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* Section of Rheumatology, Department of Medicine,
Section of Immunobiology, and Departments of
Pathology and
Dermatology, Yale School of Medicine, New Haven, CT 06520; and
¶ Renal, Electrolyte, and Hypertension Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production by both
CD4+ and CD8+ cells and increased serum
concentration of IgG2a anti-dsDNA autoantibodies. The protective
effect of IL-10 in this lupus model was further supported by the
observation that administration of rIL-10 reduced IgG2a anti-dsDNA
autoantibody production in wild-type
MRL-Faslpr animals. In summary, our
results provide evidence that IL-10 can down-modulate murine lupus
through inhibition of pathogenic Th1 cytokine responses. Modulation of
the level of IL-10 may be of potential therapeutic benefit for human
lupus. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells are required for full
penetrance of disease in MRL and other murine models of lupus, largely
via their help to autoreactive B cells (2, 3, 4).
A predominant Th1 cytokine response is associated with the pathogenesis
of lupus in both humans and mice. Increased IFN- mRNA and decreased
IL-4 gene expression are found in PBMCs taken from SLE patients
(5). Th1 cytokines, including IFN-, are present in
tissue of SLE patients with the most severe disease, with enhanced
production of this cytokine by peripheral blood cells and in the
kidneys of patients with severe lupus glomerulonephritis, compared with
individuals with milder renal disease (6, 7). Likewise, in
mice with lupus, Th1 cytokines play a dominant role in disease
pathogenesis (8). For example, disease severity in
MRL-Faslpr mice is linked to the presence
of the Th1 cytokines IFN- (9, 10, 11, 12) and IL-12
(11). IFN- gene deficiency (9, 12) or
IFN-R deletion (10) in these animals dramatically
reduces glomerulonephritis, apparently through reduced production of
IgG2a (an Ig isotype associated with the Th1 phenotype) anti-dsDNA
Abs. Similarly, IFN-R deletion prevents autoantibody production and
glomerulonephritis in lupus-prone (NZB x
NZW)F1 mice (13). Transgenic
expression of IFN- in the epidermis of nonautoimmune mice leads to
the development of inflammatory skin disease resembling lupus
erythematosus, with 30% of female mice also developing severe immune
complex-mediated glomerulonephritis (14, 15). Expression
of an IFN-R Fc fusion protein by i.m. injection of a plasmid
containing its cDNA can retard lupus development and disease
progression in MRL-Faslpr mice
(16). Consistent with these observations is the finding
that retroviral-induced IL-12 expression in the kidney elicits
autoimmune injury by fostering the accumulation of IFN--secreting T
cells within this organ (11).
IL-10 is a regulatory cytokine that inhibits Th1 cytokine production
and proliferation of CD4+ T cells via its
indirect effects on APC function or through direct effects on T cells
(17). This cytokine has been presumed to be an important
modulator of disease activity in human SLE. Patients with lupus produce
large amounts of IL-10 (18), with enhanced gene expression
in PBMCs (5), and its serum level correlates with disease
activity (19, 20, 21). While the precise role of IL-10 in the
pathogenesis of lupus remains uncertain, studies have suggested that
this cytokine is pathogenic both in humans and in mice with disease.
For example, continuous administration of anti-IL-10 delays onset
of autoimmunity in (NZW x NZB)F1 mice, an
effect perhaps mediated by up-regulation of TNF- production
(22). Moreover, it has been recently reported that
anti-IL-10 administration to six human lupus patients with active
disease, and dependent upon steroids for treatment, led to a reduction
in disease activity (23).
