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Distinct Pathways for NF-κB Regulation Are
Associated with Aberrant Macrophage IL-12
Production in Lupus- and Diabetes-Prone
Mouse Strains
Jiajian Liu and David I. Beller
J Immunol 2003; 170:4489-4496; ;
doi: 10.4049/jimmunol.170.9.4489
http://www.jimmunol.org/content/170/9/4489
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References
The Journal of Immunology
Distinct Pathways for NF-␬B Regulation Are Associated with
Aberrant Macrophage IL-12 Production in Lupus- and
Diabetes-Prone Mouse Strains1
Jiajian Liu2 and David I. Beller2
A
n unresolved issue in immunology is why autoimmune
diseases can be divided into two groups, in which pathology is mediated by either T (specifically Th1) or B
cells. It is known that IL-12 is critical for development of disease
in the former group (1–3), and blocking IL-12 blocks the development of disease in animal models of diabetes (4) and multiple
sclerosis (5). Moreover, low levels of IL-12 may play the opposite
role in Ab-mediated diseases, either indirectly, by enhancing Th2
responses (6), or directly, by inhibition of B cell function (7, 8).
Thus, aberrant regulation of IL-12 could be one mechanism by
which autoimmunity is directed toward a T or B cell-mediated
pathway.
We have previously demonstrated that IL-12 is intrinsically dysregulated in macrophages (M␾)3 from autoimmune-prone strains
in a manner consistent with the nature of disease: its production is
elevated in diabetes-prone nonobese diabetic (NOD) (9) and reduced in lupus-prone NZB/W and MRL/⫹ M␾ (10) in response to
a range of stimuli including LPS, intact bacillus Calmette-Guérin,
and CD40 ligand (9). It has also been shown that dendritic cells
Arthritis Section, Evans Department of Medicine and Clinical Research, Boston University Medical Campus, Boston, MA 02118
Received for publication December 5, 2002. Accepted for publication February
25, 2003.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants from the Juvenile Diabetes Research Foundation, the Arthritis Foundation, and the American Autoimmune Related Diseases
Association.
2
Address correspondence and reprint requests to Dr. Jiajian Liu or Dr. David Beller,
Arthritis Section, E-519, Boston University Medical Campus, 715 Albany Street,
Boston, MA 02118. E-mail addresses: [email protected] and [email protected]
Abbreviations used in this paper: M␾, macrophage; NOD, nonobese diabetic; DC,
dendritic cell; ChIP, chromatin immunoprecipitation; CIP, calf intestinal phosphatase;
siRNA, short interfering RNA.
(DC) from NOD mice have a similar propensity for elevated IL-12
production (11), suggesting that these defects are reflective of the
innate immune system in general, and not limited to M␾. We have
recently reported that unique patterns of Rel binding to the ␬B site
of the p40 promoter are consistent with the nature of IL-12 dysregulation in each autoimmune-prone strain (12). Specifically, in
in vitro EMSA assays, binding of the transactivating c-Rel/p50
heterodimer dominates in extracts from NOD M␾, whereas excess
binding of the inhibitory p50 homodimer characterizes M␾ extracts from the two lupus-prone strains.
In this study, we report that a strikingly similar pattern of ␬B/
Rel usage is seen in vivo as assessed by the chromatin immunoprecipitation (ChIP) assay; i.e., during activation of NOD M␾, the
p40 ␬B site preferentially associates with c-Rel, whereas in
NZB/W M␾, this sites binds predominantly p50. Additionally,
blocking c-Rel in these primary M␾ blocks IL-12 selectively and
normalizes the small amount of residual IL-12 production, suggesting that the defects in IL-12 production that characterize these
M␾ are governed by c-Rel. However, the mechanisms responsible
for the NF-␬B defects differ in NOD compared with NZB/W M␾.
The NOD defect appears to be limited to enhanced c-Rel phosphorylation, whereas the NZB/W defect is associated with a broad
activation of the NF-␬B pathway, including ␬B/Rel as well as I␬B
proteins. These findings reveal that the IL-12 defects in both
strains are linked to alterations in NF-␬B metabolism. Because of
the broad use of Rel in regulating gene activity in the immune
system, these findings may provide insight into not only the contribution of the innate immune system to the basic dichotomy of B
vs T cell-mediated autoimmunity, but also the regulation of a wide
range of APC defects associated with autoimmune diseases.
