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,*
* Neurocrine Biosciences, Inc., San Diego, California;
BD Biosciences, San Diego, California; and
Department of Medicine, Rheumatology Section, E550, Boston University Medical Center, Boston, Massachusetts
Correspondence: Dr. David Alleva, Neurocrine Biosciences, Inc., 10555 Science Center Dr., San Diego, CA 92121-1102. E-mail: dalleva{at}neurocrine.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; i.e., APC)
from naïve SJL mice produced levels of TNF-
, IL-1, IL-6,
IL-10, and TGF-ß within the range of six normal strains. In contrast,
SJL IL-12p40 (in addition to IL-12p70) production was consistently
five- to 20-fold greater than that of any normal strain tested, which
arose from elevated expression of the IL-12p40 but not the IL-12p35
gene, because p40 mRNA levels were eight- to 15-fold greater than those
of normal strains. This aberrancy in IL-12p40 expression appears
identical to that observed in the NOD mouse, another strain prone to
organ-specific autoimmunity. A genetically programmed bias toward
elevated expression of IL-12 in M from the SJL and NOD strains of
autoimmunity provides a conserved mechanism for the dominant Th1
development of naïve, autoantigen-specific T-cells in these
strains. This study is the first demonstration of a genetically
programmed aberrant phenotype that is intrinsically expressed within a
cell type in the SJL mouse and provides insight into its predisposition
for EAE.
Key Words: EAE • diabetes • antigen-presenting cell • multiple sclerosis • cytokines
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(IFN-)- and interleukin (IL)-2-producing cells] of
neuroantigen-specific T-cells in the SJL mouse because the B10.S strain
lodges an H2S-restricted protective Th2 response (i.e.,
IL-4-, IL-5-, and IL-10-producing cells) to the same neuroantigen
epitopes that induce disease in the SJL [13
,
14
].
The development of the pathogenic Th1 phenotype of autoantigen-specific
T-cell responses in SJL mice involves the production of IL-12, a
cytokine produced by antigen-presenting cells (APCs) such as M and
dendritic cells (DC; reviewed in [15
]). IL-12 is the
most potent Th1-inducing factor known and is required for Th1 cell
development in almost any immune response studied, including those
against neuroantigens during EAE [13
,
15
16
17
]. Its expression is up-regulated during the course
of several Th1-mediated, autoimmune diseases, including mouse, rat, and
human EAE/MS and Th1 diabetes [15
, 17
].
Furthermore, IL-12 treatment of SJL mice and the Lewis rat model of EAE
exacerbate disease, although antibody (Ab)- or antagonist-mediated
neutralization of IL-12 activity blocks disease in these rodent models
(reviewed in [15
, 17
]). It is interesting
that ex vivo IL-12 treatment of MBP-specific T-cells
obtained from B10.S mice immunized with MBP caused these cells to
develop a Th1 phenotype and to become pathogenic when transferred
adoptively into naïve hosts [13
]. Hence,
differences in endogenous IL-12 production between the SJL and B10.S
strains may contribute to differences in Th cell phenotypes that
develop in response to neuroantigens, resulting in a pathogenic Th1 or
a protective Th2 response, respectively.
Here, we determined whether SJL mice were genetically programmed
for abnormal expression of Th1-promoting cytokines, in particular
IL-12, by comparing the levels of cytokines produced by APCs (i.e.,
M) from naïve disease-free SJL mice with those of other
disease-resistant strains. Naïve disease-free mice were studied
to ensure that any aberrant cellular phenotypes observed in SJL mice
would have arisen from the genetic background of the mouse and not as a
consequence of the inducible disease process. From a panel of six
cytokines, we demonstrated that only IL-12 was expressed aberrantly in
the SJL, with a five- to 20-fold increase in expression relative to
that of six normal strains. Moreover, we showed that this SJL defect
arose from a hyperexpression of the IL-12p40 gene and appears identical
to that observed in the nonobese diabetic (NOD) mouse model of
autoimmune-mediated diabetes [18
]. This study is the
first demonstration of a genetically programmed aberrancy expressed
within a cell type from the SJL mouse and provides insight into the
genetic predisposition for autoimmunity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Reagents
Lipopolysaccharide (LPS; Escherichica coli: 0111:B4;
Sigma Chemicals, St. Louis, MO), recombinant murine IFN-
(PharMingen, San Diego, CA), and recombinant murine CD40 ligand (CD40L;
provided by Dr. Marilyn R. Kehry, Boehringer Ingelheim Pharmaceuticals,
Inc., Ridgefield, CT) were stored at -80°C and diluted in medium
immediately before use. RPMI-1640 medium was supplemented with 2 mM
L-glutamine, 0.5% HEPES (Cellgro, Herndon, VA), 5 µg/ml penicillin,
100 U/ml streptomycin (GIBCO, Grand Island, NY), and 10% fetal bovine
serum (FBS; BioWhittaker, Walkersville, MD) and was used for culturing
M. No endotoxin (<10 pg/ml) was detected using the Limulus
amoebocyte assay (BioWhittaker) in any of the reagents and media
described above.
