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Local interleukin-12 gene transfer promotes conversion of an acute arthritis to a chronic destructive arthritis.

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ARTHRITIS & RHEUMATISM
Vol. 46, No. 5, May 2002, pp 1379–1389
DOI 10.1002/art.10233
© 2002, American College of Rheumatology
Local Interleukin-12 Gene Transfer Promotes Conversion of an
Acute Arthritis to a Chronic Destructive Arthritis
Leo A. B. Joosten,1 Marleen Heuvelmans-Jacobs,1 Erik Lubberts,1 Fons A. J. van de Loo,1
Andrew C. Bakker,1 Monique M. A. Helsen,1 Carl D. Richards,2 and Wim B. van den Berg1
Objective. To determine whether local overexpression of interleukin-12 (IL-12), a pleiotropic
cytokine that promotes the development of naive T cells
into Th1 cells, could aggravate murine streptococcal
cell wall (SCW)–induced arthritis, a model of acute
arthritis.
Methods. C57BL/6 mice were injected intraarticularly with saline or with 107 plaque-forming units of
control vector (Ad5del70-3) or IL-12 vector (AdmIL12.1) into the right knee joint 1 day before intraarticular
injection of 25 ␮g of SCW fragments. The development
of joint swelling, changes in chondrocyte proteoglycan
(PG) synthesis, and joint destruction were examined
thereafter.
Results. In normal joints, high levels of IL-12 (20
ng/ml on day 1) could be detected after application of
the AdmIL-12.1 vector. After 14 days, expression of
IL-12 was still found locally, but IL-12 alone did not
induce protracted inflammation. Local expression of
IL-12, in combination with SCW, markedly aggravated
SCW-induced arthritis, as determined by enhanced
joint swelling and prolonged inhibition of chondrocyte
PG synthesis. Histologic examination on day 21 showed
a chronic inflammatory process, with persistent cartilage PG depletion, cartilage erosion, and VDIPEN neoepitope expression (indicative of metalloproteinase ac-
tivation). The mixture of IL-12 with SCW fragments did
not lead to a chronic destructive process in mice deficient for recombination-activating gene 2, indicating the
involvement of lymphocytes. In addition, systemic flare
of smoldering SCW arthritis, produced by intravenous
injection of SCW fragments, was only seen in the
AdmIL-12/SCW group.
Conclusion. These results indicate that local overexpression of IL-12 promotes conversion of an acute
arthritis to a chronic destructive immune-mediated
process, which is more susceptible to flares.
Originally described as cytotoxic lymphocyte
maturation factor and natural killer (NK) cell stimulatory factor, interleukin-12 (IL-12) is a 70-kd heterodimeric cytokine consisting of 2 subunits, a 35-kd and
a 40-kd subunit. This heterodimeric structure of IL-12 is
unique among cytokines. IL-12 is produced by phagocytic cells, dendritic cells, B lymphocytes, and NK cells
upon stimulation with lipopolysaccharide, bacteria, and
parasites (1–4).
IL-12 generates the development of naive T cells
into Th1 cells and stimulates the secretion of
interferon-␥ (IFN␥) by differentiated Th1 cells (5,6).
Recently, it was demonstrated that the newly discovered
cytokine IL-18 can act as a costimulus for IFN␥ production and NK cell cytotoxicity induced by IL-12 (7,8).
However, IL-18 itself is not a strong inducer of IFN␥ in
the absence of other cytokines. Through the induction of
IFN␥ and tumor necrosis factor ␣ (TNF␣), IL-12 plays a
crucial role in lipopolysaccharide-induced lethal shock
and the generalized Shwartzman reaction (9,10). During
bacterial infections, IL-12 is considered a principal protective cytokine, where it bridges innate resistance and
antigen-specific immunity (11). However, IL-12 may
unmask Th1-dependent autoimmune responses and may
1
Leo A. B. Joosten, PhD, Marleen Heuvelmans-Jacobs, MSc,
Erik Lubberts, PhD, Fons A. J. van de Loo, PhD, Andrew C. Bakker,
PhD, Monique M. A. Helsen, Wim B. van den Berg, PhD: University
Medical Center Nijmegen, Nijmegen, The Netherlands; 2Carl D.
Richards, PhD: McMaster University, Hamilton, Ontario, Canada.
Address correspondence and reprint requests to Leo A. B.
Joosten, PhD, Rheumatology Research Laboratory, University Medical Center Nijmegen, PO Box 9101, Nijmegen 6500 HB, The Netherlands. E-mail: l.joosten@reuma.azn.nl.
Submitted for publication September 6, 2001; accepted in
revised form December 19, 2001.
1379
1380
be a crucial intermediate in the often-suggested link
between bacterial infections and the expression of autoimmune diseases (12).