Materials and Methods
Mice
3
Copyright © 2003 by The American Association of Immunologists, Inc.
Five-week-old male mice were purchased from The Jackson Laboratory (Bar
Harbor, ME) and were housed on-site for an additional week before use.
0022-1767/03/$02.00
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One characteristic of mice prone to a variety of autoimmune diseases is the aberrant regulation of cytokine production by
macrophages (M␾), noted in cells isolated well before the onset of disease. Strikingly, the pattern of IL-12 dysregulation, in
particular, is consistent with the nature of the autoimmune disease that will develop in each strain, i.e., elevated in mice prone to
Th1-mediated organ-specific disease (nonobese diabetic (NOD) and SJL mice) and reduced in lupus-prone strains (MRL/ⴙ and
NZB/W). Mechanistically, the abnormal regulation of IL-12 in these strains was found to be strictly associated with novel patterns
of Rel binding in vitro to the unique NF-␬B site in the IL-12 p40 promoter. In this study, we report several new findings related
to these Rel-␬B interactions. Evaluation of the p40 NF-␬B site in vivo, assessed by chromatin immunoprecipitation, revealed Rel
usage patterns similar to those found in vitro using EMSA, with preferential association of the p40 ␬B site with c-Rel in NOD M␾
but with p50 in NZB/W M␾. Moreover, blocking c-Rel in primary M␾, using short interfering RNA, selectively blocked IL-12
production and normalized the minimal, residual IL-12 levels. Nuclear extracts from NOD M␾ were characterized by c-Rel
hyperphosphorylation, and dephosphorylation of nuclear proteins completely blocked binding to the ␬B site. In contrast, elevated
I␬B appears to be a likely mechanism accounting for the reduced nuclear c-Rel levels noted in NZB/W M␾. Alterations in NF-␬B
metabolism thus appear to define a pathway regulating intrinsic IL-12 defects in both diabetes- and lupus-prone strains. The
Journal of Immunology, 2003, 170: 4489 – 4496.
NF-␬B DEFECTS IN AUTOIMMUNE M␾
4490
M␾ isolation and culture
M␾ were obtained from peritoneal exudates cells by peritoneal lavage with
cold RPMI 1640 medium supplemented with 5% FBS, 1% L-glutamine,
0.5% HEPES, and 1% penicillin/streptomycin (BioWhittaker, Walkersville, MD) 4 days after i.p. injection of 2 ml of thioglycolate (REMEL,
Lenexa, KS).
EMSA
EMSA was performed as previously described (12). For phosphatase treatment, nuclear extracts were incubated with affinity-purified calf intestinal
phosphatase (CIP; catalog no. P3681; Sigma-Aldrich, St. Louis, MO) for
30 min at 37°C.
accession nos. NM009044, X15842, and XM122179). This sequence was
subjected to a basic local alignment search tool search against the NCBI
expressed sequence tag mouse database to ensure that a unique c-Rel specific sequence was targeted. The siRNA duplex was synthesized by Dharmacon Research (Lafayette, CO). The transfection was performed following the TransIT-TKO transfection reagent protocol (catalog no. MIR2154;
Mirus, Madison, WI). Briefly, 7.5 ⫻ 104 cells were seeded in 100 ␮l of
medium per well of 96-well plates overnight before transfection. Cells
were then transfected by 2.5 nM siRNA with 1 ␮l of TransIT-TKO transfection reagent in each well. After 24 h, the cells were stimulated by 100
ng/ml LPS for 16 h. Cytokine levels in supernatants were quantitated by
ELISA.
Results
The ChIP assay was performed using a kit (catalog no. 17-295; Upstate
Biotechnology, Lake Placid, NY) and following the manufacturer’s protocol, except that the sonication procedure was optimized for M␾. Briefly,
M␾ (4 ⫻ 106 in 10 ml of medium/100-mm-diameter dish) were adhered for
2 h and then vigorously washed free of nonadherent cells. The purified M␾
were stimulated with LPS (100 ng/ml) for 2 h. DNA-protein structure was
then cross-linked with 1% formaldehyde (37°C; 10 min), and cells were
scraped on ice and spun down. The cell pellet was resuspended in 400 ␮l
of SDS lysis buffer (all buffers and adsorption reagents were provided with
the kit). The resulting lysate was sonicated to shear DNA to lengths between 200 and 1000 bp using a Fisher (Pittsburgh, PA) Sonic Dismembrator 550 (power setting 5). The pellet was subjected to optimized sonication conditions: 30 times for periods of 20 s each. The sonicate was
centrifuged, and the nucleosome-containing supernatant was diluted with
10-fold excess dilution buffer. An aliquot (1% volume) of the diluted cell
supernatant was saved to quantitate the input DNA present in each strain.
