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Section for Medical Inflammation Research, University of Lund, Lund, Sweden
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-2, and IL-4 after mitogen
stimulation; however, macrophages showed a reduced TNF-
production
compared with IL-10+/- littermates. IL-10-/-
mice had an increased incidence, and a more severe CIA disease than the
IL-10+/- littermates. To study the role of IL-10 in T cell
tolerance, IL-10-/- were crossed into mice carrying the
immunodominant epitope, CII(256–270), in cartilage (MMC) or in skin
(TSC). Both IL-10-/- and IL-10+/- MMC and
TSC mice were completely tolerized against CIA, indicating that lack of
IL-10 in this context did not break tolerance. To investigate whether
IL-10 was important in the effector phase of CIA, arthritis was induced
with anti-CII Abs. Surprisingly, IL-10-/- were less
susceptible to Ab-transferred arthritis, as only 30% showed signs of
disease compared with 90% of the littermates. Therefore, IL-10 seemed
to have a protective role in CIA, but seemed to exacerbate the
arthritogenicity of anti-CII Abs. These data emphasize the
importance of studying IL-10 in a defined genetic context in vivo, to
understand its role in a complex disease like
arthritis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The IL-10 production by peripheral blood cells is generally higher in patients with autoimmune diseases such as RA, Sjögren’s syndrome, and systemic lupus erythematosus when compared with healthy controls (4). Further to this, IL-10 is also abundantly expressed in the rheumatoid synovium (5), and it has been demonstrated that T cell clones from RA synovium produce higher levels of IL-10 than T cell clones derived from peripheral blood (6).
IL-10 was originally described as a cytokine synthesis-inhibitory
factor because of its capacity to inhibit cytokine production by mouse
Th1 cells (7). Today, IL-10 is regarded as a complex
pleiotrope cytokine that is produced by Th2 cells, B cells, and
macrophages (8) and exerts both suppressive and
stimulatory effects. Suppressive effects of IL-10 include reducing
proinflammatory cytokine production in Th1 cells (7) and
macrophages, as well as down-regulating MHC II on macrophages
(9). In contrast to these suppressive effects on
macrophages, IL-10 can stimulate other macrophage functions, including
up-regulation of FcR1, which correlated with an enhanced
Ab-dependent cell-mediated cytotoxicity (10). IL-10 has
also been shown to increase the cytotoxic capacity of
CD8+ T cells (11) and up-regulate
MHC II on B cells, and is a viability factor for B cells
(12).
IL-10 is thought to have a regulatory function in RA, as neutralizing
mAbs (in vitro) led to a marked increase in the disease-associated
cytokines IL-1
and TNF- as well as up-regulation of HLA-DR,
whereas exogenous IL-10 decreased the synthesis of these molecules
(5, 13).
The protective role of IL-10 in CIA has been suggested previously, based on systemic treatment with IL-10 (14, 15, 16) and adenovirus-mediated transfer of viral IL-10 (17, 18). The immunological effect mediated by IL-10 in these systems is still being debated as multiplex and maybe contradictory results have been reported. Some authors report that IL-10 exerts a systemic effect by affecting the anti-collagen type II (CII) IgG1:IgG2a ratio (17) due to B cell stimulation, whereas others detected a decreased T cell proliferation toward CII (18). Administration of IL-10, through gene therapy or injection of recombinant protein, has been effective, however, only prophylactically or given around the time of onset (14, 15, 16, 17, 18). Therapeutic treatments have only been achieved with an extremely high dose (14) or in combination with corticosteroids (19). Neutralization of IL-10 with Abs has also been reported and led to an increased severity and an accelerated onset of CIA in DBA/1 mice, but only if given before arthritis onset (20). Treatment with neutralizing Abs could, however, have unpredictable effects on the function of the targeted cytokine, and may in addition evoke an immunological response that could affect the outcome of arthritis.