Rheumatoid arthritis (RA) is associated with a
Th1 immune response, although typical Th1 cytokines,
such as IL-2, IFN␥, and TNF␤, have been reported to be
scant in synovial fluid or tissues (13). Recently, IL-12
was detected in the synovium of RA patients, and
macrophage-like cells seem to be the main producers of
this cytokine (14). Interestingly, exogenous IL-12 was
shown to cause severe exacerbation of RA activity in an
RA patient being treated for metastatic cervical cancer,
indicating the disease-promoting role of IL-12 (15). This
is consistent with recent data showing that IL-12 levels in
both serum and synovial fluid correlated with RA disease activity (16).
In murine type II collagen–induced arthritis, an
autoimmune model of RA, it was clearly demonstrated
that exogenous, systemically applied IL-12, either alone
or in combination with IL-18, aggravated disease expression by enhancing systemic immunity against type II
collagen (17–19). Both the disease-promoting and immunostimulatory roles of IL-12 in this RA model were
corroborated by elegant studies in IL-12–deficient mice
(20). Apart from a role in autoimmune collagen arthritis,
we recently observed that arthritis induced by local
injection of streptococcal cell wall (SCW) fragments
(21–24) was also dependent on IL-12 (25). Blockade of
endogenous IL-12 during the onset of this macrophagedriven arthritis suppressed the severity of joint inflammation, apparently by the up-regulation of IL-10. However, IL-12 production was brief, and this SCW arthritis
was neither chronic nor destructive.
In the present study, we investigated whether
local overexpression of IL-12 could promote the conversion of an acute arthritis induced by injection of SCW
fragments to a chronic joint disease. To this end, synovial lining cells were transduced with murine IL-12 just
before arthritis induction. To elucidate the role of
lymphocytes in the IL-12–driven conversion, mice deficient in recombination-activating gene 2 (RAG-2) were
used. In addition, systemic exacerbation of the chronic
arthritis was performed to confirm the hyperresponsiveness of the IL-12–transduced arthritic joint to bacterial
fragments. Our findings clearly demonstrate that IL-12
can convert an acute inflammatory process into a
chronic destructive process. Targeting of IL-12 may be
protective against the conversion of arthritic processes
to chronic destructive joint diseases as can be observed
in RA in humans.
JOOSTEN ET AL
MATERIALS AND METHODS
Animals. Male C57BL/6 mice were obtained from
Charles River (Sulzfeld, Germany). Immunodeficient mice
with a targeted disruption of RAG-2 (C57BL/6/Tgh [RAG-2])
were obtained from Centre National de la Recherche Scientifique CDTA (Orleans, France) (26). The mice were housed in
filter-top cages, and water and food were provided ad libitum.
Mice were used at 10–12 weeks of age.
Materials. Bovine serum albumin was purchased from
Sigma (St. Louis, MO). RPMI 1640 medium was obtained
from Life Technologies (Breda, The Netherlands). IFN␥ (catalog no. CMC4034) enzyme-linked immunosorbent assay
(ELISA) cytosets were obtained from BioSource (Camarillo,
CA). Anti–murine IL-12 antibodies (capture 80-3891-01; detection 80-3892-01) were purchased from Genzyme (Cambridge, MA). Murine IL-12 was kindly provided by Dr. S. Wolf
(Genetic Institute, Cambridge, MA). VDIPEN antibodies
were kindly provided by Drs. I. I. Singer and E. K. Bayne
(Merck, Rahway, NJ).
SCW preparation and induction of SCW arthritis.
Streptococcus pyogenes T12 organisms were cultured overnight in Todd-Hewitt broth. Cell walls were prepared as
previously described (21). The supernatant resulting from
centrifugation at 10,000g was used throughout the experiments. These preparations contained 11% muramic acid.
Unilateral arthritis was induced by intraarticular injection of 25
␮g of SCW fragments (rhamnose content) in 6 ␮l of phosphate
buffered saline into the right knee joint of naive mice. Phosphate buffered saline alone was injected into the left knee joint
as a control. Reactivation of SCW arthritis was performed by
intravenous injection of 100 ␮g of SCW fragments on day 28
after the initial intraarticular injection of 25 ␮g of SCW
fragments (21).
Adenoviral murine IL-12 vector and intraarticular
gene transfer. Recombinant adenovirus AdmIL-12.1 was constructed with insertions of the murine IL-12 p35 and p40
subunit cDNA into early region 1 (E1) and E3, respectively
(27). The expression of each cDNA was driven by the human
cytomegalovirus immediate early gene promoter and terminated by the polyadenylation sequence of SV40. The virus was
produced by cotransfection of 293 cells with two plasmids. This
double adenovirus expressed functional heterodimeric murine
IL-12. Transfection with this adenoviral construct results in
active production of murine IL-12, both in vitro and in vivo
(18,28). As a control, we used the empty recombinant
replication-defective adenovirus Ad5del70-3.