The remainder of the supernatant was precleared with salmon sperm DNA/
protein A-agarose. Nucleosome fractions were isolated by adding anti-p50
(catalog no. sc-114x), anti-p65 (sc-372x), anti-c-Rel (sc-71x; all from
Santa Cruz Biotechnology, Santa Cruz, CA), or no Ab (as negative control), and incubating overnight at 4°C with rotation. Salmon sperm DNA/
protein A-agarose was then used to immunoprecipitate the Ab-bound nucleosomes. After washing, the immunoprecipitated nucleosomes, as well as
the saved reference aliquot, were incubated at 65°C for 4 h to reverse
protein/DNA cross-linking.
The immunoprecipitated DNA was purified by standard phenol/chloroform and ethanol precipitation, and was dissolved in 20 ␮l of H2O; 2 ␮l of
this DNA solution was used as template for the first round of PCR (25
cycles). An aliquot (1 ␮l) of this first-round PCR product was used for the
second-round PCR (20 cycles). This product was labeled by [␣-32P]dATP
(NEN Life Science Products, Boston, MA). PCR amplification was performed in a volume of 25 ␮l (PCR reagents; catalog no. M7665; Promega,
Madison, WI) under the following conditions: initial denaturation at 95°C
for 3 min; amplification cycles at 95°C for 30 s, 58°C for 1 min, and 72°C
for 1 min; and a final extension at 72°C for 10 min. Primer pairs (amplifying
a 170-bp PCR product spanning the p40 NF-␬B site) were 5⬘-AGTATCTCT
GCCTCCTTCCTT-3⬘ (sense) and 5⬘-GCAACACTGAAAACTAGTGTC-3⬘
(antisense) (13). PCR products were run on 8% polyacrylamide gel and visualized by autoradiography.
Distinct patterns of Rel binding to the p40 ␬B promoter are
found in vivo in M␾ from normal and autoimmune-prone strains
Immunoblot assay
Immunoprecipitation and immunoblotting were performed as described
previously (12). The following Abs were used in these experiments: antiphospho-Thr (catalog no. sc-5267), anti-I␬B␣ (sc-371), anti-I␬B␤ (sc-945),
anti-I␬B⑀ (sc-7155) (all from Santa Cruz Biotechnology), and anti-phosphoSer/Thr-Pro (MPM-2; catalog no. 05–368; Upstate Biotechnology).
ELISA
Levels of IL-12 p70 and p40 and TNF-␣ in M␾ culture supernatants were
measured by ELISA using selected Ab pairs (OptEIA mouse p70, catalog
no. 22661KI; p40, catalog no. 2619KI; and TNF-␣, catalog no. 555268;
BD PharMingen, San Diego, CA) following the manufacturer’s instructions, as described previously (12).
Short interfering RNA (siRNA) assay
For the siRNA assay, the following target sequence for murine c-Rel
mRNA was used: 5⬘-AACAACCGGACAUACCCGUCU-3⬘ (AA dimer
plus 19 nucleotides), which is located 129 bases downstream from the start
codon (Ref. 14 and National Center for Biotechnology Information (NCBI)
We had previously used EMSA to show that unique patterns of
M␾ nuclear Rel protein binding to the IL-12 p40 ␬B site distinguished diabetes-prone NOD and lupus-prone NZB/W mice from
each other and from several normal strains (A/J, B6, and BALB/c)
(12). Importantly, these patterns were not characterized by the simple over- or underexpression of Rel proteins. Rather, the elevated
expression of IL-12 mRNA and protein in NOD M␾ was associated with a shift from the normal balance between Rel heterodimer
(containing primarily c-Rel and p50) and p50 homodimer, toward
elevated transactivating heterodimer (9, 12). In contrast, NZB/W
and MRL/⫹ M␾, which display a markedly reduced capacity for
IL-12 expression, were characterized by dominant binding of the
inhibitory p50 homodimer (10, 12). Thus, binding to the unique
p40 ␬B half-site was consistent with the known function of the ␬B
family members (14, 15). However, whether the in vitro assay—in
which nuclear proteins bind to a truncated promoter sequence devoid of structural regulatory proteins (e.g., histones)—reflected
gene regulation in vivo remained to be determined. Therefore, we
addressed the biological significance of our initial findings using a
ChIP assay. In this procedure, the preferential association in vivo
of the p40 ␬B sequence with different Rel proteins was assessed.