M isolation and culturing
Thioglycollate-elicited peritoneal exudate M were obtained by
peritoneal lavage and isolated by adherence to plastic as previously
described [19
]. Peritoneal exudate cells
(2x105) were seeded per well of 96-well, flat-bottom
tissue culture-treated plates and allowed to adhere for 2 h, and
nonadherent cells were washed away. To ensure that equal numbers of
adherent M among strains remained after washing, nonadherent cells
from washes of single wells from each strain were counted routinely and
showed no differences among strains. M were activated with different
stimuli, and culture-conditioned medium was collected at an optimal
culture period of 16 h [19
] and stored at -20°C
for assessment of cytokines. For assessment of IL-12 production,
conditioned medium was collected in two sequential phases, 0–16 h and
16–40 h, with change of medium and fresh stimuli at 16 h,
permitting quantitation of cytokine levels in the latter time window
that are independent of levels produced during the earlier 16-h period.
IL-1 levels were assessed in M lysates because substantial IL-1 is
retained in the cell [20
]. M lysates were prepared by
adding 200 µl medium to each well of M monolayers after
conditioned medium was collected. Cells were subjected to three rapid
cycles of freeze-thawing at -70°C and 37°C, respectively, and
stored at -20°C.
Assessment of cytokine production in conditioned medium
IL-1 bioactivity in M lysates was measured in the D10.G4
bioassay as previously described [19
, 20
],
and TNF- levels in M-conditioned medium were assessed using the
WEHI-164 bioassay as previously described [21
]. Alamar
Blue solution (AccuMed International, Inc., Westlake, OH) was used to
assess cell viability in both bioassays in which one unit of IL-1 or
TNF- activity was defined as the titer of cell lysates or
culture-conditioned medium, respectively, that caused 50% of maximal
cell viability. IL-6, IL-10, IL-12, and TGF-ß levels in conditioned
medium were assessed by enzyme-linked immunosorbent assay (ELISA;
PharMingen).
Intracellular cytokine staining
Thioglycollate-elicited (6x106), adherent
peritoneal M from SJL and B10.S mice were cultured with brefeldin A
(1 µg/ml) under different conditions for 16 h. Cells were
detached by treatment with trypsin and stained using a
paraformaldehyde- and saponin-based procedure (PharMingen) with
R-phycoerythrin (PE)-conjugated, rat anti-mouse IL-12p40/p70 [C15.6,
immunoglobulin G1 (IgG1)]; PE-conjugated, rat IgG1 isotype control
(R3-34); fluorescein isothiocyanate (FITC)-conjugated, rat anti-mouse
CD11b (MAC-1; clone M1/70, IgG2b); and purified rat anti-mouse
IL-12p40/p70 (C15.6, IgG1; all Ab from PharMingen) in which
fluorescence intensity of each marker was measured per cell using flow
cytometry. A total of 10,000 cells per sample were counted in which the
macrophage population was gated based on the light-scattering
characteristics and FITC, anti-MAC-1 immunofluorescent staining. Within
the light scatter gate, >96% of cells were MAC-1+ for all samples.