Gene transfer was performed by intraarticular injection of naive mice with either 108, 107, 106, or 105 plaqueforming units (PFU) of AdmIL-12.1 or Ad5del70-3 per 6 ␮l of
saline. At different time points, patellae with adjacent tissue
were dissected, and patellae washouts were used for the
determination of IL-12 levels. To examine the effect of local
IL-12 expression on SCW-induced arthritis, AdmIL-12.1 (107
PFU) gene transfer was performed 1 day before the injection
of SCW fragments.
Measurement of joint inflammation. SCW arthritis
was quantified by the 99mTc-uptake method (29). This method
measures by external gamma counting the accumulation of
a small radioisotope at the site of inflammation due to local
increased blood flow and tissue swelling. The severity of
IL-12 GENE TRANSFER AND CHRONIC DESTRUCTIVE ARTHRITIS
inflammation was expressed as the ratio of the 99mTc-uptake in
the right (inflamed) knee joint to that in the left (control) knee
joint. All values ⬎1.10 were considered to represent inflammation.
Determination of chondrocyte proteoglycan (PG) synthesis. Patellae with minimal surrounding tissue were placed in
RPMI 1640 medium with Glutamax, gentamicin (50 ␮g/ml),
and 35S-sulfate (0.74 MBq/ml). After 3 hours of incubation at
37°C in a CO2 incubator, patellae were washed in saline 3
times, fixed in 4% formaldehyde, and subsequently decalcified
in 5% formic acid for 4 hours. Patellae were punched out of
the adjacent tissue, dissolved in 0.5 ml of LumaSolve at 65°C
(Omnilabo, Breda, The Netherlands) and after the addition of
10 ml of Lipoluma (Omnilabo), the 35S-sulfate content was
measured by liquid scintillation counting. Values are presented
as the percentage of 35S-sulfate incorporation in the left
(control) joint.
Cytokine measurements. To determine the levels of
IL-12 and IFN␥ in patellae washouts, patellae were isolated
from inflamed knee joints as previously described (22,24,25).
Patellae were cultured in RPMI 1640 medium (200 ␮l/patella)
for 1 hour at room temperature. The supernatant was then
harvested and centrifuged for 5 minutes at 1,000g. Levels of
IL-12 and IFN␥ were measured by ELISA. The sensitivity of
the ELISA for IL-12 and IFN␥ was 20 pg/ml and 10 pg/ml,
respectively.
Immunohistochemical evaluation of VDIPEN expression. For immunostaining, knee joint sections were deparaffinized, rehydrated, and digested with proteinase-free chondroitinase ABC to remove the chondroitin of the PGs.
Subsequently, sections were treated with 1% hydrogen peroxide, 1.5% normal goat serum, and affinity-purified rabbit
anti-VDIPEN IgG (kindly provided by Drs. I. I. Singer and
E. K. Bayne, Merck, Rahway, NJ). This antibody has been
characterized previously (30–32). Sections were then incubated with biotinylated goat anti-rabbit IgG, and avidin–
streptavidin–peroxidase (Elite kit; Vector, Burlingame, CA).
Sections were counterstained with orange G (Sigma).
Histologic assessment. Mice were killed by ether anesthesia, and whole knee joints were removed and fixed for
4 days in 4% formaldehyde. After decalcification in 5% formic
acid, the specimens were processed for paraffin embedding.
Tissue sections (7 ␮m) were stained with hematoxylin and eosin
or Safranin O. Histopathologic changes were scored according to
the following parameters. Cell infiltration was scored on a scale of
0–3, depending on the amount of inflammatory cells in the
synovial cavity and synovial tissues. PG depletion was determined
with Safranin O staining. The loss of PGs was scored on a scale of
0–3, ranging from fully stained cartilage to destained cartilage or
a complete loss of articular cartilage. Cartilage destruction was
scored on a scale of 0–3, ranging from no damage to fully
destroyed cartilage layers. Histopathologic changes in the knee
joints were scored in the region of the patella/femur on 5
semiserial sections of the joint, spaced 70 ␮m apart. Scoring was
performed on decoded slides by 2 observers (LABJ and MMAH),
as described elsewhere (17,31,32).
Statistical analysis. Differences between experimental
groups were tested using the Mann-Whitney U test, unless
stated otherwise.
1381
RESULTS
Local IL-12 overexpression in knee joints of
naive mice. To examine IL-12 protein overexpression in
the joint, naive mice were injected intraarticularly with
108 PFU of AdmIL-12.1 adenovirus. The highest IL-12
protein expression was found on day 1 (up to 20 ng/ml).
