (BD
Biosciences).
Adoptive transfer
Spleens from naive NF-B2-/-,
NF-B2+/-, and WT B/6 mice were harvested, and
single cell suspensions were prepared. CD3+ T
cells were purified by negative selection (cell purification columns;
R&D Systems) and adoptively transferred by i.v. injection (5 x
106 cells/mouse) into
RAG-/- mice. One day later, the mice were
infected with L. major, and the course of infection was
monitored, as described above. Mice were sacrificed at 14 wk
postinfection, and single splenocyte suspensions were prepared, as
described above.
Statistics
Significance was determined by Student’s paired t test, with a p value < 0.05 considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B2-/- mice are susceptible to L.
major infection
To assess whether NF-B2 is required for resistance to L.
major, NF-B2-/-,
NF-B2+/-, and WT B/6 mice were infected in
the hind footpad with 2 x 106 metacyclic
promastigotes, and lesion size was measured over time.
NF-B2+/- and WT BL/6 mice exhibited a
resistant phenotype, characterized by small lesions that healed by 12
wk. In contrast, NF-B2-/- mice developed
chronic nonhealing lesions (Fig. 1
A). Histopathologic
evaluation of lesions from 4-wk infected
NF-B2+/- mice showed extensive dermal
fibroblast proliferation interspersed with numerous lymphocytes, and
very few infected macrophages (Fig. 1B). In contrast,
NF-B2-/- lesions showed a loss of dermal
collagen, large numbers of infected macrophages, and few lymphocytes
(Fig. 1C). Evaluation of lesions from 2- and 6-wk infected
mice revealed a similar discrepancy between
NF-B2-/- and
NF-B2+/- footpads (data not shown). Parasite
quantitation at 2, 8, and 12 wk postinfection correlated well with
these observations (Table I). At later
time points, there was evidence for a systemic spread of the infection.
Viable amastigotes could also be recovered from the spleen and liver of
L. major-infected NF-B2-/- mice
(data not shown).
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B2-/- mice
NF-B2 is expressed at high levels in myeloid cells, and the
NF-B family of transcription factors is implicated in a large number
of effector functions, including the production of NO and reactive
oxygen intermediates (34, 35). Therefore, we asked whether
the susceptibility of NF-B2-/- mice results
from an inability of L. major-infected macrophages to
respond to IFN- and produce sufficient levels of NO necessary to
restrict parasite replication. Macrophages from
NF-B2-/- and
NF-B2+/- mice were infected with L.
major in vitro and stimulated with IFN- alone, or IFN- and
LPS for 2 or 72 h. It should be noted that the percentage of
infected macrophages ranged from 50 to 60% after the initial 2 h
and did not reveal a discrepancy in the infectivity of
NF-B2-/- and
NF-B2+/- cells.
NF-B2-/- and
NF-B2+/- macrophages produced equivalent
levels of nitrate after 72 h and were equally capable of
controlling parasite replication (Fig. 2). Based on these data, we concluded
that the immune response downstream of IFN- production is
functionally intact in NF-B2-/-
mice.
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B2-/- mice exhibit defective IFN- expression
following L. major infection
To assess the Th effector cell phenotype in L.
major-infected NF-B2-/- mice, we
stimulated splenocytes from infected mice with SLA and measured IFN-
and IL-4 levels in the culture supernatants.
NF-B2-/- splenocytes produced substantially
less IFN- than NF-B2+/- cells even when
IL-12 was added to the cultures (Fig. 3).
However, NF-B2-/- and
NF-B2+/- mice produced comparable amounts of
IL-4, demonstrating that the observed defect in IFN- production was
not the result of an overwhelming Th2 response in the absence of
NF-B2 (Fig. 3).