RNase protection assay
Thioglycollate-elicited (107), peritoneal exudate
cells were plated from each mouse strain on 75 cm2 plastic
tissue culture-treated flasks (Corning Glassworks, Corning, NY), and
M were allowed to adhere for 2 h at 37°C, 5%
CO2. Nonadherent cells were washed away, and adherent M
were cultured in the presence or absence of LPS (100 ng/ml; Sigma) or
LPS (100 ng/ml) plus IFN- (100 U/ml; PharMingen) for 3–24 h, as
indicated, at which time total RNA was extracted using the UltraSpec-II
RNA Isolation Kit (Biotecx Laboratories, Inc., Houston, TX) as
specified in the manufacturer’s instructions. RNA was quantified by UV
spectrophotometry in addition to gel electrophoresis. Total RNA (10
µg) from each sample was used in an RNase protection assay (Riboquant
mCK-2b; PharMingen) and resolved using a 0.4 mm urea-polyacrylimide gel
and visualized by autoradiography. RNA was quantified using
phosphoimaging (GS-525; Bio-Rad, Hercules, CA) and normalized to
control RNA using the Molecular Analyst 2.1 software program.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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/APC (i.e., APCs) from the SJL and a
panel of normal, disease-resistant strains, B10.S, BALB/c, A/J,
C57BL/6, C57BL/10, and C3H/OuJ. Cytokine levels for each M
population were normalized to those of BALB/c M (a strain that
produces cytokine levels intermediate to those of several normal
strains) [18
, 19
], and the mean of these
relative values from several experiments is shown (Table 1
). SJL M produced TNF-, IL-6, IL-10, and TGF-ß levels
within the range of normal strains (Table 1)
. In addition, early (i.e.,
16 h) and late (48 h) IL-1 production by M from SJL mice were
also within the normal range (Fig. 1
), indicating that relative differences in IL-1 production among
strains do not change over time. Note that we have shown previously
that A/J and C3H/OuJ M produce IL-1 within the normal range
[18
, 19
]. Differences in M cytokine
expression among all strains were not associated with a variation in
viability or metabolic activity among M from each strain, as
measured by conversion of the redox reagent, Alamar Blue (unpublished
results).
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from SJL mice showed a profound
elevation (five- to 20-fold) in total IL-12p40 production (i.e., p40
monomer and homodimer, and p70 heterodimer) compared with any normal
strain, either during the first 16 h or during 16–40 h of LPS
stimulation (Fig. 2A
). This defect appears identical to that observed in M from
young, pre-diseased NOD mice (Fig. 2A)
, a model of autoimmune-mediated
diabetes, which is also mediated by Th1 autoreactive T-cells and IL-12
[15
, 22
]. We have demonstrated recently
this observation in NOD M, in addition to demonstrating that
expression of other cytokines produced by NOD M was not elevated
aberrantly [18
].
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does not require
stimulation by IFN- from SJL and
NOD mice was manifested in the absence of IFN-, a Th1, cell-derived
cytokine that is known to potently stimulate IL-12 production by
monocytes and M activated with agents such as LPS. In fact, SJL and
NOD M production of total p40 induced by LPS alone appears close to
its maximum capacity because addition of IFN- to LPS-activated M
from these strains caused only a modest increase (i.e., twofold) in p40
production, whereas normal strains responded by a five- to tenfold
increase (Fig. 2B)
. (Maximum p40 production occurred under these
conditions because higher doses of IFN- or LPS did not further
up-regulate production of the cytokine by any strain tested.) IFN-
alone did not stimulate p40 or p70 production. Indeed, this maximum
stimulation of total p40 production by IFN- treatment in all strains
led to a normalization of cytokine production between autoimmune and
normal strains (Fig. 2B)
. It is unlikely that elevated p40 production
in the absence of IFN- in vitro is a result of in
vivo, IFN- priming of M from SJL or NOD mice because other
cytokines that are potently up-regulated by IFN-, such as TNF- ,
IL-1, and IL-6, were not aberrantly expressed (see Table 1
, Fig. 1
, and
[18
]).
Elevated expression of the biologically active form of IL-12, the
IL-12p70 heterodimer, in SJL and NOD M
The biologically active agonist heterodimer of IL-12, p70, is
comprised of the p40 and p35 subunits derived from separate genes
(reviewed in [15
]). Although the p70 heterodimer has
IL-12 receptor agonist activity that leads to Th1 cell development, the
p40 homodimer is known to antagonize this activity (reviewed in
[15
]). Therefore, we tested whether the aberrant
elevation in total p40 production manifested a concomitant elevation in
biologically active p70 using an IL-12p70-specific ELISA. Strikingly,
p70 production induced by LPS alone was elevated at least as much as
p40, in that p70 was only detectable in conditioned medium from SJL and
NOD M and was absent in M cultures of six normal strains (Fig. 2C)
, demonstrating an aberrant induction of biologically active IL-12
in these autoimmune strains. Similar to total p40 production, SJL and
NOD production of p70 was close to its maximum relative to that of
normal strains, as demonstrated by the differences in responsiveness to
IFN-. Although the autoimmune strains produced maximal p70 levels by
responding to IFN- by only threefold, normal strains absolutely
required IFN- for production of detectable p70 levels (Fig. 2C)
.