An alternative way to address the role of IL-10 in vivo is to use IL-10-deficient mice; however, these mice have been reported to spontaneously develop colitis if kept in an environment with potentially pathogenic microorganisms and with a possible contribution of 129-derived genes (21).
In the present study, we have limited these problems by using IL-10-deficient C57BL/10.Q (B10.Q) mice that spontaneously developed a less severe colitis, and when given antibiotics from birth appear healthy. The mice were genotyped and carried only an 8-cM linked fragment derived from the 129 strain. These IL-10-deficient mice and their heterozygous littermates were used to investigate the role of IL-10 in CIA and Ab-transferred arthritis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The IL-10-deficient mice, generously provided by W. Müller (Institute of Genetics, Cologne, Germany), were upon arrival on a mixed C57BL/6/129/Ola background, and therefore were backcrossed for nine generations to the C57BL/10.Q (B10.Q) strain (originally from J. Klein, University of Tübingenn, Tübingenn, Germany). The 129/Ola fragment around the IL-10 gene on chromosome 1 in the congenic B10.Q ranged from D1 Mit187 at 62 cM to D1 Mit312 at 70 cM (data not shown). All mice were kept and used in the animal facility of Medical Inflammation Research under similar conditions. The mice were kept in a climate-controlled environment with 12-h light/dark cycles, housed in polystyrene cages containing wood shavings, and fed standard rodent chow and water ad libitum in a specific pathogen-free environment (as defined in http://net.inflam.lu.se). All mice were given trimethoprim and sulfonamide (Borgal vet, Hoechst, Munich, Germany) in the drinking water throughout their entire lives to prevent them from developing enterocolitis. The MMC and TSC mice, on a C3H background (22), were backcrossed for eight generations to the B10.Q strain. The MMC and TSC strains were then further crossed with the IL-10-deficient B10.Q mice. Genomic DNA was prepared from the tip of the tail. All transgenic mice were screened using PCR technique with specific primers. The PCR products were visualized by agarose gel electrophoresis.
Genotyping
Microsatellite markers were purchased from Interactiva Biotechnology (Ulm, Germany). The order of the markers was based on the map available from The Jackson Laboratory (Bar Harbor, ME) (www.jax.org). The IL-10-/- mice were investigated with the following markers: D1Mit301, D1Mcg101, D1Mit180, D1Mit23, D1Mit132, D1Mit331, D1Mit215, D1Mit253, D1Mit216, D1Mit48, D1Mit10, D1Mit157, D1Mit136, D1Mit187, D1Mit217, D1Mit89, D1Mit218, D1Mit262, D10Mit286, D1Mit344, D1Mit193, D1Mit312, D1Mit445, D1Mit446, D1Mit194, D1Mit264, D1Mit288, D1Mit141, D1Mit101, D1Mit102, D1Mit346, D1Mit449, and D1Mit348. All markers were assayed by PCR, as follows: Genomic DNA (15 ng) was amplified in a final volume of 9 µl containing dNTP (200 µM), MgCl2 (1.5 mM), primer (1.5 pmol, of each), and Taq GOLD DNA polymerase (0.2 U) (Perkin Elmer Biosystems, Norwalk, CT). The forward primer was labeled with a fluorescent dye. Amplification conditions were as follows: 95°C for 1.1 min, followed by 33 cycles of 95°C for 30 s, 55°C for 75 s, 72°C for 75 s, and a final extension at 72°C for 7 min. The reactions were performed using an ABI 877 Integrated Thermal cycler (Applied Biosystems, Foster City, CA), followed by an automatic pooling of the products. The PCR products were size fractionated on 4% polyacrylamide gels, on an ABI 377 sequencer, and the size of the fragments was determined using the Genescan software version 3.1 (Applied Biosystems) with TAMRA GS-350 or 500 as internal size standard.
Histology
Tissue samples for light microscopy were processed according to standard procedures. Briefly, the tissue samples were fixed in 4% paraformaldehyde for 24 h, dehydrated and embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin and erythrosin. All sections were evaluated blindly.