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B2-/- T cells can mediate resistance to
L. major
One possible explanation for the susceptibility of
NF-B2-/- mice to L. major is an
inability of T lymphocytes to differentiate into Th1 effectors. To
address this, we cultured splenocytes from
NF-B2-/- and
NF-B2+/+ mice under neutral or Th1 (IL-12,
anti-IL-4) conditions in the presence of anti-CD3. After 4
days, the cells were harvested and stained for intracellular IFN-
expression. NF-B2-/- T cells proliferated
normally and produced similar levels of IFN- as
NF-B2+/+ lymphocytes under both culture
conditions (Fig. 4A). We also
examined whether polarized NF-B2-/-
splenocytes could reveal a defect in Th cytokine production upon
restimulation. To do this, we cultured
NF-B2-/- and
NF-B2+/+ splenocytes under neutral, Th1, or
Th2 (anti-IFN-, IL-4) conditions for 7 days. The cells were then
harvested and stimulated under neutral conditions for an additional 3
days. IFN- and IL-4 levels were comparable in supernatants from
NF-B2-/- and
NF-B2+/+ cultures, demonstrating that T
lymphocytes cannot only become Th1 effectors but also effectively
differentiate into Th2 cells in the absence of NF-B2 (Fig. 4B). This observation contrasts with
NF-B1-/- T cells, which are deficient in
their ability to undergo Th2 cell polarization (18).
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B2 to
mediate resistance to L. major. To definitively address this
issue, we asked whether reconstitution of
RAG-/- mice with
NF-B2-/- T cells could confer protection to
L. major. As expected, B/6 T cells were able to control
lesion development in RAG-/- mice, whereas
unreconstituted RAG-/- mice remained
susceptible and developed severe cutaneous leishmaniasis (Fig. 5A). More importantly, the
adoptive transfer of NF-B2-/- T cells also
conferred a healing phenotype to RAG-/- mice.
The healing observed in these mice correlated with the development of a
Th1 response. Thus, when RAG-/- recipients were
sacrificed at 14 wk of infection, splenocytes were stimulated with SLA
or anti-CD3, and the number of IFN--producing cells was
assessed. As seen in Fig. 5B, similar numbers of cells from
RAG-/- mice that received either
NF-B2-/- or control splenocytes produced
IFN- in response to both SLA and anti-CD3. These results
demonstrate that NF-B2-/- T cells can
differentiate into Th1 effector cells to promote healing in vivo and
provide compelling evidence that the susceptibility of
NF-B2-/- mice to L. major is not
explained by an intrinsic defect in T cell function.
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B2-/- mice to L.
major is associated with a defect in IL-12 production
IL-12 stimulates the production of IFN- from lymphoid
cells, and NF-B has been implicated in the regulation of IL-12
synthesis (36, 37). To address a potential defect in IL-12
production during L. major infection of
NF-B2-/- mice, we measured IL-12 p40
production by SLA-stimulated splenocytes from 4- and 6-wk infected
animals. NF-B2-/- splenocytes produced
significantly less IL-12 p40 than NF-B2+/-
cells at both time points (Fig. 6). To
determine whether the susceptibility of
NF-B2-/- mice is linked to this defect in
IL-12 production, we administered exogenous IL-12 to
NF-B2-/- mice and measured lesion size over
time. As reported previously, IL-12-treated BALB/c mice resisted
L. major and controlled infection (23, 24).
IL-12 treatment of NF-B2-/- mice also
provided protection against disease, resulting in the development of
small lesions that had healed by 6 wk postinfection (Fig. 7A). Viable promastigotes were
recovered at >10-8 dilutions for untreated
NF-B2-/- mice. In contrast,
NF-B2-/- mice treated with IL-12 exhibited
3- to 4-log fewer parasites (data not shown).
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B2-/- mice is explained by a
deficiency in IL-12 production, and yet these animals prevent
progressive lesion development. As shown in Fig. 6
, NF-B2-/- mice still produce low levels of
IL-12 during infection with L. major. To address whether the
NF-B2-independent production of IL-12 may account for the chronic
nature of this infection, we treated
NF-B2-/- mice with anti-IL-12 and
measured lesion size over time. In the complete absence of this
cytokine, NF-B2-/- mice were unable to
contain lesion growth and developed a course of infection similar to
that observed in IL-12 or CD40-/- mice (Fig. 7B). Viable promastigotes were recovered at
>10-17 dilutions for anti-IL-12-treated
NF-B2-/- and
NF-B2+/- mice, at
10-10–10-11 dilutions
for untreated NF-B2-/- mice, and at
10-1 for untreated
NF-B2+/- mice (data not shown).
CD40-induced IL-12 production is impaired in the absence of
NF-B2
The development of a protective cell-mediated immune response to
leishmaniasis is dependent on CD40-induced IL-12 production by
macrophages and dendritic cells (39, 40, 41). In an effort to
explain why IL-12 levels are reduced in L. major-infected
NF-B2-/- mice, we asked whether CD40
signaling is impaired in the absence of this transcription factor.