Considerable amounts of IL-12 could be detected in
synovial washouts up to day 14 after AdmIL-12.1 gene
transfer (Figure 1A).
To determine whether overexpression of IL-12 in
naive joints leads to inflammation or inhibition of chondrocyte function, a dose-range experiment was performed with 108, 107, 106, or 105 PFU of AdmIL-12.1
virus particles. On day 2, we measured local levels of
IL-12, the degree of joint inflammation, and levels of
chondrocyte PG synthesis. As shown in Figure 1B, there
was a clear dose-range effect of the virus concentration
on the amount of IL-12 produced. IL-12 produced by the
108 PFU dose induced a mild joint inflammation on day
2, but histologic examination on day 7 showed no
persistent inflammation (data not shown). Analysis of
chondrocyte function on day 2 revealed that injection of
108 PFU induced a 30% inhibition of chondrocyte PG
synthesis. Chondrocyte PG synthesis was completely
restored on days 4 and 7 after injection of the AdmIL12.1 virus. The 107 PFU dose of AdmIL-12.1 resulted in
significant local levels of IL-12 without any signs of
inflammation or inhibition of chondrocyte synthetic
function. This dose was used for IL-12 gene transfer in
combination with SCW arthritis.
Aggravation of SCW-induced arthritis by IL-12
gene transfer. To investigate the disease-promoting effect of local IL-12 expression, we injected 107 PFU of
AdmIL-12.1 adenovirus intraarticularly 1 day before
induction of SCW arthritis in the same joint. As shown
in Figure 2A, there was markedly enhanced joint swelling during the first 2 weeks in the AdmIL-12.1–injected
group compared with the group injected with
Ad5del70-3 or with saline. Significantly increased joint
swelling was still noted on day 21. Intraarticular injection
of 107 PFU of Ad5del70-3 control virus did not significantly influence joint swelling.
Inhibition of chondrocyte PG synthesis is one of
the deleterious effects of joint inflammation on cartilage. Severe inhibition of matrix synthesis can be seen in
the first stages of SCW-induced arthritis (Figure 2B). At
later time points (days 7 and 14), an overshoot phenomenon can be observed. Injection of Ad5del70-3 control
adenoviral vector before arthritis induction showed re-
1382
JOOSTEN ET AL
IFN␥ to get insight into the local production of these
cytokines after induction of arthritis. At several time
points, synovial tissue washouts were analyzed for these
Figure 1. Intraarticular transfer of murine interleukin-12 (IL-12) gene.
A, To determine the duration of IL-12 overexpression, patellae with
surrounding tissue were isolated at different time points after intraarticular gene application with 108 plaque-forming units (PFU) of AdmIL-12.1
adenoviral vector. Patellae were cultured for 1 hour in RPMI 1640
medium, and IL-12 levels were determined by enzyme-linked immunosorbent assay. Values are the mean ⫾ SD of 6 patellae washouts. B, Levels
of IL-12 overexpression after injection of different doses of AdmIL-12.1
adenoviral vector. On day 2 after gene transfer, patellae were isolated,
washouts were prepared, and IL-12 levels were measured as above.
Values are the mean and SD of at least 5 mice per group.
sults similar to those with saline injection. In contrast,
long-lasting inhibition of chondrocyte PG synthesis was
seen in patellar cartilage after AdmIL-12.1 injection.
Severe disturbance of chondrocyte function was noted
up to day 21.
We also determined protein levels of IL-12 and
Figure 2. Deterioration of streptococcal cell wall (SCW)–induced
arthritis by interleukin-12 (IL-12) gene transfer. A, Effect of IL-12
overexpression on joint swelling. On day ⫺1, saline or 107 plaqueforming units of either Ad5del70-3 or AdmIL-12.1 was injected into
the right knee joint of naive mice. One day later, arthritis was induced
in the same joint by intraarticular injection of 25 ␮g of SCW fragments.
Joint swelling was determined at different time points after induction
of arthritis. Values are the mean and SD of at least 6 mice per group.
B, Prolonged inhibition of patellar cartilage chondrocyte proteoglycan
synthesis. At different time points, patellae were isolated from the
inflamed (right) and control (left) knee joints and chondrocyte synthetic function was determined by 35S-sulfate incorporation. Values
are the mean and SD percentage of proteoglycan (PG) synthesis in the
inflamed joint compared with the control joint in at least 6 mice per
group. The experiment was repeated twice, with similar results. ⴱ ⫽
P ⬍ 0.05 versus Ad5del70-3, by Mann-Whitney U test.
IL-12 GENE TRANSFER AND CHRONIC DESTRUCTIVE ARTHRITIS
Table 1.