Tissue preparation and in vitro activation
Lymph nodes (cervical, axillary, mesenteric, paraaortic, and inguinal lymph nodes) were suspended in DMEM, HEPES, 2-ME, penicillin, streptomycin, glutamine, and 10% FCS (cell medium). A total of 1.5 x 106 cells/ml was cultured in cell medium with either the superantigen Staphylococcus aureus enterotoxin A (SEA, 175 ng/ml; kindly provided by A. Sundstedt at Pharmacia, Lund, Sweden) or the lectin Con A (5 µg/ml; Pharmacia, Uppsala, Sweden) for 2–6 days in 5% CO2 at 37°C. The cells were restimulated with 50 ng/ml PMA and 1 µg/ml ionomycin for 6 h in the presence of 3 µM of the protein transport inhibitor monensin (all from ICN Pharmaceuticals, Costa Mesa, CA).
Antibodies
For staining of surface epitopes, the following mAbs were used:
anti-CD4 (H129.19) and anti-CD8 (53.6.7), purchased from BD
PharMingen (San Diego, CA); anti-B220 (RA3-6B2), anti-CD4
(CT-CD4), and anti-CD8 (CT-CD8), all three from Caltag Laboratories
(Burlingame, CA); anti-FcRII/III (2.4.G2.) and anti-CD3
(145-2C11), purified from culture supernatants by affinity
chromatography on protein G-Sepharose and conjugated. For intracellular
cytokine detection, the following mAbs were used: anti-IFN-
(AN18 and R4-6A2) and anti-IL-2 (S4B6), all three purified and
conjugated; anti-IL-4 (11B11) and anti-IL-10 (JES5-16E3), both
purchased from BD PharMingen; FITC and PE conjugate mAbs, with the same
isotype as the anti-cytokine Abs, used as negative controls.
Intracellular immunofluorescence staining of cytokines
Cells were washed and resuspended in staining buffer (SB)
containing 0.5% BSA (Sigma, St. Louis, MO) and 0.01%
NaN3 in PBS. A total of 1–2 x
106 was stained for various surface epitopes for
20 min at 4°C. An anti-FcRII/III Ab was used to inhibit Ab
binding to the FcR. The cells were fixated in 1%
paraformaldehyde/PBS overnight at 4°C. For permeation and to inhibit
unspecific Ab binding, the cells were incubated with 5% normal rat
serum/1% saponin (Sigma, St. Louis, MO) in SB for 30 min at 4°C. The
cells were washed with an intracellular staining buffer (ISB)
containing 0.025% digitonin (Sigma), 1% saponin, 2% BSA, 0.01%
NaN3, and PBS before intracellular staining.
Intracellular staining was performed in ISB at 4°C for 20 min; the
cells were then washed twice with ISB. Finally, the cells were washed
with SB and resuspended in PBS for flow cytometry analysis.
A typical forward and side scatter gate for activated lymphocytes was set to exclude dead cells and aggregates. A total of 104 events from the lymphocyte gate was collected and analyzed using a FACSort (Becton Dickinson, San Jose, CA) and Becton Dickinson software. Quadrant and histogram statistics were placed on the basis of the staining of the negative controls. Less than 0.5% positively stained cells was not regarded as significant.
Macrophage assays
Peritoneal macrophages were recovered by peritoneal lavage. The
cells were washed in DMEM and allowed to adhere to the plastics for
1 h, and the nonadherent cells were subsequently washed off. The
cells were put in the incubator overnight and washed once before
stimulation with LPS (10 ng/ml) or LPS plus IFN- (100 U/ml) for
24–48 h. To the control wells, only medium was added. Supernatants for
TNF- ELISA were recovered after 24 h. TNF- was measured
using the dissociation-enhanced lanthanide fluoroimmunoassay (Delfia)
system based on the time-resolved fluoroimmunoassay technique with
europium-labeled streptavidin (Wallac, Turku, Finland). Plates
(Fluoronunc; Nunc Nalgene International, Roskilde, Denmark) were coated
with 6 µg/ml capture anti-TNF- mAb (G281-2626; BD PharMingen).