Note that M from normal and autoimmune strains produced 100–300
times more total p40 than the p70 heterodimer (Fig. 2B
vs. C
),
demonstrating that a large excess of free p40 (i.e., monomers and
dimers) is produced relative to that of p70 in normal and autoimmune
strains, an observation that others have demonstrated
[15
, 23
]. Although there has been no
explanation of why these excess levels of free IL-12p40 do not
antagonize the biologically active IL-12p70, these ratios of free
IL-12p40 to IL-12p70 levels are not substantially different between
normal and autoimmune strains.
Induction of the IL-12 expression defect in SJL M by the
activated T-cell surface protein, CD40L
Although LPS is commonly used to activate M, there is no direct
evidence that it plays a role in EAE. Therefore, we determined whether
the elevated expression of IL-12 in SJL M could be induced with the
T-cell-derived surface molecule, CD40L, which has been shown to induce
IL-12 production potently during EAE and other Th1-mediated responses
and is highly expressed on activated T-cells during
antigen-presentation (reviewed [24
]). Similar to LPS
alone, soluble CD40L stimulated an elevated expression of total p40 in
M from SJL mice relative to the disease-resistant B10.S strain
(Fig. 3A
). We have also demonstrated that soluble CD40L induces a similar
aberrancy in NOD M [18
]. This elevated, IL-12
expression induced by LPS and CD40L is also induced in spleen cells
from SJL mice relative to the B10.S (Fig. 3B)
. Hence, this aberrant
expression of total IL-12p40 is not limited to a particular anatomical
source of M/APC nor to a single activation agent, indicating diverse
possibilities for elevated IL-12p40 and IL-12p70 expression to modulate
immune function in the SJL mouse. Collectively, these results
demonstrate that M/APC from SJL mice do not display a global
dysregulation of cytokine production but rather display a selective
enhancement of total IL-12p40 and IL12p70 production.
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and enhanced IL-12 synthesis per cell may be a consequence of an increased percentage of
IL-12-producing cells in the M population or enhanced production on
a per-cell basis. We dealt with this issue by assessing the
intracellular levels of p40 produced per M in SJL versus B10.S mice
by intracellular cytokine staining and flow cytometry. Adherent M
from SJL or B10.S mice were activated for 16 h with LPS alone or
with LPS plus IFN- in the presence of brefeldin A, which blocks
secretion of cytokines and allows cytokines to accumulate within the
cell. By analyzing the total adherent peritoneal M population
(<98% MAC-1+), activation with LPS induced p40 production
in a greater percentage of cells in the M population from SJL mice
compared with that of the B10.S mice (i.e., 30.9% vs. 19.5%,
respectively; Fig. 4C
and D
). LPS was required for p40 production because
the unactivated M populations from either strain had <1%
p40-producing cells (Fig. 4A
and 4B)
. In addition, IFN- treatment
normalized the percentage of p40-producing M between the two strains
by increasing the LPS-stimulated, p40-producing population in the B10.S
(Fig. 4E
and 4F)
, which is consistent with IFN--induced effects on
secreted p40 levels (see Fig. 2B
).
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population produced more p40 per cell than
did the B10.S (i.e., 148 vs. 89 mean fluorescent units per cell,
respectively), and IFN- treatment normalized these values by
enhancing only B10.S p40 production per cell (i.e., 131 vs. 156 mean
fluorescent units per cell, respectively). Subtraction of base-line
values in unstimulated SJL and B10.S M populations (38 and 40 mean
fluorescent units per cell, respectively) from the respective values
from LPS-activated cultures demonstrates that SJL M expressed
roughly twofold greater IL-12p40 levels per cell than B10.S M after
LPS activation (i.e., 110 vs. 49 mean fluorescent units per cell,
respectively). The results obtained with intracellular cytokine
staining demonstrate an approximate 3.5-fold increase in p40 expression
by SJL M (i.e., the 50% increase in the percentage of
p40-expressing cells and a 2.3-fold increase in mean fluorescence).