1383
IL-12 and IFN␥ levels in transduced knee joints with SCW arthritis*
Day 2
Saline
Ad5del70-3
AdmIL-12.1
Day 7
Day 14
IL-12
IFN␥
IL-12
IFN␥
IL-12
IFN␥
120 ⫾ 20
140 ⫾ 30
580 ⫾ 40†
40 ⫾ 20
80 ⫾ 20
290 ⫾ 80†
⬍20
⬍20
439 ⫾ 70†
⬍10
⬍10
170 ⫾ 40†
⬍20
⬍20
410 ⫾ 170†
⬍10
⬍10
70 ⫾ 10†
* Local levels of interleukin-12 (IL-12) and interferon-␥ (IFN␥) were determined in patellae
washouts at several time points after induction of arthritis. One day before the injection of 25 ␮g
of streptococcal cell wall (SCW) fragments, knee joints were transduced with 107 plaque-forming
units of Ad5del70-3 or AdmIL-12.1. IL-12 and IFN␥ levels were measured by enzyme-linked
immunosorbent assay (sensitivity 20 pg/ml and 10 pg/ml, respectively). Values are the mean ⫾ SD
pg/ml in 6 patellae washout samples per time point.
† P ⬍ 0.01 versus Ad5del70-3, by Mann-Whitney U test.
Th1-directing cytokines. As shown in Table 1, during the
early stage (day 2), both IL-12 and IFN␥ could be
detected in synovial washouts of mice injected with
saline, Ad5del70-3, and AdmIL-12.1. However, in the
AdmIL-12.1 group, markedly higher levels of IL-12 and
IFN␥ were found at this time point. At later stages, only
AdmIL-12.1–injected animals expressed detectable levels of IL-12 and IFN␥. The results indicate that local
overexpression of IL-12 generates prolonged production
of proinflammatory cytokines.
Induction of cartilage damage by local overexpression of IL-12. Histologic analysis of whole knee joint
sections revealed that IL-12 gene transfer leads to a
chronic destructive SCW arthritis. As shown in Table 2,
Table 2.
the number of inflammatory cells in the synovial tissue
was significantly enhanced compared with control virus
Ad5del70-3 or with saline on days 7 and 21.
Detailed analysis of cellular influx revealed that
predominately lymphocytes were present in the synovial
tissues of the IL-12-transduced joints (Figure 3). In
addition, loss of matrix PG due to the inflammatory
process was strongly deteriorated after IL-12 overexpression. Furthermore, cartilage destruction, determined as chondrocyte death and cartilage erosions, was
increased in the AdmIL-12.1–injected knee joints, especially on day 21 (Table 2). Figure 4 illustrates the
increased number of dead chondrocytes in the cartilage
due to overexpression of IL-12.
Joint changes on day 21 of SCW arthritis after IL-12 gene transfer*
Day 7
Saline
Ad5del70-3
AdmIL-12.1
Day 21
Saline
Ad5del70-3
AdmIL-12.1
Infiltration
of cells
Chondrocyte
death
Proteoglycan
depletion
Cartilage
erosion
0.8 ⫾ 0.3
1.1 ⫾ 0.3
2.4 ⫾ 0.4†
0.0 ⫾ 0.0
0.0 ⫾ 0.0
0.2 ⫾ 0.3
1.0 ⫾ 0.6
1.6 ⫾ 0.5
2.8 ⫾ 0.4†
0.0 ⫾ 0.0
0.0 ⫾ 0.0
0.0 ⫾ 0.0
0.1 ⫾ 0.1
0.0 ⫾ 0.2
0.9 ⫾ 0.3†
0.0 ⫾ 0.0
0.1 ⫾ 0.1
1.2 ⫾ 0.4†
0.0 ⫾ 0.0
0.3 ⫾ 0.4
1.5 ⫾ 0.6†
0.0 ⫾ 0.0
0.0 ⫾ 0.0
0.4 ⫾ 0.1†
* Histopathologic features of arthritic knee joints after gene transfer with Ad5del70-3 or
AdmIL-12.1 virus. On day ⫺1, saline or 107 plaque-forming units of the adenovirus was injected
intraarticularly into the right knee joint. One day later, 25 ␮g of streptococcal cell wall (SCW)
fragments was injected into the same knee joint. On days 7 and 21, knee joints were obtained and
processed for histologic analysis. Histopathologic features were scored on a scale of 0–3, as
indicated in Materials and Methods. Values are the mean ⫾ SD of at least 6 knee joints per group.
IL-12 ⫽ interleukin-12.
† P ⬍ 0.01 versus Ad5del70-3, by Mann-Whitney U test.