We first confirmed the unique patterns of Rel binding to the specific p40 ␬B sequence noted previously by EMSA. M␾ from the
different strains were stimulated by LPS for 2 h, and nuclear extracts were prepared. The results (shown in Fig. 1A) demonstrate
the characteristic pattern of elevated c-Rel-containing heterodimer
binding in NOD M␾ extracts, compared with elevated p50 homodimer binding in NZB/W extracts. Identity of the Rel proteins
in these complexes was earlier determined by supershifting (12)
and confirmed in these experiments (data not shown). Additional
cultures of stimulated M␾ were used to prepare nuclear extracts in
which the DNA was sheared into short segments of 200-1000 bp.
These segments were immunoprecipitated with selected anti-Rel
Abs (as noted in Materials and Methods) to isolate c-Rel-, p65-,
and p50-associated nucleosomes (16). From the selected DNA, a
sequence spanning the p40 ␬B site was amplified by PCR. The
results (shown in Fig. 1B) reveal that, in vivo, the p40 ␬B sequence
is preferentially associated with c-Rel in NOD M␾ but with p50 in
NZB/W M␾. These results were quantitated, as displayed in Fig.
1C. As an internal control, the level of p40 ␬B-associated Rel A
(p65) was also assessed and was found to be similar in all three
strains, as we had previously noted in vitro (12). Finally, the relative association of c-Rel with the p40 ␬B site in A/J, NOD, and
NZB/W M␾, assessed by both in vitro and in vivo assays, was
compared (Fig. 1D). Our findings reveal both a quantitative and
qualitative similarity between the EMSA and ChIP assays.
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ChIP assay
The Journal of Immunology
4491
c-Rel is critical in establishing the aberrant regulation of IL-12
in M␾ from autoimmune-prone strains
Phosphorylation of c-Rel is required for binding to the p40
NF-␬B site
FIGURE 1. Distinct patterns of Rel protein binding to the NF-␬B site of
the murine IL-12 p40 promoter in normal and autoimmune-prone strains
are seen both in vitro and in vivo. A, EMSA showing unique patterns of
elevated p50/c-Rel heterodimer in NOD and elevated p50 homodimer in
NZB/W M␾. Identity of the bands was determined by supershift (Ref. 12
and data not shown). B, ChIP assay. Upper left panel, Semiquantitative
PCR product showing that the PCR was run in a linear range (template
input from left to right of each strain represents 5-fold dilutions), as well
as that the input of three M␾ sources was similar. Upper right panel,
Negative control for each strain (mock immunoprecipitated without Ab
during ChIP procedure). Lower panel, Anti-p50, anti-p65, and anti-c-Rel
were used to isolate nucleosomes associated with specific Rel proteins.
It is known that phosphorylation of ␬B/Rel proteins is one mechanism by which both their DNA binding and transactivating functions are controlled (20 –22). Additionally, we had earlier shown
that increased Tyr phosphorylation on c-Rel, rather than an augmented c-Rel protein level, was associated with increased c-Rel
binding to the p40 ␬B site in nuclear extracts from NOD M␾ (12).
We have extended these studies and, in this report, show that nuclear c-Rel in NOD M␾ appears to be differentially phosphorylated
on selected epitopes, because no increase was noted in the overall
level of Thr phosphorylation. However, there is a substantial
(⬃3.5-fold) increase in phosphorylation of those Ser and/or Thr
that neighbor Pro residues (Fig. 3, A and B). Thus, the increase in
phosphorylation of c-Rel in NOD M␾ has been independently verified using an Ab of unique specificity, and the differences in c-Rel
phosphorylation between A/J and NOD M␾ have been shown to
be restricted to a subset of phosphorylation sites on this protein.