Despite the evidence suggesting that IL-10 might promote disease in
lupus, we hypothesized that it might also play a suppressive role,
given its effects on T cell expansion and its potential to modulate
potentially pathogenic Th1 responses. To address this hypothesis, we
produced and analyzed MRL-Faslpr mice
genetically deficient in this cytokine. Compared with wild-type
MRL-Faslpr mice, animals deficient in
IL-10 developed exacerbated disease, with earlier appearance of skin
lesions, increased lymphadenopathy, more severe glomerulonephritis, and
earlier and enhanced mortality. These findings were associated with
enhanced production of the Th1 cytokine IFN- by
CD4+ T cells and by CD8+ T
cells, along with greater synthesis of potentially pathogenic IgG2a
anti-dsDNA Abs. The latter were increased in IL-10-deficient mice
early in disease, a finding mirrored by the administration of IL-10 to
wild-type MRL-Faslpr mice with resultant
suppression of these Abs, again early in disease. These findings
suggest that IL-10 regulates murine lupus, particularly early in the
disease course, and that its effect is at least in part mediated by
suppressing pathogenic Th1 responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B6.129.P2-Il10tm1Cgn (C57BL/6-Il10tm1Cgn, B6 IL-10-/-) mice obtained from The Jackson Laboratory (Bar Harbor, ME) were backcrossed to the MRL-Faslpr background for 10 generations (N10; >99.9% MRL background genes) to obtain MRL-Faslpr IL-10+/- mice, which were then intercrossed to generate three groups of animals: IL-10 intact (IL-10+/+), IL-10 heterozygous (IL-10+/-), and IL-10 deficient (IL-10-/-). Genotypes were determined by PCR screening of tail DNA according to a protocol from The Jackson Laboratory and were confirmed at time of sacrifice: LPS-stimulated splenocytes lacked IL-10 synthesis as determined by intracellular staining after brefeldin A blockade (see Intracellular cytokine staining). B6 IL-10-/- mice were also crossed twice with B6.MRL-Tnfrsf6lpr (B6.MRL-Faslpr, B6-Faslpr; The Jackson Laboratory) to obtain control B6-Faslpr IL-10-/- mice. All animals were maintained under specific pathogen-free conditions at the Yale University School of Medicine (New Haven, CT).
IL-10 treatment of MRL-Faslpr mice
Wild-type MRL-Faslpr mice (6 wk old) were administered rIL-10 (a kind gift from Schering-Plough Research Institute, Kenilworth, NJ) in PBS (200 ng/mouse/day for 6 days, n = 20 mice) via i.p. injection, or, as a control, were given PBS alone (n = 12 mice). Mice were bled by retroorbital puncture under anesthesia before the initiation of IL-10 or control injections, and again at ages of 10 and 14 wk, and just before the mice were sacrificed at age 17 wk. Sera were stored at -20°C until assays were performed for autoantibodies and their isotypes as described below.
ELISA for autoantibodies and their isotypes
Serum anti-dsDNA IgG Abs were measured by ELISA using S1 nuclease-treated DNA linked to plastic wells by methylated BSA as described previously (9, 24). Simply, methylated BSA in carbonate buffer (pH = 9.6) was coated on ELISA plates (Costar, Cambridge, MA) at room temperature, followed by overnight coating with dsDNA at 4°C. Mouse sera were diluted 1/100 in 3% FBS in PBS and incubated at 37°C for 2 h, with the bound IgG detected with HRP-conjugated anti-mouse IgG (Sigma-Aldrich, St. Louis, MO). After adding the tetramethylbenzidine peroxidase substrate and sequential stop solution (Kirkegaard & Perry Laboratories, Gaithersburg, MD), the plates were read in an ELISA reader at wavelength of 450 nm. For analysis of anti-dsDNA IgG isotypes, after incubating the mouse serum as above, biotin-labeled anti-mouse IgG1, IgG2a, IgG2b, and IgG3 (BD PharMingen, San Diego, CA) were added at a dilution of 1/2000 to the plates at 37°C for 1 h, followed with streptavidin-HRP. Plates were then developed and measured by using tetramethylbenzidine substrate as above. A positive serum sample from a 5-mo-old wild-type MRL-Faslpr mouse was used as an internal control in all assays. The reaction was stopped according to the OD value of the standard serum in all assays to assure day to day consistency.