1384
JOOSTEN ET AL
demonstrate the destructive character of the chronic
inflammation caused by the IL-12 overexpression, sections were stained for this neoepitope. VDIPEN was
highly expressed throughout the patellar cartilage layer
in the AdmIL-12.1–treated group (Figure 5C). In contrast, no VDIPEN expression was noted in the
Figure 3. Histopathologic features of streptococcal cell wall (SCW)–
induced arthritis in combination with interleukin-12 (IL-12) gene
transfer. Mouse knee joints were injected with A, saline, B, Ad5del70-3
(107 plaque-forming units [PFU]), or C, AdmIL-12.1 (107 PFU) and
SCW fragments and were harvested and examined on day 21 after
arthritis induction. Note the chronic inflammation and the cellular
infiltrate in the AdmIL-12.1–injected group. (Hematoxylin and eosin
stained; original magnification ⫻ 400.)
VDIPEN neoepitope is a marker of metalloproteinase-mediated cleavage of aggrecan, the major PG
of articular cartilage. Previous studies have revealed that
the VDIPEN neoepitope was more abundant at sites
and stages of advanced damage (31,32). To further
Figure 4. Chondrocyte damage after IL-12 gene transfer. Mouse knee
joints were injected with A, saline, B, Ad5del70-3, or C, AdmIL-12.1 as
in Figure 3. Note the severe chondrocyte death in the cartilage layers
of the patella and femur in the AdmIL-12.1–injected group. (Hematoxylin and eosin stained; original magnification ⫻ 100.) See Figure 3
for definitions.
IL-12 GENE TRANSFER AND CHRONIC DESTRUCTIVE ARTHRITIS
1385
Ad5del70-3–injected (Figure 5B) or the saline-injected
(Figure 5A) groups.
Lymphocyte dependence of the IL-12 gene
transfer–induced conversion to chronic destructive arthritis. To investigate whether a local immune response
is involved in the process of switching from an acute
SCW arthritis to a chronic destructive arthritis, we
performed experiments in RAG-2–deficient mice. These
mice do not establish immune responses to antigens
because their T cells and B cells are not functional (26).
Figure 6. Role of functional lymphocytes in the conversion of an
acute arthritis to a chronic streptococcal cell wall (SCW)–induced
arthritis. Wild-type (C57BL/6) and recombination-activating gene 2
(RAG-2)–deficient mice were injected intraarticularly injected 107
plaque-forming units of AdmIL-12.1 virus. One day later, 25 ␮g of
SCW fragments was injected in the same joint. Joint swelling was
determined on days 3, 14, and 21. Values are the mean and SD of at
least 7 mice per group. ⴱ ⫽ P ⬍ 0.05 versus wild-type mice, by
Mann-Whitney U test.
Figure 5. Metalloproteinase activity as visualized by VDIPEN staining. Shown are immunostained paraffin sections of mouse knee joints
injected with A, saline, B, Ad5del70-3, and C, AdmIL-12.1 (see Figure
3 for details). VDIPEN neoepitope expression was seen only in
cartilage layers of the AdmIL-12.1–injected group. (Original magnification ⫻ 200.)
The results (Figure 6) demonstrated that IL-12 overexpression leads to a local immune-mediated arthritic
process. Overexpression of IL-12 in C57BL/6 mice induced a chronic inflammatory joint disease after injection of SCW fragments, whereas RAG-2–deficient mice
did not develop chronic arthritis. The initial arthritis was
equivalent in the two mouse strains, as determined on
day 3. The results of histologic assessments corroborated
the finding that RAG-2–deficient mice did not generate
a chronic destructive arthritis (Table 3).
Systemic reactivation of chronic SCW arthritis.
In line with the above findings, we investigated whether
the local chronic inflammatory process induced by IL-12
gene transfer could be experiencing a flare. Earlier
studies have shown that chronically inflamed joints
bearing a specific T cell infiltrate can be reactivated with
small amounts of homologous antigen. To this end, mice
were challenged intravenously with 100 ␮g of SCW
fragments on day 28. SCW arthritis in mice of the
saline-injected and the Ad5del70-3–injected groups did
not show a significant flare (Figure 7). In contrast,
intravenous administration of SCW fragments reactivated inflammation in the AdmIL-12.1–injected group
(Figure 7).
These data indicate that local overexpression of
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JOOSTEN ET AL
Table 3.
Joint changes in C57BL/6 and RAG-2⫺/⫺ mice after IL-12 gene transfer*
C57BL/6 mice
RAG-2⫺/⫺ mice
Infiltration
of cells
Chondrocyte
death
Proteoglycan
depletion
Cartilage
erosion
1.0 ⫾ 0.3
0.2 ⫾ 0.1†
1.1 ⫾ 0.5
0.1 ⫾ 0.2†
1.4 ⫾ 0.4
0.2 ⫾ 0.3†
0.5 ⫾ 0.2
0.0 ⫾ 0.0†
* Histopathologic changes in C57BL/6 mice and recombination-activating gene 2 (RAG-2)–
deficient mice after gene transfer with AdmIL-12.1 virus in combination with streptococcal cell wall
(SCW) fragments. On day ⫺1, 107 plaque-forming units of the adenovirus was injected intraarticularly into the right knee joint. One day later, 25 ␮g of SCW fragments was injected into the
same knee joint. On day 21, knee joints were obtained and processed for histologic analysis.