To address the role of phosphorylation more directly, EMSA
was performed before and after enzymatic dephosphorylation of
the nuclear extracts, using an affinity-purified CIP as phosphatase.
The results (Fig. 3C, upper panel) reveal that phosphorylation is
absolutely required for binding of the p50/c-Rel heterodimer to the
p40 ␬B site. The lower panel shows that neither the amount nor
This pull-down in turn provided DNA for the PCR which produced a
170-bp product spanning the IL-12 p40 ␬B site. The data represent one of
three independent experiments that showed similar results. The ChIP p40
␬B PCR product was quantified by densitometry (C) and compared as ratio
of PCR product from c-Rel vs p50 pull down (ChIP), to ratio of c-Rel
containing heterodimer to p50 homodimer (EMSA) (D).
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Given the substantial evidencefor a critical role for c-Rel in IL-12
expression in both M␾ and DC (12, 15, 17, 18), we attempted to
determine the importance of c-Rel in expression of the IL-12 defects by transfecting M␾ with siRNA specific for c-Rel. M␾ from
each strain were divided into three groups: untreated, mock transfected with an irrelevant oligomer, or actively transfected with a
21-mer duplex siRNA representing a sequence unique to c-Rel.
After 16 h, the cultures were stimulated with LPS for an additional
24 h, and conditioned medium was collected to assess cytokine
production. The results of these experiments confirm that transfection with the c-Rel-specific siRNA produced profound inhibition of both IL-12 p40 and p70 production (Fig. 2). As a control,
TNF-␣, whose promoter preferentially binds p65 rather than c-Rel
(19), was also tested. In marked contrast to IL-12, TNF-␣ production was virtually unaffected, demonstrating the specificity of the
siRNA treatment. Note that the characteristic pattern of TNF-␣
produced by M␾ from these strains (A/J ⬎ NOD ⬎ NZB/W) is
clearly distinct from the pattern of IL-12 production (9). These
results provide novel confirmation of the importance of c-Rel in
IL-12 p40 regulation. More importantly, they reveal that, when
c-Rel is inhibited, the minimal residual levels of IL-12 expressed
appear to be similar in all strains, presumably as a result of the
similar levels of NF binding to the IL-12 p40 promoter Ets and
CEBP/␤ sites in these strains (12). These findings suggest that
c-Rel is the critical factor in establishing the aberrant IL-12 levels
characteristic of M␾ from autoimmune-prone strains, and that the
aberrant regulation of IL-12 p40 results from preferential ␬B/Rel
protein usage.
4492
mobility of c-Rel (assessed by immunoblotting) is altered by the
enzyme treatment.
NZB/W M␾ are characterized by increased cytoplasmic levels of
Rel proteins
We had previously shown that, in contrast to NOD M␾, in NZB/W
M␾, the nuclear levels of both p50 and c-Rel were sufficient to
explain the observed bias in Rel binding to the p40 ␬B site (12). To
characterize the events by which aberrant nuclear ␬B/Rel levels
were established in NZB/W M␾, as well as to evaluate the possibility of additional defects in NOD M␾, cytoplasmic Rel levels
were assessed in A/J, NOD, and NZB/W M␾ by immunoblotting.