Urea nitrogen measurement
Serum urea nitrogen levels were performed by the Clinical Chemistry Laboratory of Yale New Haven Hospital.
Intracellular cytokine staining
To assess T cell cytokine production, splenocytes (2 x
106/ml) were activated with anti-CD3 and
anti-CD28 in the presence of brefeldin A for 6 h, and cells
were washed, fixed, permeabilized, and stained with
FITC-anti-IFN- and PE-anti-IL-4 or PE-anti-IL-10 (see
genotypic analysis of cohorts above) as described
(25). Meanwhile, cells were stained with
CyChrome-anti-CD4 or anti-CD8 and biotin-B220 followed with
avidin-allophycocyanin. After gating on
CD4+B220- or
CD8+B220- cells (Fig. 6
B), data were displayed as dot plot of FITC
(x-axis) and PE (y-axis) fluorescence (log
scale). Quadrant markers were positioned to include >99% of control
Ig-stained cells in the lower left quadrant (data not
shown).
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Spleen and cervical lymph nodes (LN) were weighed, homogenized,
and cleared of erythrocytes by Red Blood Cell Lysing Buffer
(Sigma-Aldrich). Phenotypic analysis was performed by using four-color
FACS analysis (FACSCalibur; BD Biosciences, San Jose, CA) using the
following Abs: biotinylated anti-mouse CD45R/B220 (RA3-6B2, rat
IgG2a) followed by avidin-allophycocyanin, FITC anti-mouse CD3
(145-2C11, hamster IgG), PE or CyChrome anti-mouse CD4 (H129.19,
rat IgG2a), and CyChrome anti-mouse CD8 (53-6.7, rat IgG2a), all
purchased from BD PharMingen. Cells were labeled with FITC
anti-CD3, PE anti-CD4, CyChrome anti-CD8, and biotin-B220
followed by avidin-allophycocyanin. After gating on
CD3+B220+ or
CD3+B220- cells,
CD4+ and CD8+ cells were
then analyzed.
Histopathology
Renal disease and skin lesions were assessed by light microscopy as described (9, 24). Briefly, formalin-fixed tissues (kidney or skin) were stained with H&E. Individual kidney specimens were read blindly by Dr. M. Madaio (University of Pennsylvania School of Medicine, Philadelphia, PA) and scored on a scale of 0 to >4+ as described in detail previously (26, 27, 28). Skin samples were read blindly by Dr. J. McNiff (Department of Dermatology, Yale University).
Statistics
Statistical significance was evaluated by two-tailed, unpaired Student’s t test or nonparametric analysis if SD were significantly different between the two compared groups, using software InStat 2.03 for Macintosh (GraphPad, San Diego, CA). Survival curves and analysis of the penetrance of skin lesions were determined using log rank test, performed by GraphPad Prism Version 3.0a for Macintosh (GraphPad).
Throughout the text and figures, the following terminology was used to denote statistical significance: *, p < 0.0001 or p < 0.001; **, p < 0.01; ***, p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We initially asked whether the genetic absence of IL-10 affected
mortality of MRL-Faslpr mice. In this
study, three groups of animals, all housed together in the same room
and followed for up to 17 wk of age, were analyzed:
MRL-Faslpr IL-10+/+
(n = 13), MRL-Faslpr
IL-10+/- (n = 24), and
MRL-Faslpr
IL-10-/- (n = 10). Notably,
MRL-Faslpr
IL-10-/- mice demonstrated substantially
increased mortality compared with their IL-10+/+
or IL-10+/- counterparts: the survival rate for
MRL-Faslpr
IL-10-/- mice was only 50% at 17 wk of age (5
of 10 mice survived until this time point), with death beginning as
early as 12 wk, compared with a 100% survival rate in
MRL-Faslpr IL-10+/+
animals and a 90% survival rate in the
MRL-Faslpr IL-10+/-
group (Fig. 1; p <
0.0001, survival of MRL-Faslpr
IL-10-/- mice compared with either the
IL-10+/- and IL-10+/+
groups). By contrast, none of the cohort of
B6-Faslpr IL-10-/-
mice died by age 28 wk (n = 6; data not shown). These
results suggest that IL-10 plays an important role in down-modulation
of disease in MRL-Faslpr mice.