Histopathologic features were scored on a scale of 0–3, as indicated in Materials and Methods.
Values are the mean ⫾ SD of at least 6 knee joints per group. IL-12 ⫽ interleukin-12.
† P ⬍ 0.01 versus C57BL/6 mice, by Mann-Whitney U test.
IL-12 leads to a chronic T cell–dependent hyperresponsive state. Histologic sections obtained before and after
reactivation revealed increased numbers of inflammatory cells, such as polymorphonuclear granulocytes and
monocytes, in the synovial cavity and tissue of the
AdmIL-12.1–injected group that had been rechallenged
with SCW fragments (Figure 8).
Figure 7. Systemic reactivation of SCW-induced arthritis after IL-12
gene transfer. Mice were injected with saline, Ad5del70-3 (107 PFU),
or AdmIL-12.1 (107 PFU) 1 day before the induction of SCW arthritis.
On day 28, joint swelling was examined by a 99mTc-uptake method. An
arthritis flare was then induced by intravenous (i.v.) injection of 100 ␮g
of SCW fragments, and 24 hours later (day 29), joint swelling in the
inflamed (right) and control (left) knee joints was measured. Values
are the mean and SD of at least 6 mice per group. ⴱ ⫽ P ⬍ 0.05 versus
the Ad5del70-3 group, by Mann-Whitney U test. The experiment was
repeated, and similar results were obtained. See Figure 3 for other
definitions.
DISCUSSION
In the present study, we demonstrated that local
overexpression of IL-12 changed an acute joint inflammation induced by bacterial fragments to a chronic
destructive arthritic process. This conversion was shown
to be mediated by lymphocytes, since RAG-2–deficient
mice did not develop a chronic stage of arthritis after
IL-12 gene transfer.
RA is a disease that is characterized by Th1associated pathologic changes. IL-12 is expressed in the
synovial tissue of RA patients and may be involved in the
IFN␥-dominant cytokine production by infiltrating T
cells in joints with chronic arthritis. As previously demonstrated, the major producers of IL-12 in human RA
synovium are mainly CD68-positive macrophages (18,33).
Several cell types, including macrophages and NK cells,
produce IL-12 after in vitro triggering with a range of
stimuli (1–3). Bacteria or fragments of bacteria are the
most potent inducers of IL-12 production by monocyte/
macrophages. Interestingly, gram-positive bacteria are potent inducers of IL-12 production, whereas gram-negative
bacteria are inducers of IL-10 (34).
IL-12 alone, as well as in synergy with IL-18,
stimulates IFN␥ production. It has been demonstrated
that bacteria, in combination with IL-12 or IFN␥, induce
a strong elevation of TNF␣ and nitric oxide production
by macrophages (35). Recently, we demonstrated that
fragments of gram-positive streptococci generated enhanced levels of TNF␣, IL-1, IL-12, and IFN␥ when
injected intraarticularly into the knee joints of naive
mice (25).
Apart from bacterial cell wall fragments, intraarticular application of CpG motifs of bacterial DNA into
the mouse knee joint can induce arthritis. Analysis of
cytokine messenger RNA (mRNA) expression revealed
IL-12 GENE TRANSFER AND CHRONIC DESTRUCTIVE ARTHRITIS
1387
Figure 8. Influx of inflammatory cells after reactivation of chronic arthritis induced by interleukin-12 (IL-12) gene transfer. A,
Ad5del70-3–injected joint on day 28. B, Ad5del70-3–injected joint on day 29 (1 day after reactivation of arthritis). C,
AdmIL-12.1–injected joint on day 28. D, AdmIL-12.1–injected joint on day 29 (1 day after reactivation of arthritis) (see Figure
7 for details). Note the influx of predominantly polymorphonuclear cells in the synovial tissue and joint cavity in the
AdmIL-12.1–injected knee after rechallenge (day 29). (Hematoxylin and eosin stained; original magnification ⫻ 100.)
that IL-12 was up-regulated for several days after injection of CpG oligonucleotide (36). Furthermore, murine
synovial explants cultured for 24 hours in vitro with CpG
oligonucleotides showed an up-regulation of mRNA
levels for IL-12, IL-15, and IL-18 (Joosten LAB, et al:
unpublished observations). These data indicate that
bacterial cell wall or DNA fragments can induce the
production of Th1-directing cytokines in synovial tissues.