A biotinylated anti-TNF- (1 µg/ml; MP6-XT3; BD PharMingen) and
europium-conjugated avidin (1/1000 in assay buffer; Wallac) were used
for detection. Finally, enhancement buffer (Wallac) was added, and the
plates were analyzed using a Wallac 1420 workstation.
Nitrate concentration was measured in the supernatants after 48 h using Griess method. Briefly, 1% sulfonylic acid/5% H3PO4 and 0.1% naphthylethylenediamine were mixed 1:1 (Griess reagent). An equal volume of Griess reagent and the samples was added to ELISA plates (Costar, Corning, Corning, NY). NaNO2 at various concentrations was used as control. The plates were read in a SpectramaxPlus (Molecular Devices, Sunnyvale, CA) at 540 nm.
Collagen preparations
Rat collagen was extracted from rat Swarm chondrosarcoma after pepsin digestion, as described previously (23), or from lathyritic chondrosarcoma, as described earlier (24). The collagen was dissolved in and stored in 0.1 µM acetic acid.
Induction of arthritis
Male mice between 8 and 12 wk of age were immunized intradermally at the base of the tail with 100 µg rat collagen emulsified in CFA (Difco, Detroit, MI). The mice were boosted at day 35 with 50 µg rat type II collagen (RCII) emulsified in immunofluorescence assay (IFA; Difco). All mice were bled at the peak of the anti-CII Ab production, which is 35 days after immunization with the current protocol. The levels of anti-collagen IgG were determined using quantitative ELISA (25). Clinical scoring was performed, as described earlier (2): briefly, each inflamed toe or knuckle gives one point, whereas an inflamed wrist or ankle gives five points, resulting in a score of 0–15 (5 toes + 5 knuckles + 1 wrist/ankle) for each paw and 0–60 points for each mouse.
Proliferation assay and IFN- ELISA
Mice were immunized in each hind paw with 50 µg RCII
emulsified in CFA, and the draining popliteal lymph nodes were removed
10 days later. The lymph node cells were cultured in triplicates
together with medium alone or the Ags, lathyritic collagen, or the two
RCII peptides CII256–270 and GalCII256–270 for 72 h, and then
labeled with [3H]thymidine (0.5 µCi)
and harvested 15–18 h later. The supernatants were removed before
harvesting and analyzed for IFN- using quantitative ELISA
(26).
Ab transfer
CII-specific mAbs M2139 (IgG2b) and C1 (IgG2a) were purified
from culture supernatants using -bind plus affinity matrix
(Pharmacia). Four- to five-month-old male
IL-10+/- and IL-10-/-
littermates were injected i.v. with a combination of two arthritogenic
mAbs (M2139 and C1) at a total concentration of 3 mg per mouse. Control
mice received an equivalent volume of PBS. On day 5, LPS (50 µg/mice)
was injected i.p. to all the mice. Arthritis was scored for a minimum
of 21 days post-Ab transfer.
Statistical analysis
Mann-Whitney U test, Kruskal-Wallis, or ANOVA tests were used to compare the groups of animals. Differences between strains were regarded as significant if the p value was less than 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice carrying the IL-10-deleted gene on a mixed C57BL/6 x 129/Ola background were backcrossed to B10.Q for nine generations and subsequently checked with genetic markers. The remaining 129/Ola genes were found to be located around the IL-10-encoding gene, and ranged from D1Mit187 at 62 cM to D1Mit312 at 70 cM on chromosome 1 (data not shown). The IL-10-/- and their littermates were given antibiotics in the drinking water throughout their entire lives to prevent development of enterocolitis. No clinical signs of colitis or inflammatory abnormalities, as determined by histopathology of the colon or the small bowel, were observed in the IL-10-/- mice or the IL-10+/- littermates (data not shown). The mice looked healthy, had a normal life span, and showed normal weight development (data not shown).