As anticipated, the cytoplasmic levels of p50 and c-Rel in A/J and
NOD reflected their nuclear levels (Fig. 4). However, we found
that not only were levels of p50 elevated, but also c-Rel was substantially higher in the cytosol of NZB/W M␾ than would have
been predicted from its nuclear level. Thus, it became clear that the
relatively low level of c-Rel found in NZB/W nuclear extracts did
not arise as a consequence of low cytoplasmic levels of this pro-
FIGURE 3. The level of c-Rel binding to the NF-␬B site in NOD nuclear extracts correlates with the level of c-Rel phosphorylation on Ser-Pro
and Thr-Pro residues, but not with overall Thr phosphorylation. Moreover,
phosphorylation is critical for p50/c-Rel binding to the ␬B site. A, Upper
panel, A/J and NOD nuclear proteins were immunoprecipitated (IP) with
anti-c-Rel, and then immunoblotted (IB) with anti-p-Thr. Middle panel, the
same membrane was stripped and immunoblotted with anti-p-Ser/Thr-Pro
Abs recognizing phosphorylated Ser-Pro and Thr-Pro residues. Lower
panel, The same membrane was stripped again and immunoblotted with
anti-c-Rel showing similar c-Rel levels in both strains. B, Bands in A were
quantified by densitometry and normalized to c-Rel (shown in A, lower
panel). A/J and NOD nuclear extracts contained nearly identical levels both
of c-Rel and of Thr phosphorylated c-Rel. In contrast, NOD M␾ display an
⬃3.5-fold higher level of Ser/Thr-Pro-phosphorylated c-Rel than normal
A/J M␾. C, To address the role of phosphorylation more directly, EMSA
was performed before and after enzymatic dephosphorylation of the nuclear extracts, using an affinity-purified CIP. The results (upper panel)
reveal that phosphorylation is absolutely required for binding of c-Rel/p50
heterodimer to the p40 NF-␬B site. The lower panel shows that the amount
and mobility of c-Rel (assessed by immunoblotting) is not altered by the
enzyme treatment. The data represent one of three or four independent
experiments that showed similar results.
tein. Therefore, we explored the possibility that c-Rel was not
being efficiently transported to the nucleus in NZB/W M␾.
Reduced nuclear c-Rel in NZB/W M␾ is associated with
increased levels of I␬B␤ and I␬B⑀ in the cytosol
The family of I␬B molecules (␣, ␤, and ⑀) is responsible for retention of Rel proteins in the cytosol, controlling their function by
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FIGURE 2. Treatment of primary M␾ with c-Rel siRNA blocks IL-12
production and normalizes residual IL-12 content. M␾ from A/J, NOD, and
NZB/W mice were transfected with c-Rel siRNA (or left untreated, or
mock-transfected with an irrelevant oligo, as indicated). Twenty-four hours
later, cells were stimulated with 100 ng/ml LPS for 16 h. IL-12p70 (A), p40
(B), and TNF-␣ (C) levels in supernatants were determined by ELISA, and
the results were normalized to the A/J values. IL-12 levels were dramatically reduced by siRNA treatment. Of note, the minimal residual IL-12
levels were similar in all strains, indicating that c-Rel is critical for establishing the defect in IL-12 expression noted in the autoimmune-prone
strains. TNF-␣ was used as a control; it is regulated primarily by p65rather than c-Rel-containing NF-␬B complexes, and was resistant to c-Rel
siRNA treatment.
NF-␬B DEFECTS IN AUTOIMMUNE M␾
The Journal of Immunology
4493
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FIGURE 4. Levels of cytoplasmic p50, but not c-Rel, are consistent
with nuclear ␬B/Rel levels in NZB/W M␾. A, A/J, NOD, and NZB/W
cytoplasmic p50 and c-Rel levels were detected by immunoblotting using
anti-p50 and c-Rel Abs. B, The bands in the panels in A were quantified by
densitometry and normalized for actin content. The data showed that the
cytoplasmic p50 level in NZB/W is about 2.5-fold higher than that in both
A/J and NOD, consistent with the nuclear p50 levels among these three
strains as we reported earlier (12). However, the cytoplasmic c-Rel level in
NZB/W is about 3-fold higher than in both A/J and NOD, the opposite of
the nuclear c-Rel levels found in M␾ from these three strains. The data
represent one of three independent experiments that showed similar results.
restricting nuclear import (23). Levels of the three I␬B proteins
were therefore assessed to determine whether they might contribute to the discordance between cytoplasmic (Fig. 4) and nuclear
(12) levels of c-Rel found in NZB/W M␾. Cytoplasmic levels of
I␬B␣, I␬B␤, and I␬B⑀ were determined by immunoblotting (Fig.