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To define the effects of IL-10 deficiency upon end-organ
autoimmune disease, and to attempt to ascertain the cause of
accelerated mortality in the MRL-Faslpr
IL-10-/- group, we evaluated kidneys at 17 wk
of age from a second cohort of 23 animals:
MRL-Faslpr IL-10+/+
(n = 6), MRL-Faslpr
IL-10+/- (n = 11), and
MRL-Faslpr
IL-10-/- (n = 6). We also
assessed renal disease in mice from the first cohort, described above
in the mortality curves, that survived to 17 wk of age:
MRL-Faslpr IL-10+/+
(n = 5, randomly selected of the 13 animals),
MRL-Faslpr IL-10+/-
(n = 11, randomly selected of the 21 survivors), and
MRL-Faslpr
IL-10-/- (n = 4). Kidney
specimens were blindly evaluated by light microscopy and scored on a
scale of 0 to >4+ as described in detail previously (26, 27). MRL-Faslpr
IL-10-/- mice consistently developed more
severe disease than IL-10+/+ animals with
significantly enhanced glomerulonephritis (Fig. 2A, upper panel,
mean score 3.7 in IL-10-/- mice vs 2.4 in
IL-10+/+ animals, p < 0.001) and
interstitial nephritis with large multifocal infiltrates (Fig. 2A, lower panel, mean score 3.35 for
IL-10-/- mice vs 1.95 for
IL-10+/+ animals, p = 0.0074; see
examples in Fig. 2B). Although
IL-10+/- mice had significantly worse
glomerulonephritis (p < 0.01) and interstitial
nephritis than wild-type mice (p < 0.05) and
less severe disease than IL-10-/- animals, the
comparison to the latter group did not reach statistical significance
(Fig. 2A). Of note, kidney abnormalities were not observed
in B6-Faslpr
IL-10-/- mice when they were sacrificed at 28
wk of age (n = 6, data not shown).
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C). Moreover, at age 17 wk, urea
nitrogen levels were significantly higher in
IL-10-/- mice than in
IL-10+/+ mice. IL-10 suppresses skin lesions in MRL-Faslpr mice
MRL-Faslpr mice typically develop
skin lesions of back, neck, and ears by age 5–6 mo (29, 30). To determine whether IL-10 is associated with the
development of the skin lesions that characterize
MRL-Faslpr mice, we also blindly analyzed
our initial cohort of 47 mice, described in the mortality assessment in
Fig. 1, for skin involvement. MRL-Faslpr
IL-10-/- mice had skin lesions at an earlier
age, compared with the IL-10+/+ or
IL-10+/- controls. By age 13 wk, 50% of
IL-10-/-
MRL-Faslpr mice developed skin lesions,
with 80% penetrance of these manifestations by 14 wk of age, in
comparison to 25% penetrance in the IL-10+/-
group and in comparison to the absence of skin lesions in
IL-10+/+ mice (Fig. 3A, p < 0.01
for IL-10-/- compared with
IL-10+/+ and IL-10+/- mice
at 13 wk of age and compared with IL-10+/- mice
at 14 wk of age, and p < 0.0001 for
IL-10-/- compared with
IL-10+/+ mice at 14 wk of age; see representative
examples in Fig. 3B). At age 16 wk, only 30% of the
IL-10+/+ cohort had skin lesions, although 60%
of these mice developed skin lesions at 17 wk of age (Fig. 3A). Overall, IL-10-/- mice had a
significantly higher penetrance of skin lesions in comparison to both
IL-10+/- and IL-10+/+
mice, and IL-10+/- had more such lesions than
the IL-10+/+ group (p <
0.05 for both comparisons, log rank test). Skin lesions were absent in
the B6-Faslpr
10-/- controls at age 28 wk (data not
shown).