As previously shown, IL-12 is involved in SCWinduced arthritis. Blockade of endogenous IL-12 by use
of anti–murine IL-12 antibodies suppressed joint swelling in SCW-induced arthritis. In addition, reduced levels
of IFN␥ and IL-1 were found in washouts of synovial
tissue explants (25). IL-1 is a pleiotropic cytokine that
exerts a direct cartilage-destructive property as well as
an immune-stimulatory activity. It has been shown that
IL-1␤ is a potent costimulus of IFN␥ production by a
subset of NK cells following infectious insult (37). The
important role of IL-1 in the development of chronic
arthritis was nicely demonstrated in IL-1␣/␤ deficient
mice (23). It is unlikely that IL-12 plays a direct role in
the production of IL-1, since IL-12 administration to
mice did not result in elevated serum levels of IL-1␣ or
IL-1␤ (38).
Gene transfer by adenoviral vectors has been
shown to be an effective way to generate overexpression
of certain cytokines or cytokine inhibitors (14,39). Synovial cells can be targeted by adenoviral constructs to
achieve high levels of cytokines, such as IL-4, IL-10, or
IL-17 (40,41). In the present study, we showed that high
levels of IL-12 were produced up to day 14 after local
application of 1 ⫻ 107 PFU of AdmIL-12.1. Interestingly, apart from IL-12, enhanced levels of IFN␥ were
found in transduced knee joints (Table 1).
IL-12 is thought to play a pivotal role in inducing
Th1 cell–mediated organ-specific autoimmune diseases,
1388
as described previously (42). The role of IL-12 in the
induction of chronic Th1-mediated diseases, such as
experimental colitis and arthritis, is well known. IL-12
exposure or the use of IL-12–deficient mice has revealed
that IL-12 is a crucial immune-stimulatory cytokine for
the development of experimentally induced arthritis
(13,15,16). This is the first study showing that local
overexpression of IL-12 in combination with an acute
inflammation generates a chronic immune-mediated destructive process. It has recently been shown that bacteria or fragments of bacteria activate nuclear factor ␬B
directly and, via induction of cytokines, activate signal
transducer and activator of transcription 1 (STAT-1)
and STAT-3 (43) in macrophages. This is consistent with
our previous findings that SCW fragments generate the
production of cytokines, such as TNF␣, IL-1, IL-12,
IL-18, and IFN␥, when injected directly into the mouse
knee joint (25,44).
To corroborate the finding that local lymphocytes
were involved, we performed experiments in immunodeficient mice with a targeted disruption in the
recombination-activation gene 2, which is expressed only
in lymphocytes (26). These mice lack functional T and B
cells, as shown in several models of experimental autoimmune diseases (45,46). We found that local overexpression of IL-12 did not convert acute SCW-induced
arthritis to a chronic arthritis in the RAG-2–deficient
mice, indicating that T and B cells are involved in the
generation of the chronic destructive arthritis (Figure 6).
Involvement of a local IL-12–induced hyperreactive
immune response was further investigated by reactivation of the arthritic process by intravenous challenge
with SCW fragments (Figures 7 and 8). We demonstrated previously that this latter reactivation was
dependent on lymphocytes, since antilymphocyte treatment abrogated the exacerbation of arthritis (21). Taken
together, these findings demonstrate that IL-12 can
convert modest joint inflammation to a T cell–mediated
destructive joint pathology.
Whether IL-12 contributes to the development of
the chronic destructive process in human RA is still not
known, since anti–IL-12 treatment is not performed in
the clinic. However, it is known that several bacterial
infections correlate with arthritis. Apart from septic
arthritis, arthritis occurs in patients with Lyme disease
and infections of the throat and gastrointestinal tract.
IL-12 levels are elevated in patients with infections, and
it might be that IL-12 is the important cytokine that
generates a local autoimmune disease. It has been
postulated that a possible cause of joint destruction in
septic arthritis is the presence of CpG motifs in the joint
JOOSTEN ET AL
(47). CpG motifs can induce local production of IL-12,
and the acute bacterial-induced inflammation could be
transformed to a destructive arthritis. Furthermore,
CpG motifs present in Chlamydia can act as an adjuvant
to trigger immune responses to self antigens, causing
heart diseases (48). Enhanced local levels of IL-12, due
to the presence of bacterial fragments or CpG motifs,
may be the driving force responsible for the development of an autoimmune response to antigens, such as
PGs, type II collagen, or human cartilage gp-39.
IL-12 may be a novel target in human RA, since
IL-12 may promote the spreading of autoimmune processes to other joints. However, IL-12 inhibition should
be limited to the joint to obtain an efficient innate
immune response.
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