It has previously been described that IL-10 could suppress
proinflammatory cytokine production by Th1 cells (7). To
study whether lack of IL-10 in this genetic context affected the
production of IL-2, IL-4, and IFN-, lymph node cells were cultured
with the lectin Con A or the superantigen SEA for 6 days, and the
number of IL-2-, IL-4-, and IFN--producing
CD4+ or CD8+ T cells were
investigated using flow cytometry.
The CD8+ T cells from the
IL-10-/- mice showed decreased production of
IFN- (p < 0.05) compared with the heterozygous
littermates after culture with Con A, but not after SEA culture (Table I
). No significant differences were
detected in the number of CD4+ or
CD8+ T cells producing IL-2 and IL-4 between the
IL-10-/- and the
IL-10+/- littermates after in vitro culture
(Table I). These data suggest that there is no Th1 shift in the
IL-10-deficient mice, but rather a decreased tendency to produce
IFN-.
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IL-10 has been reported to decrease the TNF- production and
increase the NO production in macrophage cell lines (9, 27). To investigate whether lack of IL-10 affected the
proinflammatory cytokine TNF-, peritoneal macrophages were activated
with LPS for 24 h, and the supernatants were investigated for
TNF-. The IL-10-/- mice showed an almost
50% reduced production of TNF- compared with the
IL-10+/- (p < 0.05; Fig. 1a). This raised the question
as to whether other macrophage functions, such as the NO production,
were affected in the IL-10-deficient mice. The nitrate levels in
supernatants from IL-10-/- and
IL-10+/- macrophage cultures were measured after
LPS stimulation for 48 h, as an indirect measurement of NO
production. No difference in the nitrate levels between
IL-10-/- and IL-10+/-
was detected (Fig. 1b).
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It has earlier been described that neutralization of IL-10 with
Abs led to an increased severity and an accelerated onset of CIA in
DBA/1 mice (20). To investigate whether a similar disease
pattern was seen in the B10.Q IL-10-deficient mice, male
IL-10-/- mice (n = 43) and
their IL-10+/- littermates (n =
54) at an age of 8–12 wk were immunized with RCII emulsified in CFA.
After 35 days, all mice were boosted with RCII emulsified in IFA. The
IL-10-deficient mice showed an increased incidence of arthritis (Fig. 2a; p < 0.05),
whereas the mean day of onset was similar in the IL-10-deficient mice
as compared with the IL-10+/- littermates. The
severity of the disease, seen as increased numbers of affected joints,
was also markedly increased in the IL-10-deficient mice compared with
the IL-10+/- littermates (Fig. 2b;
p < 0.05). This confirms the previous finding that lack of
IL-10 increases the severity of CIA, but we did not see any effect on
the onset.
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The effect of IL-10 on the anti-CII IgG1/IgG2a ratio has been
debated (17). To investigate the Ab production against CII
in the IL-10-/- and the
IL-10+/- littermates, sera were obtained from
the mice at the peak of the Ab response, day 35. The levels of
CII-specific IgG Abs were measured using quantitative ELISA. However,
anti-CII Abs of the IgG2a isotype could not be measured, as the
IgG2a-encoding gene is deleted in strains such as the C57BL/10, which
carries the Igh1-b allele (28). No significant
variations were seen in the total levels of anti-CII-specific IgG
or IgG isotypes between the IL-10-/- and the
IL-10+/- littermates (Fig. 3), suggesting that the lack of IL-10
does not affect the anti-CII Ab response.
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As described above, lack of IL-10 did not increase the number of
Th1-producing cells after in vitro culture. To investigate whether the
T cell response against RCII was affected by IL-10 deficiency,
IL-10-/- and the
IL-10+/- littermates were immunized with RCII
emulsified in CFA in their hind paws. Ten days later, the draining
lymph node cells from individual mice were investigated for the
proliferative response toward denatured RCII or RCII peptides. There
was no significant difference between the
IL-10-/- and the
IL-10+/- littermates in the IFN- production
upon activation or in the proliferative response to any of the Ags
(Fig. 4).