Several labs have shown that NF-B is activated in response to CD40,
and one study provides evidence for its specific involvement in
anti-CD40-induced IL-12 transcription (13). To address
whether NF-B2 is required in the CD40 signaling pathway to produce
IL-12, we stimulated NF-B2-/- and
NF-B2+/- macrophages with anti-CD40 or
LPS. Importantly, CD40 expression was comparable between both cell
types (data not shown). IL-12 levels were not impaired in response to
LPS; however, anti-CD40-induced IL-12 production was defective in
the absence of NF-B2 (Fig. 8A). We also addressed CD40
signaling defects in NF-B2-/- dendritic
cells but failed to see a reproducible deficiency in IL-12 (data not
shown). This is consistent with data suggesting that NF-B-inducing
kinase-deficient dendritic cells, which have very little NF-B2
activation, produce normal levels of IL-12 in response to anti-CD40
(42, 43). To confirm our macrophage results in vivo, we
administered anti-CD40 or LPS to
NF-B2-/- and
NF-B2+/- mice and quantitated serum IL-12 p40
levels at 3 and 24 h posttreatment.
NF-B2-/- mice produced higher levels of
IL-12 than NF-B2+/- mice in response to LPS
(Fig. 8B). Importantly, however, CD40-induced IL-12 levels
were reduced in NF-B2-/- mice. These results
suggest that NF-B2 is required for optimal anti-CD40-induced
IL-12 production.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B2 is dispensable for T
cell proliferation and polarization into Th subsets. We use the
L. major model of infection to show in vivo that
NF-B2-/- T cells can promote resistance to a
disease that requires Th1 cell development for effective healing. This
is intriguing considering that numerous studies have shown NF-B
activation in Th cells and a role for this transcription factor in
normal T cell function (44). Because NF-B2 appears to
be of minimal importance to T lymphocytes during L. major
infection, the susceptibility of NF-B2-/-
mice must be explained by a defect in another cell lineage. We show
that macrophages require NF-B2 for optimal CD40-stimulated IL-12
production, and provide evidence that the failure of
NF-B2-/- mice to resist cutaneous
leishmaniasis results from a deficiency in IL-12 during infection.
Importantly, NF-B2 is not essential for all aspects of macrophage
function. Parasite killing and NO production are intact in the absence
of this transcription factor.
We were interested in addressing a role for NF-B2 in T cells because
gene deletion studies have defined roles for other NF-B family
members in modulating T cell function. For example, T cells lacking
c-Rel cannot effectively proliferate in response to mitogenic stimuli
(45). In addition, the susceptibility of
RelB-/- mice to T. gondii is
explained by an intrinsic defect in T cell-induced IFN- production
(9). Most recently, Th2 cell development was shown to
require NF-B1, potentially through its role in inducing GATA-3
expression (18). It is surprising that T cells are not
more affected by the absence of NF-B2, considering the structural
similarity between this and other NF-B family members. One
explanation is that compensation by other NF-B components masks a
potential role for NF-B2. Indeed, evidence for partial redundancy of
NF-B1 and NF-B2 functions has already been shown using mice
deficient in both family members (11). It is also possible
that NF-B2 is not essential in T lymphocytes because its activation
patterns are tightly regulated. Research demonstrates that NF-B1 is
ubiquitously expressed in mouse tissues, while NF-B2 activation is
largely restricted to myeloid cells of the immune system (46, 47).
To determine why NF-B2-/- mice fail to
resist L. major, we addressed potential defects in two
myeloid cell functions known to involve NF-B, inducible NO synthase
expression, and IL-12 production. The susceptibility of
c-Rel-/- mice to L. major is
correlated with impaired NO induction and parasite killing
(48). However, despite an association between c-Rel and
NF-B2 in some tissues (15), NF-B2 is not essential
for macrophage effector cell function. Our results indicate that a more
likely explanation for the susceptibility of
NF-B2-/- mice is a deficiency in IL-12
production during infection. Given that CD40-CD40 ligand (CD40L)
ligation provides the primary stimulus of IL-12 during leishmaniasis,
we investigated potential defects in CD40 signaling to produce IL-12 in
the absence of NF-B2 (39, 40, 41, 49). Although this
signaling pathway is involved in a number of immunological processes,
including the maturation and survival of dendritic cells and the
production of Ab, IL-12 production appears to be its only required
function for resistance to L. major (50).