Although five- to 20-fold increases were observed in secreted p40
levels between SJL and normal strains, the more modest difference
between these strains observed using intracellular cytokine staining
may be a result of the effects of the protein transport inhibitor,
brefeldin A, which would ultimately limit the amount of cytokine
synthesized as intracellular protein levels increase. In fact, the ten-
to 20-fold induction by IFN- on secreted p40 by normal strains (see
Fig. 2B
) was also reduced using this intracellular, cytokine-staining
technique. Nevertheless, this technique clearly showed that SJL mice
have a significant elevation in the percentage of p40-expressing cells
and in p40 expression per cell.
Elevated expression of IL-12p40 mRNA in SJL M
Because IL-12p70 is secreted as a heterodimer of p40 and p35
subunits, we determined whether elevated p70 production by SJL M
might be a result of elevated transcript levels of one or both
subunits. Using the RNase protection assay, p40 and p35 mRNA levels
were assessed in control (C57BL/6) and SJL M at 3, 8, 16, and
24 h after LPS activation (Fig. 5A
). LPS was required for expression of either subunit mRNA by both
strains and induced their expression as early as 3 h, which became
maximal around 8 h (see normalized values in Fig. 5A
). Whereas
C57BL/6 and SJL M expressed similar kinetic and quantitative
expression patterns of p35 mRNA at each time point (Fig. 5A)
, C57BL/6
M showed eight- to 15-fold lower p40 mRNA levels than those of SJL
M at 3 and 8 h and completely lost expression by 16 h.
Strikingly, high levels of p40 mRNA expression by SJL M were
maintained at 16 h, levels which exceeded the highest levels of
p40 mRNA expressed by C57BL/6 M at any time point tested (Fig. 5A)
.
Consistent with cytokine secretion data (see Table 1
and Fig. 1
), mRNA
levels of other cytokines (i.e., IL-1, IL-ß, IL-6, and IL-18) were
expressed at similar levels in SJL and B6 M (Fig. 5B)
. It is
interesting that IFN- treatment strongly up-regulated LPS-induced,
p40 mRNA expression, which surpassed p35 mRNA expression levels at
16 h, resulting in normalized LPS-induced, p40 mRNA expression
between both strains (Fig. 5A)
. This activity of IFN- is consistent
with cytokine secretion data above and suggests that a large excess of
the p40 subunit is required for secretion of substantial levels of the
p70 heterodimer. Because 16 h after LPS activation demonstrated
the greatest contrast of p40 mRNA expression between SJL and C57BL/6
M, this time point was used to show the p40 mRNA expression
aberrancy of SJL M relative to M from the B10.S and its parental
strain, C57BL/10 (B10) (Fig. 5C)
. Strikingly, M from NOD mice also
displayed a similar, if not identical, defect in p40 gene expression
(Fig. 5C)
. These results demonstrate that the SJL and NOD strains have
a genetically programmed predisposition for p40 gene expression, which
leads to an elevated expression of free p40 and the heterodimer p70. In
turn, this event may be responsible for the innate tendency of these
strains to develop Th1 responses to self-antigens.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This cellular aberrancy in IL-12p40 gene expression may well be
involved in promotion of a pathogenic, Th1-mediated, autoimmune
response in these organ-specific diseases. Although the
elevated-expression defect in the p40 gene in SJL and NOD mice leads to
elevated production of the biologically active p70 heterodimer,
elevated levels of free p40 are also apparent, which may have a
negative impact on the biological activity of p70. It should be noted
that 5–20% of secreted, free p40 are in the homodimer
(p40)2 form [15
], which is known to display
antagonist activity toward IL-12p70 by binding the IL-12 receptor ß1
subunit (reviewed in ref. 15
). Although the antagonist activity of
elevated (p40)2 production could be substantial in the SJL
mouse, the following observations support the contention that elevated
expression of the p40 gene does not lead to a dominant antagonist
activity under physiological conditions but rather promotes the
pathogenic Th1 response in these mice. 1) As has been observed by
others [15
, 23
], activated M from normal
strains produce 100- to 300-fold more free p40 than p70 (see Fig. 2
),
suggesting that a large excess of free p40 does not interfere with Th1
cell development under normal physiological conditions. 2) Although
IFN- is known to induce biologically active p70 to potentiate Th1
cell development (reviewed in ref. 15
), this cytokine induced greater
p40 gene expression relative to p35 gene expression in normal and
autoimmune strains (see Fig. 5A
) and did not appear to change the ratio
of free p40 to p70 production substantially in any strain (compare Fig. 2B
with C). 3) Free p40 itself may play a potent role in the induction
and propagation of Th1-mediated EAE because p35 gene-deficient C57BL/6
mice, which cannot generate p70, still acquire EAE induced with
injection of MOG(35–55) in CFA [27
].