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C). In both IL-10-/- and
IL-10+/- tissues, there was marked epidermal
hyperplasia, hyperkeratosis, and interface change of both epidermis and
hair follicles consisting of basal cell degeneration and necrotic
keratinocytes. There was also mononuclear infiltration in the dermis
with dermal fibrosis. These changes are characteristic of lupus
erythematosus in the skin of the mouse (30). In contrast,
histological analysis of skin of IL-10+/+ mice
was normal, with samples revealing a thin two- to three-cell layer
epidermis, delicate keratin layer, and no inflammation in the
dermis. IL-10 inhibits production of Th1-type autoantibodies
Sera from the initial, larger cohort of 47 mice at ages 6, 10, 14,
and 17 wk (terminal bleed at sacrifice) were analyzed for
anti-dsDNA IgG and IgG isotype levels. At the age of 10 wk, the
mean level of total anti-dsDNA IgG was significantly higher in sera
of IL-10-/-
MRL-Faslpr animals than in the
IL-10+/+ group (p <
0.01), with sera from the IL-10+/- mice
containing levels between the IL-10-/- and
IL-10+/+ groups (Fig. 4A). Upon further analysis of
the IgG anti-dsDNA isotypes in sera of mice of the same age (10
wk), significant differences in the levels of IgG2a and IgG2b were
found between the IL-10-/- and
IL-10+/+ animals (Fig. 4B,
p < 0.05). Levels of these isotypes in the
IL-10+/- cohort were intermediate between the
levels in IL-10-/- and
IL-10+/+ animals, although these differences were
not statistically significant. Levels of IgG1 and IgG3 anti-dsDNA
Abs in sera were not statistically different among all three groups of
mice. Notably, total IgG anti-dsDNA and anti-dsDNA IgG isotypes
also were not significantly different among the three groups of animals
at 14 and 17 wk of age (Fig. 4A and data not shown). Here,
we cannot exclude the possibility that these latter results at later
time points might be affected by death of sickest animals.
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C,
p < 0.05); however, effects on IgG2a anti-dsDNA,
or effects on other isotypes, were not observed at ages 14 or 17 wk,
again analogous to the finding in the mice genetically deficient in
IL-10 compared with controls. This observation supports the notion that
IL-10 may down-modulate the relatively early production of potentially
pathogenic IgG2a autoantibodies; however, the effects of administration
of recombinant cytokine are not prolonged. Moreover, we did not
identify any significant improvement of renal pathology in the IL-10
treatment group in comparison to control mice (data not shown). While
we do not know the reasons for this finding, it is possible that it
reflects the timing, duration, or amount of IL-10 treatment. IL-10 deficiency increases lymphadenopathy in MRL-Faslpr mice
Wild-type MRL-Faslpr mice, like other
murine models of lupus, develop Th1 cytokine deviation (8, 9, 10, 12, 13). The former mice also develop lymphadenopathy and
splenomegaly, phenotypes largely the consequence of accumulation of
so-called double negative T cells that have down-regulated their
coreceptors, primarily CD8, and up-regulated B220, with a resultant
cell surface phenotype that is
TCR+CD3+CD4-CD8-B220+
(31) compared with conventional
CD3+D4+B220- or
CD3+CD8+B220- T cells.
Thus, we next assessed the effects of IL-10 deficiency upon these T
cell phenotypes, using the mice from both cohorts that had also been
analyzed for kidney disease. First, spleen and cervical LN from each
mouse were weighed at time of sacrifice at 17 wk, and total cell
numbers counted with cell composition were analyzed by flow cytometry.