Administration of exogenous IL-12 to CD40L knockout mice confers
protection against disease (41).
Signaling via CD40 is mediated in part by TNFR-associated factor family
members and results in the subsequent activation of mitogen-activated
protein kinases, I-B kinases, and NF-B (51, 52, 53). The
defect in CD40-induced IL-12 production by
NF-B2-/- macrophages is similar to that
observed in human PBMCs lacking NF-B essential modulator (or I-B
kinases) activation (54). Importantly, LPS signaling is
intact in the absence of either NF-B2 or NF-B essential
modulator, highlighting a signal-specific requirement for these
molecules in mediating IL-12 production. However, recent work in these
laboratories demonstrates that, in contrast to NF-B2, NF-B1 is
dispensable for normal IL-12 production via CD40 ligation (D. Artis,
manuscript in preparation).
The distinct requirement for NF-B2 in CD40-stimulated IL-12
production may also explain why IL-12 levels are normal during acute
T. gondii infection of NF-B2-/-
mice (10). In contrast to L. major infections,
the CD40-CD40L interaction is not essential for early IL-12 production
and innate immunity to toxoplasmosis (55). However, a
requirement for this pathway has been defined recently in the control
of parasite replication during the chronic phase of infection
(55). CD40L-/- mice are
increasingly defective in IL-12 as the disease progresses
(55). A similar reduction in IL-12 observed during chronic
T. gondii infection of NF-B2-/-
mice was previously associated with the reduced capacity of these mice
to produce IFN- (10). Thus, this diminution in IL-12
may instead be explained by a defect in CD40-induced IL-12 production
in the absence of NF-B2. Interestingly, infection of
NF-B2-/- mice with T. gondii was
accompanied by a massive increase in lymphocyte apoptosis
(10). Although we found no evidence of enhanced cell death
in the spleens of L. major-infected
NF-B2-/- mice (data not shown), an increase
in apoptosis may provide an explanation for the reduced lymphocytic
infiltrate in the footpad lesions of these animals.
Another possibility for the low numbers of lymphocytes observed in
NF-B2-/- lesions is a defect in T lymphocyte
recruitment by cells at the site of infection. While many adhesion
molecules and chemokines require NF-B family members for their
induction and activity, a specific role for NF-B2 has not been
defined (56, 57). Endothelial cells lacking NF-B2 may
reveal a defect in E-selectin expression, for example, which could
impair the ability of T cells to home to the infected footpad.
Alternatively, if NF-B2 is involved in cutaneous T cell-attracting
chemokine production, its absence might prevent efficient trafficking
of lymphocytes to skin lesions (58). Further studies will
be required to determine how enhanced lymphocyte apoptosis or impaired
recruitment may influence the susceptibility of
NF-B2-/- mice to L. major.
We propose a role for NF-B2 in the production of the IL-12 required
to heal L. major infection and suggest that a defect in CD40
signaling may explain the in vivo deficiency in this cytokine. However,
it is important to note that anti-CD40-induced IL-12 production is
not completely abrogated in the absence of NF-B2. This may explain
why NF-B2-/- mice still produce low levels
of IL-12 and prevent uncontrolled parasite replication. Indeed,
treatment of NF-B2-/- mice with
anti-IL-12 results in progressive lesion development, a
manifestation of disease similar to that observed in IL-12- or
CD40-deficient mice (38, 39). It is possible that NF-B2
acts in concert with other transcription factors to stimulate maximal
IL-12 promoter activity. Alternatively, NF-B2 may be indirectly
required for the synthesis of elements, such as IFN regulatory factor
1, which regulate IL-12 gene transcription (59).
Defining the role of NF-B2 in mediating IL-12 production aids us in
viewing the NF-B family as a set of individual transcription
factors, each with unique roles in modulating immune function. The
selectivity of NF-B2 in CD40-induced IL-12 production highlights
this NF-B component as a potential drug target in the treatment of
diseases resulting from a hyperactive Th1 response.
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Acknowledgments |
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B2-/- mice. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Phillip Scott, Department of Pathobiology, University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA 19104-6008. E-mail address: pscott{at}vet.upenn.edu
3 Abbreviations used in this paper: RAG, recombination-activating gene; BMM, bone marrow macrophage; CD40L, CD40 ligand; CTCM, complete tissue culture medium; SLA, soluble leishmanial Ag; WT, wild type.
Received for publication October 24, 2001. Accepted for publication February 22, 2002.
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