Surprisingly, MOG(35–55)-specific Th1 cell development and
disease were blocked in these p35-deficient mice by administration of
neutralizing anti-IL-12p40 mAb, suggesting that free p40 plays a role
in the initiation of disease and in pathogenic Th1 cell development.
Indeed, free p40 homodimers stimulate IFN- production in CD8+
T-cells [28
], which may shed light on how this IL-12
complex plays a dual role in EAE. 4) MS patients have a strikingly
similar cytokine-expression phenotype to that of SJL mice; relative to
normal individuals, patients with chronic progressive MS show increased
expression of the p40 but not the p35 gene in peripheral blood
mononuclear cells [29
]. In addition, analysis of
cerebral spinal fluid from patients with chronic progressive MS showed
an elevation in production of free p40 but not in p70, and this was
associated with a Th1 cytokine profile (i.e., IFN- production)
[30
]. In addition, TNF- and IL-1 levels were not
elevated in these patient samples [30
], a pattern also
consistent with that observed in SJL mice (see Table 1
and Fig. 1
). In
conclusion, it is clear that the SJL strain possesses a genetically
programmed defect in the p40 gene, which leads to substantially
elevated production of p70 and free p40, both of which appear to play a
role in promoting EAE and perhaps MS under physiological conditions.
The genetically programmed, elevated expression of M-derived
IL-12 in the SJL mouse is consistent with the known functions and
expression of IL-12 during the course of EAE [15
,
17
] and appears to explain differences in development of
neuroantigen-specific T-cell phenotypes (i.e., Th1 vs. Th2) between the
SJL and its MHC-congenic, disease-resistant strain, B10.S. Although MHC
haplotypes are associated with EAE susceptibility, it appears that the
predominant role of these haplotypes is to bind and present
immunodominant epitopes of neuroantigens to potentially pathogenic
T-cells (reviewed in [31
]). However, without the
disease-associated non-MHC genes, as is the case with the B10.S and
other disease-resistant strains, these neuroantigen-specific T-cell
responses develop into a protective Th2 phenotype [14
].
The non-MHC genetic contribution to disease in the SJL appears, in
part, to include elevated expression of p40 because B10.S mice have low
p40 expression in vivo during their immune response to
neuroantigens and because ex vivo IL-12 treatment of
B10.S-derived neuroantigen (i.e., MBP)-specific T-cells induced
development of a Th1 phenotype and pathogenicity when adoptively
transferred into naïve hosts [13
]. Because this
elevated expression of the p40 gene in SJL M was induced by LPS or
CD40L in the absence of the Th1 cell-derived cytokine, IFN-, the
abnormally elevated IL-12 levels produced by SJL APCs would be
stimulated by CD40L during naïve autoreactive, T-cell
activation before substantial IFN- levels would be produced.
Therefore, these overpowering and prematurely elevated IL-12 levels in
the SJL would prevent development of a disease-resistant, Th2 phenotype
of neuroantigen-specific T-cells that naturally develops in the B10.S
mouse [14
]. Indeed, activated T-cell-derived CD40L is a
major inducer of APC p40 production, leading to a Th1 response
[22
], which is a major pathway for elevated production
of IL-12p40 in MS patients [32
]. Our results suggest
that a greater intrinsic capacity of the M/APC to produce IL-12
during antigen-presentation to naïve autoreactive T-cells is
characteristic of EAE susceptibility and sheds light on the genetic
predisposition of the SJL for other Th1-mediated autoimmune diseases
such as Theiler’s virus-mediated encephalitis [26
].