Both IL-10-/- (n = 10) and
IL-10+/- (n = 22)
MRL-Faslpr mice had higher spleen and LN
weights compared with their IL-10+/+
(n = 11) counterparts (Fig. 5A; p < 0.01
for spleen and LN weights from IL-10-/- vs
IL-10+/+ mice, and p < 0.05 for
weights from IL-10+/- vs
IL-10+/+ animals). By contrast, there was no
significant difference in weights between the
IL-10-/- and IL-10+/-
groups (p > 0.05). Notably, cell numbers per
gram of lymphoid organ were not different among the three groups (data
not shown).
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T cells from spleen was
significantly higher in IL-10-/-
(n = 10) mice than that in
IL-10+/- (n = 22) or
IL-10+/+ (n = 11) controls (Fig. 5B, lower panel, p < 0.01,
comparing phenotypes in IL-10-/- vs
IL-10+/- or IL-10+/+
animals). This increase was mirrored by an apparently compensatory
decrease in the typical double negative
TCR+CD3+CD4-CD8-B220+
T cells that accumulate in MRL-Faslpr mice
(Fig. 5B, upper panel, p <
0.01). Given that the average cell numbers per gram of spleen
(
500 x 106) were not different among
three groups, we calculated the mean number of conventional
CD3+B220- T cells as
210 x 106, 125 x
106, and 100 x 106
for IL-10-/-, IL-10+/-,
and IL-10+/+ animals, respectively. By contrast,
the mean number of double negative
CD3+B220+ cells was 30
x 106, 60 x 106, and
45 x 106 for
IL-10-/-, IL-10+/-, and
IL-10+/+ animals, respectively. However, the
percentages of conventional
CD4+B220- and
CD8+B220- T cells, of the
total CD3+ cells (Fig. 5C), as well as
CD3-B220+ traditional B
cells (data not shown), were not statistically different among the
three groups, indicating expansion of both lineages of T cells
in IL-10-deficient mice. Cellular composition of LN from
IL-10-/- mice revealed a similar pattern to
that from spleen, with increased numbers of conventional T cells and
decreased
CD4-CD8-B220+
double negative T cells (data not shown). Thus, IL-10 deficiency
apparently leads to expansion of both conventional
CD4+ and CD8+ T cells in
secondary lymphoid tissues in
MRL-Faslpr mice. IL-10 strongly down-modulates Th1 response in lupus mice
Because CD4+ T cell responses in
MRL-Faslpr mice are characterized by Th1
deviation (8, 9), with evidence that this response is
pathogenic (8, 9, 10, 11, 12), and because IL-10 has been
demonstrated to inhibit Th1 cytokine responses (17), we
hypothesized that IL-10 may down-modulate autoantibody production and
end-organ disease in lupus via inhibition of Th1 cytokine production.
To test the effect of IL-10 upon the pattern of cytokine responses in
MRL-Faslpr mice, cells from spleen and LN
of the second cohort of 23 animals were analyzed for intracellular
IFN- and IL-4 production as described in Materials and
Methods. Both CD4+ and
CD8+ T cells from wild-type
MRL-Faslpr mice produced more IFN- than
IL-4 (data not shown), consistent with previous findings
(8). In IL-10-/- mice, splenic and
LN CD4+ and CD8+ cells had
strongly enhanced IFN- production: the percentage of
cytokine-secreting cells in spleens and LN of
IL-10-/- mice (n = 6) was 5-
and 20-fold higher for CD4+ T cells and 2- and
24-fold higher for CD8+ T cells, respectively, in
comparison to cells from IL-10+/+
(n = 6) and IL-10+/-
(n = 11) mice (Fig. 6A, p <
0.001; see representative example in Fig. 6B). Moreover, the
percentage of CD4+IFN-+
cells from spleens and LN of IL-10+/- mice was
significantly greater than that from IL-10+/+
animals (Fig. 6, A and B, p <
0.01), although the percentage of
CD8+IFN-+ T cells was
similar between these two groups.