5A). Although levels of I␬B␣ were similar in M␾ from all three
strains, levels of both ␤ and ⑀ were about 3-fold higher in NZB/W
M␾ than in M␾ from A/J or NOD (Fig. 5B). Thus, there is a
selective elevation of I␬B proteins in NZB/W M␾. Additionally, it
is known that I␬B proteins are present constitutively, and are rapidly degraded after cell activation (24 –26). Therefore, the levels of
I␬B␤ and I␬B⑀ were evaluated kinetically to determine whether
the differences noted reflected intrinsic differences between
NZB/W and other strains in expression of these proteins, or
whether they arose in response to stimulation. The data shown in
Fig. 5C reveal that, before stimulation, levels of both I␬B␤ and
I␬B⑀ in A/J and NOD M␾ were within ⬃30% of NZB/W M␾
levels (I␬B␤: A/J, 30% ⬍ NZB/W; NOD, 3% ⬎ NZB/W; I␬B⑀:
A/J, 31% ⬍ NZB/W; NOD, 13% ⬍ NZB/W). In contrast, 2 h after
activation with LPS, the levels of I␬B␤ and I␬B⑀ in NZB/W M␾
were 335% and 285%, respectively, of A/J levels, and 271% and
197%, respectively, of the levels in NOD M␾. Similar differences
were seen at 1 h. While degradation of I␬B␤ was nearly complete
in all strains at 30 min, reduction in I␬B⑀ levels appeared to be less
pronounced within the parameters of the time points selected.
Thus, the significant elevation noted in the levels of cytoplasmic
I␬B␤ and I␬B⑀ in NZB/W M␾ arises as a consequence of
activation.
FIGURE 5. Cytoplasmic levels of both I␬B␤ and I␬B⑀, but not I␬B␣,
are higher in NZB/W compared with A/J and NOD M␾. A, A/J, NOD, and
NZB/W cytoplasmic I␬B␣, I␬B␤, and I␬B⑀ were detected by immunoblotting. B, The bands in A were quantified by densitometry and normalized to actin levels. The amounts of I␬B␤ and I␬B⑀ in NZB/W are, respectively, 2.7- and 2.5-fold greater than in A/J and 3.0- and 2.5-fold
greater than in NOD. No significant difference in the amount of I␬B␣ was
noted among the strains. The data represent one of three independent experiments that showed similar results. C, Kinetic evaluation of the changes
in I␬B levels, demonstrating that the differences in I␬B␤ and I␬B⑀ arise as
a consequence of M␾ activation.
Additionally, it has been reported that there is a preferential
association of c-Rel with I␬B␤ and I␬B⑀, but not I␬B␣ (23, 27). If
true in M␾, the preferential elevation of ␤ and ⑀ in NZB/W M␾
could help explain why c-Rel appears to be retained in the cytosol,
thus leading to the lower nuclear levels noted. Therefore, the levels
of c-Rel bound to individual I␬B proteins were determined by
immunoblotting for c-Rel after immunoprecipitation of I␬B␣,
I␬B␤, and I␬B⑀. Our findings revealed elevated levels of ␤- and
NF-␬B DEFECTS IN AUTOIMMUNE M␾
4494
⑀-associated c-Rel in the cytosol of NZB/W M␾ (Fig. 6, A and B).
This contrasted to the nearly identical levels of p50 bound to each
I␬B protein in M␾ from each strain (Fig. 6, C and D). A possible
explanation for the lack of correlation between total I␬B and p50associated I␬B is that with the elevation of both I␬B␤/⑀ and c-Rel
in NZB/W M␾, the amount of residual I␬B␤/⑀ left to bind in a
lower affinity interaction with p50 may be similar in NZB/W M␾
compared with M␾ from other stains. In any case, the elevation in
expression of I␬B␤/⑀, and the preferential association of these proteins with Rel as compared with p50, may provide one basic mechanism by which c-Rel is selectively excluded from the nucleus of
NZB/W M␾, resulting in lowered potential for IL-12 production.
Discussion
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FIGURE 6. In NZB/W M␾, I␬B␤ and I␬B⑀ preferentially retain c-Rel
in the cytosol compared with p50. A/J, NOD, and NZB/W cytoplasmic
proteins were immunoprecipitated with anti-I␬B␣, -I␬B␤, and -I␬B⑀, and
then immunoblotted with anti-c-Rel (A) or anti-p50 (C). Results were quantified by densitometry (B and D). The data show that the amount of c-Rel
associated with cytoplasmic I␬B␤ and I␬B⑀ is substantially greater in
NZB/W compared with A/J and NOD M␾, in keeping with the elevated
levels of the I␬B proteins in the former. In contrast, the level of p50 associated with I␬B␤ and I␬B⑀ is similar in each strain. Thus, the increased
levels in NZB/W M␾ of I␬B␤ and I␬B⑀ result in selective retention (cytoplasmic binding) of c-Rel and thus provide a possible mechanism for the
reduced nuclear c-Rel levels characteristic of this strain. The data represent
one of three independent experiments that showed similar results.