The fact that two apparently different strains of organ-specific autoimmunity, the SJL and NOD mice, share an almost identical defect in intrinsic p40 gene expression strengthens the functional significance of this observation. These two strains may have originated from a common murine stock, providing not only genes that cause an overexpression of p40 but also a unique series of mutations in the IL-2 gene [33 ] whose functional significance remains to be determined. It is interesting to note that the NOD mouse is also a strain highly susceptible to EAE upon administration of MOG(35–55) peptide, which requires the MHC IAg7 haplotype [14 ]. Similar to the relationship between the SJL and B10.S strains, an EAE-resistant mouse (i.e., III) congenic for IAg7 lodges a Th2 immune response to MOG(35–55) without showing any signs of disease, demonstrating that non-MHC genes are necessary for EAE in the NOD mouse [14 ].
Although the genetic basis for the p40 defect in the SJL is not known,
the p40 gene itself may harbor the underlying mutation responsible for
its elevated expression because location of the gene on chromosome 11
maps close to the disease-associated locus, Eae6b, in the
SJL mouse [34
] and a similar locus, Idd4, in
the NOD mouse [22
]. However, Teuscher et al.
[34
] have determined that the IL-12p40 mRNA sequence in
the SJL and B10.s strains is identical, thus ruling out a structural
abnormality in the IL-12p40 protein expressed in SJL mice but leaves
the possibility of mutations in noncoding regions that affect gene
expression. Therefore, our initial attempts to identify a mutation in
the p40 gene of SJL and NOD mice entailed sequencing of its promoter
region. Although we did not find any sequence polymorphisms unique to
SJL and NOD mice in the 800-bp sequence upstream from the transcription
start site when compared with at least eight normal murine strains
(unpublished results), mutations could occur in other regions known to
regulate the expression of this gene. Alternatively, mutations in genes
encoding transcription factors that preferentially regulate the
IL-12p40 gene but not those of TNF-, IL-1, IL-6, IL-10, or TGF-ß
are also candidate genes underlying this cellular aberrancy. Indeed,
our finding of a common, aberrant phenotype between SJL and NOD mice is
consistent with the common sequence polymorphism in the IL-2 gene
previously identified in these strains [33
] and supports
that these two strains arose from a common ancestral stock.
The balance of Th1/Th2 responses has been recognized as a critical
factor in the development of immune pathology associated with models of
autoimmunity that occur spontaneously or that are experimentally
induced and with ones that are organ-specific (i.e., SJL and NOD;
reviewed in [15
]) or systemic (i.e., lupus-prone mice,
MRL and NZB/W F1) [35
] in nature. By performing a
rigorous analysis of genetically programmed M cytokine production
profiles in organ-specific [18
] and systemic
[19
] autoimmune-prone murine strains, we have identified
that defective IL-12p40 production appears predictive of the Th
response associated with the course of organ-specific or systemic
autoimmunity. The elevated IL-12 levels readily produced by SJL and NOD
M may contribute to the dominant and pathogenic Th1 response that
mediates EAE and IDDM, respectively, whereas reduced IL-12
levels noted in M from systemic, autoimmune-prone NZB/W and MRL mice
[19
] are consistent with the Th2-mediated, B-cell
responses characteristic of lupus. Moreover, we have found a reduction
in the ability of M from BALB/c mice to produce p40 levels, a strain
that is unable to lodge a protective Th1 response to infection by
Leishmania major [19
]. Note that the
"normal range" of genetically programmed cytokine levels was
defined by the relatively large range displayed by a panel of eight
normal strains used in most of our studies, which constitutes a
rigorous criterion for defining aberrant expression in the
autoimmune-prone strains. Using this criterion, we have also identified
a substantial aberrancy (i.e., reduction) in TNF- production among
the lupus-prone strains and one that is relatively modestly expressed
in the NOD strain [18
]. Nevertheless, the association of
intrinsic defects in M IL-12p40 production with polarized,
pathogenic Th responses in different disease-prone murine strains is
currently the most apparent genetically programmed cellular phenotype
that characterizes these strains. These observed aberrancies in
IL-12p40 gene expression should provide insight into the cellular basis
of immune deviation and may contribute to identification of specific
genetic elements contributing to autoimmune disease.
|
ACKNOWLEDGEMENTS |
|---|
Received August 15, 2000; revised November 13, 2000; accepted November 16, 2000.
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