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T regulatory 1 cells, defined by a high expression level of IL-10
and its low proliferate capacity, have been described to be important
in peripheral regulation of autoreactive T cells (29). To
investigate whether IL-10 deficiency may abrogate tolerance to RCII, we
used the earlier described MMC and TSC mice. Transgenic C3H.Q mice
carrying the immunodominant RCII epitope CII (256–270) in the
cartilage (MMC) or in skin (TSC) are partially or completely tolerized
to CIA, respectively (22). We backcrossed these to the
B10.Q background (with eight backcrossing generations) and found that
both B10.Q MMC and B10.Q TSC were almost completely protected against
arthritis. To investigate whether lack of IL-10 could break this
tolerance, MMC and TSC were crossed with IL-10-deficient mice to obtain
IL-10-/- MMC and
IL-10-/- TSC mice. The
IL-10-/- MMC and
IL-10-/- TSC mice together with their
littermates were immunized with RCII in CFA to induce arthritis. The
mice were examined for arthritis, as described above, and the
experiment was ended after 95 days. Both the
IL-10-/- MMC and
IL-10-/- TSC mice were protected against
arthritis, and the anti-CII Ab response was not significantly
increased compared with the IL-10+/- MMC and
IL-10+/- TSC (Table II). TSC mice did not have a T cell
response against RCII, according to the report of
Malmström et al. (22), suggesting that T
cells recognizing RCII are clonally deleted. This could explain why
IL-10 did not affect the tolerance in this system. T cells from MMC
mice, on the other hand, were only partially tolerized
(22), as they could mount a low IFN- response when
stimulated with RCII. However, IL-10 is probably not important for
maintaining tolerance in the MMC system, as the
IL-10-/- MMC were protected against
arthritis.
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To investigate whether the observed increased severity in
IL-10-deficient mice was related to effects in the effector phase of
arthritis, a newly established anti-CII Ab treatment protocol to
passively induce arthritis was used (our unpublished data). In
this protocol, a combination of two different arthritogenic mAbs,
against the immunodominant B cell epitopes on CII in cartilage, was
injected i.v. into IL-10-/- and
IL-10+/- mice. To enhance the development of
arthritis, LPS was injected i.p. 5 days after immunization
(30). Surprisingly, only 30% of the
IL-10-/- developed arthritis as compared with
90% of the IL-10+/- (p <
0.05; Fig. 5), indicating that IL-10 have
a disease-promoting effect in the effector phase of arthritis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production. In addition, TNF-
production by LPS-activated macrophages was decreased, whereas NO
production was unaffected. The severity of arthritis was
increasedin the IL-10-deficient mice, as expected, but the
onset of arthritis was not affected. The increased susceptibility to
CIA in the IL-10-/- was not due to disruption
of tolerance toward CII. We were also able to show that contrary to the
enhanced incidence of CIA in IL-10-deficient mice, the lack of IL-10
seemed to protect against Ab-induced arthritis. Systemic and locally administered IL-10 has earlier been reported to have protective effect in CIA (14, 15, 16, 17, 18, 20), even though the mechanisms by which IL-10 exerts its mechanism have been debated. The protective role of IL-10 in other autoimmune animal models, such as experimental allergic encephalomyelitis (EAE), and insulin-dependent diabetes mellitus (IDDM) in the nonobese diabetic (NOD) mouse, is also controversial.
IL-10 have been proposed to have disease-promoting effects in the NOD
mouse, as transgenically expressed IL-10 in the pancreatic or
cells increased the severity of diabetes (31, 32), and
injections of neutralizing Abs prevented the disease (33).