Intrinsic dysregulation of M␾ cytokine production characterizes
many autoimmune-prone mouse strains comprising one of the
most broadly expressed functional defects among animal models
of multigenic autoimmunity (9, 10, 28 –31). Among these defects,
aberrant regulation of IL-12 may be the most instructive in its
potential for explaining the course of autoimmune pathology, because the potential for IL-12 production is substantially elevated
in models of organ-specific autoimmunity, e.g., NOD and SJL
(9, 28), and dramatically impaired in mice (MRL/⫹, NZB/W,
and NZM2410) that develop systemic autoimmunity (Ref. 10; D.
Alleva and D. Beller, unpublished observations). In the strains
studied here and in a previous study (12) (NOD, NZB/W, and
MRL/⫹), this conserved defect appears to arise from a common
basis: aberrant regulation of the NF-␬B signaling pathway. However, the precise mechanism accounting for the NOD M␾ defect is
distinct from that noted in NZB/W and MRL/⫹ M␾. Both in vitro
and in vivo, NOD M␾ have a preferential association of the functional c-Rel/p50 heterodimer with the p40 ␬B site, and this in turn
appears to arise from excessive phosphorylation of a subset of
potential phosphorylation sites and enhanced DNA binding capacity. Strikingly, the two lupus strains studied share a nearly identical
characteristic of elevation of both (p50)2 binding and nuclear p50
levels (12). In NZB/W M␾, studied further here, the modulation of
␬B appears to reflect a more pervasive mechanism than in NOD:
whereas nuclear c-Rel levels are clearly reduced (12), cytoplasmic
levels of c-Rel, p50, I␬B␤, and I␬B⑀ are all significantly elevated,
suggesting a generic activation of the ␬B pathway. This observation may be relevant to the wide range of genes regulated by NF␬B, and the broad spectrum of Ags targeted—and resultant broader
pattern of initial pathology—in lupus compared with organ-specific diseases like diabetes or MS.
The role of IL-12 in Th1-mediated organ-specific autoimmunity
is well established (1–5). The unexpected finding that IL-12 (as
well as IFN-␥)-deficient NOD mice do not develop diabetes appears to be due to the time of onset of the blockade in the knockout
and development of compensatory regulatory pathways that do not
function in intact mice (32–34). Conversely, a lack of IL-12 would
be expected to promote Th2 dominance and the B cell hyperactivity characteristic of lupus. A role for Th2 dominance has been
reported in the HgCl2 model of inducible lupus (6), but not in
spontaneous lupus. The lack of demonstration of a role for IL-12
in spontaneous lupus may be due to the complexity of the disease;
i.e., at some point, IFN-␥, produced by Th1 (or NK) cells, would
likely be required to shift the autoantibody repertoire to a complement-fixing isotype to generate pathology. However, IL-12 may
also work independently of the Th1/Th2 axis, by directly inhibiting
B cell function (7, 8). Thus, IL-12 defects may be seen as defining
a crossroad, where a commitment is made to either B or T cellmediated pathology.
The unique patterns of ␬B/Rel binding to the IL-12 p40 ␬B site
of normal and autoimmune-prone M␾, noted earlier in vitro (12),
were found to mirror the binding events in vivo. The extent to
which the EMSA data were predictive of the binding of ␬B/Rel to
the p40 promoter in the ChIP assay is striking. These findings
The Journal of Immunology
may be sufficient to explain the reduced nuclear c-Rel content of
NZB/W M␾, there may, of course, be additional mechanisms
involved.
Taken together, these findings suggest that NF-␬B defects play
a central role in the development of both organ-specific and systemic autoimmunity. The defective regulation of IL-12 may be a
critical step in the development of autoimmunity— contributing to
the commitment to an organ-specific or systemic pathway— but
ultimately may prove to be just one example of the faulty immune
regulation caused by underlying defects in the pervasive NF-␬B/
Rel pathway.
Acknowledgments
We thank Drs. Matthew Fenton and Thomas Gilmore for helpful
discussions.
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suggest that neither tertiary conformation of the DNA, nor association of structural proteins (e.g., histones), nor differences in the
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