By contrast, IL-4 levels were significantly lower in
IL-10-/- mice in comparison to
IL-10+/+ and IL-10+/- mice
(Fig. 6B). It is unclear at present whether IL-10 has a
direct effect on IL-4 production in this model or an indirect effect
via enhancement of IFN- production.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To elucidate more precisely the role of IL-10 in the pathogenesis of
murine lupus, IL-10 gene-deficient (IL-10-/-)
MRL-Faslpr mice were generated and their
phenotype was analyzed in comparison to IL-10+/-
and IL-10+/+ littermates from the same colony.
Backcrosses to the MRL background were done for 10 generations, so that
MRL background genes were >99.91%. The most striking finding from
this experiment was that deletion of IL-10 resulted in an exacerbated
disease phenotype, with increased mortality (Fig. 1), more severe renal
disease (Fig. 2), and skin lesions (Fig. 3), and augmented autoantibody
production early in the disease course, particularly potentially
pathogenic IgG2a anti-dsDNA Abs (Fig. 4). However, we did not
notice significant differences in disease penetrance between female and
male mice in our two cohorts.
What is the linkage between IL-10 deficiency and the severe lupus
phenotype? The logical explanation resulting from our studies is that
IL-10 suppressed murine lupus through inhibition of pathogenic IFN-
production (Fig. 6), especially at the early stages of disease
development. Studies using both IFN-- and IFN-R-deficient
lupus-prone mice have revealed a pathogenic role for IFN- in lupus,
especially in its initiation (9, 10, 12, 13). However,
IFN- seems not to be required for later stages of disease, a notion
supported by the failure of anti-IFN- Ab administration to
ameliorate disease in 12-wk-old MRL-Faslpr
mice (34). Our results are consistent with such a notion.
IL-10 deficiency enhanced IFN- production in the CD4 and CD8
lineages (Fig. 6), and that, in turn, was associated with increased
production of IgG2a anti-dsDNA Abs, an IFN--related pathogenic
isotype (Fig. 4). Notably, the effect on autoantibody production was
only significantly different in IL-10-deficient animals at early time
points (9–10 wk of age), but not at later stages of disease. The early
development of skin lesions and increased early mortality further
support the role of IL-10 in suppressing murine lupus at an early
stage, presumably effects mediated in part through inhibition of
IFN- production. Administration of rIL-10 reduced IgG2a
anti-dsDNA Ab production, again early in disease, supporting the
role of a specific effect of IL-10 in disease amelioration.
We would add that IL-10 deficiency only exacerbated lupus phenotypes in the MRL background that contains the genes necessary for full disease penetrance, because there was no apparent consequence of its loss in Fas deficiency in the B6 background, with B6-Faslpr IL-10-/- mice lacking features of lupus end-organ disease. The finding that MRL-Faslpr IL-10+/- mice had a more severe phenotype than MRL-Faslpr IL-10+/+ mice, in terms of kidney pathology, skin lesions, and lymphadenopathy, also intrigued us, suggesting that the number of competent alleles may determine IL-10 expression level in vivo. To our knowledge, a clinical effect of heterozygosity at the IL-10 locus has not been previously reported, raising the possibility that the phenotypes seen in our IL-10+/- mice, in comparison to IL-10+/+ controls, became manifest only under the conditions imposed by the disease-promoting genes in the MRL-Faslpr background. This issue is the subject of ongoing studies. Notably, we did not observe the development of clinical enterocolitis in our MRL-Faslpr IL-10-/- colony that has been previously reported in intrinsically nonautoimmune IL-10-deficient mice (35). This observation deserves further study to determine the role, if any, of the MRL background genes and/or Fas deficiency in protection against the enterocolitis of IL-10 deficiency.