However, the picture has become more complex, as IL-10 deficiency in
the NOD mouse did not alter the incidence of IDDM (34),
and delivery of IL-10 plasmid DNA reduced the incidence of insulitis in
the NOD (35). In EAE, on the other hand, IL-10 has been
proposed to be an important factor for remission, as increased IL-10
mRNA levels have been detected during recovery (36) and
IL-10-deficient mice never recovered from the disease (37, 38). The potential of IL-10 to prevent EAE is controversial, as
IL-10 transgenic mice and mice transferred with IL-10-producing
PLP-specific memory T cells were protected from the disease
(39). On the contrary, there was no effect detected in
rats treated with IL-10 or with Abs to IL-10 in EAE induced with
MBP-specific T cells, and injections of IL-10 i.v. even exacerbated the
disease (40).
Some of the opposing findings in experimental models for autoimmune
diseases, such as CIA, EAE, and IDDM, can to some extent be dependent
on the genetic heterogeneity, when using genetically engineered mice.
Large genetic fragments from a donor (embryonic stem cells or a mouse
strain) could include disease-modifying alleles that affect the outcome
of disease and explain the differences found in different experiments.
In our IL-10-/- mouse, there is an
approximately 8-cM fragment from the 129/Ola strain. Therefore, it
cannot be excluded that this fragment harbors genes that affect the
susceptibility to CIA or immunological functions like the TNF-
production in macrophages, Ab isotype switches, and TCR-mediated T cell
proliferation. Nevertheless, it is unlikely that there is a strong
factor other than the deleted IL-10 gene that could explain the
influence on arthritis in the linked fragment, since no locus
associated with development of CIA has been found in this region
(3, 41, 42, 43).
The genetic background of different strains could also affect the response to IL-10, as the production and most likely also the responsiveness to IL-10 seem to be genetically controlled (44, 45, 46, 47). Furthermore, a complex disease like CIA could present different types of pathogenesis depending on the strain. The DBA/1 strain, in which most of the IL-10 experiments have been performed to date, is an example. The DBA/1 develop a very complex and, in contrast to B10.Q, a partly T cell-independent arthritis process (48, 49, 50), and have a genetic control of CIA that differs distinctly from the B10.Q (43). Another complicating issue in studying the effects of IL-10 in vivo is the fact that IL-10-deficient mice in certain genetic backgrounds develop severe colitis (21, 51, 52), suggesting a dramatic and genetically controlled impact on the immune system as well as the inflammatory response. An ongoing inflammation in the bowel could very likely affect phenotypes in the immune system, such as cytokine production of T cells and macrophages as well as the Ab synthesis by plasma cells, and thereby explain some of the controversial observations that are found in different IL-10-deficient mouse strains.
Interestingly, it has recently been reported that IL-10 deficiency on the C57BL/10 background allows the induction of CIA without affecting the severity of the disease (53). The difference of these and the results from our experiments with the B10.Q strain could be due to variant 129 fragments in the background or secondary influences, such as the occurrence of colitis.
The use of different experimental systems could also add to the complexity of the arthritis-modifying properties of IL-10. A problem with systemic injections of IL-10, for example, is that repeatedly high and nonphysiological doses are needed, which makes it difficult to exclude if IL-10 has exerted a direct effect on the specific arthritogenic mechanisms or operated through secondary effects via other biological pathways. Adenoviral vectors could give rise to a similar problem, as in one report, systemic expression of viral IL-10 (vIL-10) could be detected up to 8 days after injection (18). Adenoviral vectors could, however, give rise to additional problems, as they are thought to exert cell-mediated and humoral immune responses at different locations (54, 55, 56, 57) other than the area of interest, such as joint. Contradictory findings have been reported with vIL-10-expressing vectors in CIA. In one report, there was no effect with systemic injections of IL-10 viral vectors (58), whereas others saw a clear effect if treatment started before onset of CIA (17, 18). An immune response against adenoviral vectors that express vIL-10 could divert an immune response and thereby explain some of the controversial results found in the CIA model. Other strategies for delivering IL-10, such as i.p. injections of human IL-10 plasmid, have been tested, and a suppression of CIA after onset was reported (59). However, the human IL-10 plasmid was spread all over the mice, and a general suppression of the immune system could not be excluded in this case either. Nevertheless, these experiments have shown a clear therapeutic potency of IL-10.
Neutralization of IL-10 with Abs, in Ag-specific responses, has also given important insight into the role of IL-10 in CIA. Treatment with anti-IL-10 Abs has been reported to aggravate CIA and accelerate the onset of arthritis (20), indicating that IL-10 could function as a modulator of CIA. A drawback with neutralizing Abs, however, is that an immunologic response against the Abs could take place, and thereby affects the outcome of arthritis. Another problem is that a rapid drop in the IL-10 levels, due to neutralization of IL-10, could produce an acute imbalance in the cytokine milieu. A skewing of the immune response toward a Th1 profile would likely affect the development of arthritis. The use of IL-10-deficient mice overcomes some of these problems, and makes it possible to study how IL-10 deficiency affects certain parts of Ag-specific immune response, such as T cell activation, B cell responses, and macrophage functions.
B cells and anti-CII Abs are known to play an important role in the development of CIA; this is demonstrated by the fact that B cell-deficient mice are resistant to CIA (26) and mAbs against CII epitopes can induce an acute form of arthritis in mice (30). IL-10 is regarded as a potent B cell stimulator in vitro (12), but the effect on anti-CII Abs after systemically administered IL-10 either as rIL-10 or viral vectors has been debated. It has been reported that there was an increase in the IgG1:IgG2a ratio after injection of vIL-10-expressing vectors (17), and a decrease in total anti-CII IgG was observed after repeated high doses of murine rIL-10 in rats (14). However, others did not see any such effects when using similar systems (15, 16, 18, 58, 60). These discrepancies could perhaps be explained by the dose and timing of IL-10 treatment, indicating that much more effort must be put in to investigate how variations in IL-10 levels affect different functions of the immune system.
In contrast to the protective effect of IL-10 in our CIA system, IL-10
seemed to have a disease-promoting effect in the arthritogenicity of
anti-CII Abs, as the IL-10-/- mice
developed anti-CII Ab-transferred arthritis to a lower extent than
the IL-10+/- littermates. A possible explanation
stems from the observation that IL-10 has been reported to increase
expression of FcRI on macrophages (10), which could be
directly involved in the Ab-mediated arthritis. An additional factor
could be that the macrophages in the IL-10-/-
showed a reduced production of the proinflammatory cytokine TNF-. On
the other hand, when the whole immune system is engaged, as after
induction of CIA, the protective effect of IL-10 deficiency on the
macrophage level could be put out of play by other pathways involving
both Ag-specific B cell and T cell activation.
Cytokines are redundant, and this makes it difficult to study their functions. However, it is important to sort out the functions exerted by different cytokines to understand mechanisms involved in autoimmune diseases such as RA and CIA. The present study indicates some of the functional complexities of a cytokine like IL-10. The effect of IL-10 in clinical trials is difficult to predict, and more knowledge about the functions and genetic control of IL-10 is needed before it could be safely tested in patients with autoimmune diseases. To gain further knowledge about IL-10 in autoimmune disease models, we believe it will be important to study effects of IL-10 in a defined genetic and environmental context. Further understanding in how other genes interact with IL-10 functions in different individuals and at different phases of the arthritic process may provide the possibility for formulating a tailored therapeutic window that is applicable to human RA.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Åsa C. M. Johansson, Medical Inflammation Research, I11, BMC, 221 84 Lund, Sweden. E-mail address: Asa.Johansson{at}inflam.lu.se
3 Abbreviations used in this paper: RA, rheumatoid arthritis; C. A, collagen-induced arthritis; CII, type II collagen; EAE, experimental allergic encephalomyelitis; IDDM, insulin-dependent diabetes mellitus; IFA, immunofluorescence assay; ISB, intracellular staining buffer; NOD, nonobese diabetic; RCII, rat type II collagen; SB, staining buffer; SEA, Staphylococcus aureus enterotoxin A; vIL-10, viral IL-10.
Received for publication April 12, 2001. Accepted for publication July 6, 2001.
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