How can the conflicts between our results and the current notion that
IL-10 has a disease-promoting effect in animal models and patients with
lupus be reconciled? For instance, earlier work using continuous
administration of anti-IL-10 demonstrated a delay in onset of
autoimmunity in NZB/W F1 mice (22).
In a more recent report, anti-IL-10 mAb administered to six
steroid-dependent patients with SLE was shown to have a beneficial
effect on disease activity (23). These studies, combined
with the observations that levels of IL-10 in serum are
characteristically elevated in patients with active lupus compared with
controls (19, 20, 21), suggest that this cytokine may promote
disease. Yet, IL-10 can be secreted by many cell types, with multiple
biological effects (17). For example, IL-10 can inhibit
IFN- production and inflammatory responses (36) as well
as promote B cell proliferation and Ab production (19).
Accordingly, IL-10 may have multiple effects in lupus depending on the
stage of the disease. For example, IFN- and pathogenic autoantibody
isotypes (e.g., IgG2a) are necessary for full disease penetrance
(9, 11, 12, 13), and blocking Th1 cytokine responses may
down-modulate lupus when applied in the setting of early, perhaps
preclinical, disease. Our results using gene-deficient mice to remove
endogenous IL-10 clearly exacerbated disease, particularly in young
mice, an effect mirrored with IL-10 administration, supporting the
hypothesis that IL-10 plays a down-regulatory role at an early stage of
development of lupus. Therefore, one has to consider the dual effects
of IL-10 for clinical application of either IL-10 or anti-IL-10 to
treat lupus patients. As a parallel, IL-12 has also been demonstrated
to have dual effects in collagen-induced arthritis, with a stimulatory
role in early arthritis expression and a suppressive role in the
established phase of disease (37).
Further support for the regulatory role of IL-10 in lupus comes from the finding that this cytokine promotes activation-induced death of T cells from patients with SLE, a process mediated by Fas ligand (38). Notably, our MRL-Faslpr mice that were IL-10 deficient had expansion of conventional CD3+CD4+B220- and CD3+CD8+B220- T cells, also suggesting a regulatory role of this cytokine in controlling the number of conventional T cells, including those that promote autoantibody production and renal disease. Indeed, recent studies with B cell-deficient or autoantibody-deficient MRL-Faslpr mice (39, 40) have suggested that disease can occur in the absence of B cells and autoantibodies; i.e., T cells presumably can promote disease independently of B cell help. However, the finding that MRL-Faslpr IL-10-/- mice had T cell expansion suggests that IL-10 can regulate T cell expansion apart from Fas-Fas ligand interactions. Further studies are needed to determine the mechanisms of such T cell expansion, including the role of IL-10 in controlling the CD4-CD8-B220+ T cells that characteristically predominate in cytokine-intact lpr mice.
Our results suggest the following roles for IL-10 in murine
lupus, and perhaps in patients with SLE: during the initial phases of
disease, IFN- and its induced IgG2a promote autoimmunity. IL-10
appears to be needed to suppress such pathogenic Th1 responses,
including IFN--mediated autoantibody production and renal
inflammation. By contrast, at later phases of disease, excessive
amounts of IL-10 production may lead to enhanced autoantibody
production and subsequent formation of pathogenic autoantibody-Ag
complexes. Therefore, anti-IL-10 Ab plus immunosuppression may have
a beneficial effect on disease outcome when supplied later in disease.
Further studies are needed to test this assumption by using staged
IL-10 therapy in murine models of disease.
In conclusion, we demonstrate that IL-10 plays a critical role in
down-modulating murine lupus through inhibition of IFN- production.
These results have significant implications for the pathogenesis and
treatment of human SLE.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Joe Craft, Yale School of Medicine, Box 208031, 610 Laboratory of Clinical Investigation, 333 Cedar Street, New Haven, CT 06520-8031. E-mail address: joseph.craft{at}yale.edu
3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; LN, lymph node.
Received for publication April 5, 2002. Accepted for publication June 11, 2002.
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